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Sample records for phytochelatin synthase pcs

  1. Heteroexpression of the wheat phytochelatin synthase gene (TaPCS1) in rice enhances cadmium sensitivity

    Institute of Scientific and Technical Information of China (English)

    Feijuan Wang; Zhubing Wang; Cheng Zhu

    2012-01-01

    Phytochelatin synthase (PCS) (EC 2.3.2.15) catalyzes the final step of phytochelatins (PCs) biosynthesis.PCs are a family of cysteine-rich thiol-reactive and heavy metalbinding peptides that play an important role in sequestration and detoxification of heavy metals in plants.Previous studies have indicated that plants that overexpressed PCS displayed contrasting phenotypes,ranging from enhanced cadmium (Cd) tolerance to Cd hypersensitivity in Arabidopsis thaliana.In this study,the wheat phytochelatin synthase gene,TaPCS1,was heteroexpressed in wildtype rice (Oryza sativa L.,cv.Zhonghua 11) to evaluate the relationship between synthesis of PCs and Cd tolerance in rice.Data showed that the heteroexpression of TaPCS1 in rice enhanced Cd sensitivity and significantly increased Cd accumulation in shoots,but not in roots.Additionally,the PCS line exhibited a much higher content of PCs and non-protein thiols (NPTs) in shoots.Prominent changes in NPT composition led to reduced glutathione pool depletion and higher Cd content in cell organelles in shoots,followed by higher oxidative stress,which might result in Cd sensitivity.Therefore,the heteroexpression of TaPCS1 in rice is capable of increasing Cd accumulation in rice shoots and enhancing Cd sensitivity.

  2. Functional characterization of an unusual phytochelatin synthase, LjPCS3, of Lotus japonicus.

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    Ramos, Javier; Naya, Loreto; Gay, Marina; Abián, Joaquín; Becana, Manuel

    2008-09-01

    In plants and many other organisms, phytochelatin synthase (PCS) catalyzes the synthesis of phytochelatins from glutathione in the presence of certain metals and metalloids. We have used budding yeast (Saccharomyces cerevisiae) as a heterologous system to characterize two PCS proteins, LjPCS1 and LjPCS3, of the model legume Lotus japonicus. Initial experiments revealed that the metal tolerance of yeast cells in vivo depends on the concentrations of divalent cations in the growth medium. Detailed in vivo (intact cells) and in vitro (broken cells) assays of PCS activity were performed with yeast expressing the plant enzymes, and values of phytochelatin production for each metal tested were normalized with respect to those of cadmium to correct for the lower expression level of LjPCS3. Our results showed that lead was the best activator of LjPCS1 in the in vitro assay, whereas, for both assays, arsenic, iron, and aluminum were better activators of LjPCS3 and mercury was similarly active with the two enzymes. Most interestingly, zinc was a powerful activator, especially of LjPCS3, when assayed in vivo, whereas copper and silver were the strongest activators in the in vitro assay. We conclude that the in vivo and in vitro assays are useful and complementary to assess the response of LjPCS1 and LjPCS3 to a wide range of metals and that the differences in the C-terminal domains of the two proteins are responsible for their distinct expression levels or stabilities in heterologous systems and patterns of metal activation.

  3. Heterologous expression of Ceratophyllum demersum phytochelatin synthase, CdPCS1, in rice leads to lower arsenic accumulation in grain.

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    Shri, Manju; Dave, Richa; Diwedi, Sanjay; Shukla, Devesh; Kesari, Ravi; Tripathi, Rudra Deo; Trivedi, Prabodh Kumar; Chakrabarty, Debasis

    2014-07-22

    Recent studies have identified rice (Oryza sativa) as a major dietary source of inorganic arsenic (As) and poses a significant human health risk. The predominant model for plant detoxification of heavy metals is complexation of heavy metals with phytochelatins (PCs), synthesized non-translationally by PC synthase (PCS) and compartmentalized in vacuoles. In this study, in order to restrict As in the rice roots as a detoxification mechanism, a transgenic approach has been followed through expression of phytochelatin synthase, CdPCS1, from Ceratophyllum demersum, an aquatic As-accumulator plant. CdPCS1 expressing rice transgenic lines showed marked increase in PCS activity and enhanced synthesis of PCs in comparison to non-transgenic plant. Transgenic lines showed enhanced accumulation of As in root and shoot. This enhanced metal accumulation potential of transgenic lines was positively correlated to the content of PCs, which also increased several-fold higher in transgenic lines. However, all the transgenic lines accumulated significantly lower As in grain and husk in comparison to non-transgenic plant. The higher level of PCs in transgenic plants relative to non-transgenic presumably allowed sequestering and detoxification of higher amounts of As in roots and shoots, thereby restricting its accumulation in grain.

  4. The Pb-hyperaccumulator aquatic fern Salvinia minima Baker, responds to Pb(2+) by increasing phytochelatins via changes in SmPCS expression and in phytochelatin synthase activity.

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    Estrella-Gómez, N; Mendoza-Cózatl, D; Moreno-Sánchez, R; González-Mendoza, D; Zapata-Pérez, O; Martínez-Hernández, A; Santamaría, J M

    2009-03-01

    The relationship between accumulation of Pb(2+) and the activation of chelation and metal sequestration mechanisms mediated by phytochelatins (PC) was analyzed in the Pb(2+) hyperaccumulator aquatic fern Salvinia minima, after exposure to 40microM Pb(NO(3))(2). The tissue accumulation pattern of lead and the phytochelatin biosynthesis responses were analyzed in both, S. minima submerged root-like modified fronds (here named "roots"), and in its aerial leaf-like fronds ("leaves"). S. minima roots accumulated a significantly higher concentrations of Pb(+2) than leaves did. Accumulation of Pb(2+) in roots was bi-phasic with a first uptake phase reached after 3h exposure and a second higher uptake phase reached after 24h exposure. In leaves, a single delayed, smaller uptake phase was attained only after 9h of exposure. In roots lead accumulation correlated with an increased phytochelatin synthase (PCS) activity and an enhanced PC production. A higher proportion of polymerized PC(4) was observed in both tissues of exposed S. minima plants relative to unexposed ones, although a higher concentration of PC(4) was found in roots than in leaves. PCS activity and Pb(2+) accumulation was also higher in roots than in leaves. The expression levels of the S. minima PCS gene (SmPCS), in response to Pb(2+) treatment, were also evaluated. In S. minima leaves, the accumulation of Pb(2+) correlated with a marked increase in expression of SmPCS, suggesting a transcriptional regulation in the PCS activation and PC accumulation in this S. minima tissue. However, in roots, the basal expression of SmPCS was down-regulated after Pb(2+) treatment. This fact did not correlate with the later but strong increase in both, PCS activity and PC production; suggesting that the PC biosynthesis activation in S. minima roots occurs only by post-translational activation of PCS. Taken together, our data suggest that the accumulation of PC in S. minima is a direct response to Pb(2+) accumulation, and

  5. Molecular cloning and characterization of a phytochelatin synthase gene, PvPCS1, from Pteris vittata L.

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    Dong, Ruibin; Formentin, Elide; Losseso, Carmen; Carimi, Francesco; Benedetti, Piero; Terzi, Mario; Schiavo, Fiorella Lo

    2005-12-01

    Pteris vittata L. is a staggeringly efficient arsenic hyperaccumulator that has been shown to be capable of accumulating up to 23,000 microg arsenic g(-1), and thus represents a species that may fully exploit the adaptive potential of plants to toxic metals. However, the molecular mechanisms of adaptation to toxic metal tolerance and hyperaccumulation remain unknown, and P. vittata genes related to metal detoxification have not yet been identified. Here, we report the isolation of a full-length cDNA sequence encoding a phytochelatin synthase (PCS) from P. vittata. The cDNA, designated PvPCS1, predicts a protein of 512 amino acids with a molecular weight of 56.9 kDa. Homology analysis of the PvPCS1 nucleotide sequence revealed that it has low identity with most known plant PCS genes except AyPCS1, and the homology is largely confined to two highly conserved regions near the 5'-end, where the similarity is as high as 85-95%. The amino acid sequence of PvPCS1 contains two Cys-Cys motifs and 12 single Cys, only 4 of which (Cys-56, Cys-90/91, and Cys-109) in the N-terminal half of the protein are conserved in other known PCS polypeptides. When expressed in Saccharomyces cerevisae, PvPCS1 mediated increased Cd tolerance. Cloning of the PCS gene from an arsenic hyperaccumulator may provide information that will help further our understanding of the genetic basis underlying toxic metal tolerance and hyperaccumulation.

  6. Expression of Caenorhabditis elegans PCS in the AtPCS1-deficient Arabidopsis thaliana cad1-3 mutant separates the metal tolerance and non-host resistance functions of phytochelatin synthases.

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    Kühnlenz, Tanja; Westphal, Lore; Schmidt, Holger; Scheel, Dierk; Clemens, Stephan

    2015-11-01

    Phytochelatin synthases (PCS) play key roles in plant metal tolerance. They synthesize small metal-binding peptides, phytochelatins, under conditions of metal excess. Respective mutants are strongly cadmium and arsenic hypersensitive. However, their ubiquitous presence and constitutive expression had long suggested a more general function of PCS besides metal detoxification. Indeed, phytochelatin synthase1 from Arabidopsis thaliana (AtPCS1) was later implicated in non-host resistance. The two different physiological functions may be attributable to the two distinct catalytic activities demonstrated for AtPCS1, that is the dipeptidyl transfer onto an acceptor molecule in phytochelatin synthesis, and the proteolytic deglycylation of glutathione conjugates. In order to test this hypothesis and to possibly separate the two biological roles, we expressed a phylogenetically distant PCS from Caenorhabditis elegans in an AtPCS1 mutant. We confirmed the involvement of AtPCS1 in non-host resistance by showing that plants lacking the functional gene develop a strong cell death phenotype when inoculated with the potato pathogen Phytophthora infestans. Furthermore, we found that the C. elegans gene rescues phytochelatin synthesis and cadmium tolerance, but not the defect in non-host resistance. This strongly suggests that the second enzymatic function of AtPCS1, which remains to be defined in detail, is underlying the plant immunity function.

  7. Cloning and characterization of a Phragmites australis phytochelatin synthase (PaPCS and achieving Cd tolerance in tall fescue.

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    Cuizhu Zhao

    Full Text Available The production of phytochelatins (PCs provides an important means for plants to achieve tolerance to cadmium (Cd toxicity. A reed gene encoding PC synthase (PaPCS was isolated and its function tested through its heterologous expression in a strain of yeast sensitive to Cd. Subsequently, the Cd sensitive and high biomass accumulating species tall fescue was transformed either with PaPCS or PaGCS (a glutamyl cysteine synthetase gene of reed on their own (single transformants, or with both genes together in the same transgene cassette (double transformant. The single and double transformants showed greater Cd tolerance and accumulated more Cd and PC than wild type plants, and their Cd leaf/root ratio content was higher. The ranking in terms of Cd and PC content for the various transgenic lines was double transformants>PaGCS single transformants>PaPCS single transformants>wild type. Thus PaGCS appears to exert a greater influence than PaPCS over PC synthesis and Cd tolerance/accumulation. The double transformant has interesting potential for phytoremediation.

  8. Transgenic Indian mustard (Brassica juncea) plants expressing an Arabidopsis phytochelatin synthase (AtPCS1) exhibit enhanced As and Cd tolerance.

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    Gasic, Ksenija; Korban, Schuyler S

    2007-07-01

    Phytochelatins (PCs) are post-translationally synthesized thiol reactive peptides that play important roles in detoxification of heavy metal and metalloids in plants and other living organisms. The overall goal of this study is to develop transgenic plants with increased tolerance for and accumulation of heavy metals and metalloids from soil by expressing an Arabidopsis thaliana AtPCS1 gene, encoding phytochelatin synthase (PCS), in Indian mustard (Brassica juncea L.). A FLAG-tagged AtPCS1 gDNA, under its native promoter, is expressed in Indian mustard, and transgenic pcs lines have been compared with wild-type plants for tolerance to and accumulation of cadmium (Cd) and arsenic (As). Compared to wild type plants, transgenic plants exhibit significantly higher tolerance to Cd and As. Shoots of Cd-treated pcs plants have significantly higher concentrations of PCs and thiols than those of wild-type plants. Shoots of wild-type plants accumulated significantly more Cd than those of transgenic plants, while accumulation of As in transgenic plants was similar to that in wild type plants. Although phytochelatin synthase improves the ability of Indian mustard to tolerate higher levels of the heavy metal Cd and the metalloid As, it does not increase the accumulation potential of these metals in the above ground tissues of Indian mustard plants.

  9. Arabidopsis thaliana phytochelatin synthase 2 is constitutively active in vivo and can rescue the growth defect of the PCS1-deficient cad1-3 mutant on Cd-contaminated soil.

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    Kühnlenz, Tanja; Schmidt, Holger; Uraguchi, Shimpei; Clemens, Stephan

    2014-08-01

    Phytochelatins play a key role in the detoxification of metals in plants and many other eukaryotes. Their formation is catalysed by phytochelatin synthases (PCS) in the presence of metal excess. It appears to be common among higher plants to possess two PCS genes, even though in Arabidopsis thaliana only AtPCS1 has been demonstrated to confer metal tolerance. Employing a highly sensitive quantification method based on ultraperformance electrospray ionization quadrupole time-of-flight mass spectrometry, we detected AtPCS2-dependent phytochelatin formation. Overexpression of AtPCS2 resulted in constitutive phytochelatin accumulation, i.e. in the absence of metal excess, both in planta and in a heterologous system. This indicates distinct enzymatic differences between AtPCS1 and AtPCS2. Furthermore, AtPCS2 was able to partially rescue the Cd hypersensitivity of the AtPCS1-deficient cad1-3 mutant in a liquid seedling assay, and, more importantly, when plants were grown on soil spiked with Cd to a level that is close to what can be found in agricultural soils. No rescue was found in vertical-plate assays, the most commonly used method to assess metal tolerance. Constitutive AtPCS2-dependent phytochelatin synthesis suggests a physiological role of AtPCS2 other than metal detoxification. The differences observed between wild-type plants and cad1-3 on Cd soil demonstrated: (i) the essentiality of phytochelatin synthesis for tolerating levels of Cd contamination that can naturally be encountered by plants outside of metal-rich habitats, and (ii) a contribution to Cd accumulation under these conditions.

  10. Evolution and function of phytochelatin synthases.

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    Clemens, Stephan

    2006-02-01

    Both essential and non-essential transition metal ions can easily be toxic to cells. The physiological range for essential metals between deficiency and toxicity is therefore extremely narrow and a tightly controlled metal homeostasis network to adjust to fluctuations in micronutrient availability is a necessity for all organisms. One protective strategy against metal excess is the expression of high-affinity binding sites to suppress uncontrolled binding of metal ions to physiologically important functional groups. The synthesis of phytochelatins, glutathione-derived metal binding peptides, represents the major detoxification mechanism for cadmium and arsenic in plants and an unknown range of other organisms. A few years ago genes encoding phytochelatin synthases (PCS) were cloned from plants, fungi and nematodes. Since then it has become apparent that PCS genes are far more widespread than ever anticipated. Searches in sequence databases indicate PCS expression in representatives of all eukaryotic kingdoms and the presence of PCS-like proteins in several prokaryotes. The almost ubiquitous presence in the plant kingdom and beyond as well as the constitutive expression of PCS genes and PCS activity in all major plant tissues are still mysterious. It is unclear, how the extremely rare need to cope with an excess of cadmium or arsenic ions could explain the evolution and distribution of PCS genes. Possible answers to this question are discussed. Also, the molecular characterization of phytochelatin synthases and our current knowledge about the enzymology of phytochelatin synthesis are reviewed.

  11. Characterization of the phytochelatin synthase of Schistosoma mansoni.

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    Debalina Ray

    2011-05-01

    Full Text Available Treatment for schistosomiasis, which is responsible for more than 280,000 deaths annually, depends exclusively on the use of praziquantel. Millions of people are treated annually with praziquantel and drug resistant parasites are likely to evolve. In order to identify novel drug targets the Schistosoma mansoni sequence databases were queried for proteins involved in glutathione metabolism. One potential target identified was phytochelatin synthase (PCS. Phytochelatins are oligopeptides synthesized enzymatically from glutathione by PCS that sequester toxic heavy metals in many organisms. However, humans do not have a PCS gene and do not synthesize phytochelatins. In this study we have characterized the PCS of S. mansoni (SmPCS. The conserved catalytic triad of cysteine-histidine-aspartate found in PCS proteins and cysteine proteases is also found in SmPCS, as are several cysteine residues thought to be involved in heavy metal binding and enzyme activation. The SmPCS open reading frame is considerably extended at both the N- and C-termini compared to PCS from other organisms. Multiple PCS transcripts are produced from the single encoded gene by alternative splicing, resulting in both mitochondrial and cytoplasmic protein variants. Expression of SmPCS in yeast increased cadmium tolerance from less than 50 µM to more than 1,000 µM. We confirmed the function of SmPCS by identifying PCs in yeast cell extracts using HPLC-mass spectrometry. SmPCS was found to be expressed in all mammalian stages of worm development investigated. Increases in SmPCS expression were seen in ex vivo worms cultured in the presence of iron, copper, cadmium, or zinc. Collectively, these results indicate that SmPCS plays an important role in schistosome response to heavy metals and that PCS is a potential drug target for schistosomiasis treatment. This is the first characterization of a PCS from a parasitic organism.

  12. Phytochelatin synthase activity as a marker of metal pollution

    Energy Technology Data Exchange (ETDEWEB)

    Zitka, Ondrej; Krystofova, Olga; Sobrova, Pavlina [Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno (Czech Republic); Adam, Vojtech [Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno (Czech Republic); Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, CZ-616 00 Brno (Czech Republic); Zehnalek, Josef; Beklova, Miroslava [Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno (Czech Republic); Kizek, Rene, E-mail: kizek@sci.muni.cz [Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno (Czech Republic); Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, CZ-616 00 Brno (Czech Republic)

    2011-08-30

    Highlights: {yields} New tool for determination of phytochelatin synthase activity. {yields} The optimization of experimental condition for determination of the enzyme activity. {yields} First evaluation of K{sub m} for the enzyme. {yields} The effects of cadmium (II) not only on the activity of the enzyme but also on K{sub m}. -- Abstract: The synthesis of phytochelatins is catalyzed by {gamma}-Glu-Cys dipeptidyl transpeptidase called phytochelatin synthase (PCS). Aim of this study was to suggest a new tool for determination of phytochelatin synthase activity in the tobacco BY-2 cells treated with different concentrations of the Cd(II). After the optimization steps, an experiment on BY-2 cells exposed to different concentrations of Cd(NO{sub 3}){sub 2} for 3 days was performed. At the end of the experiment, cells were harvested and homogenized. Reduced glutathione and cadmium (II) ions were added to the cell suspension supernatant. These mixtures were incubated at 35 {sup o}C for 30 min and analysed using high performance liquid chromatography coupled with electrochemical detector (HPLC-ED). The results revealed that PCS activity rises markedly with increasing concentration of cadmium (II) ions. The lowest concentration of the toxic metal ions caused almost three fold increase in PCS activity as compared to control samples. The activity of PCS (270 fkat) in treated cells was more than seven times higher in comparison to control ones. K{sub m} for PCS was estimated as 2.3 mM.

  13. The role of subcellular distribution of cadmium and phytochelatins in the generation of distinct phenotypes of AtPCS1- and CePCS3-expressing tobacco.

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    Wojas, Sylwia; Ruszczyńska, Anna; Bulska, Ewa; Clemens, Stephan; Antosiewicz, Danuta Maria

    2010-08-15

    Exposure to Cd2+ leads to activation of phytochelatin synthase (PCS) and the formation of phytochelatins (PCs) in the cytosol. Binding of Cd by PCs and the subsequent transport of PC-Cd complexes to the vacuole are essential for Cd tolerance. Attempts to improve Cd detoxification by PCS overexpression have resulted in contrasting plant phenotypes, ranging from enhanced Cd tolerance to Cd hypersensitivity. In the present paper, changes in the subcellular phytochelatin, glutathione, gamma-glutamylcysteine and cadmium vacuolar and cytosolic distribution underlying these phenotypes were examined. Cadmium and PCs levels were determined in protoplasts and vacuoles isolated from leaves of Nicotiana tabacum expressing either of two phytochelatin synthase genes, AtPCS1 and CePCS (differing in their level of Cd tolerance; being Cd hypersensitive or more Cd-tolerant as compared to wild-type plants, respectively). We showed that Cd hypersensitivity of AtPCS1-expressing tobacco results from a significant decrease in both the cytosolic and vacuolar pool of PCs, indicating a decreased cadmium detoxification capacity. By contrast, enhanced Cd tolerance of CePCS plants was accompanied by an increased cytosolic and vacuolar SH of PC/Cd ratio, suggesting more efficient Cd detoxification. Surprisingly, the substantially reduced level of PCs did not influence Cd accumulation in vacuoles of AtPCS1-transformed tobacco (relative to the wild-type), which suggests the important role of mechanisms other than PC-Cd transport in Cd translocation to the vacuole. Our data suggest that the key role of the PCs in Cd tolerance is temporary binding of Cd2+ in the cytosol, and contrary to the current view, their contribution to cadmium sequestration seems to be less important.

  14. Deletion of Phytochelatin Synthase Modulates the Metal Accumulation Pattern of Cadmium Exposed C. elegans

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    Yona J. Essig

    2016-02-01

    Full Text Available Environmental metal pollution is a growing health risk to flora and fauna. It is therefore important to fully elucidate metal detoxification pathways. Phytochelatin synthase (PCS, an enzyme involved in the biosynthesis of phytochelatins (PCs, plays an important role in cadmium detoxification. The PCS and PCs are however not restricted to plants, but are also present in some lower metazoans. The model nematode Caenorhabditis elegans, for example, contains a fully functional phytochelatin synthase and phytochelatin pathway. By means of a transgenic nematode strain expressing a pcs-1 promoter-tagged GFP (pcs-1::GFP and a pcs-1 specific qPCR assay, further evidence is presented that the expression of the C. elegans phytochelatin synthase gene (pcs-1 is transcriptionally non-responsive to a chronic (48 h insult of high levels of zinc (500 μM or acute (3 h exposures to high levels of cadmium (300 μM. However, the accumulation of cadmium, but not zinc, is dependent on the pcs-1 status of the nematode. Synchrotron based X-ray fluorescence imaging uncovered that the cadmium body burden increased significantly in the pcs-1(tm1748 knockout allele. Taken together, this suggests that whilst the transcription of pcs-1 may not be mediated by an exposure zinc or cadmium, it is nevertheless an integral part of the cadmium detoxification pathway in C. elegans.

  15. Deletion of Phytochelatin Synthase Modulates the Metal Accumulation Pattern of Cadmium Exposed C. elegans

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    Essig, Yona J.; Webb, Samuel M.; Stürzenbaum, Stephen R.

    2016-01-01

    Environmental metal pollution is a growing health risk to flora and fauna. It is therefore important to fully elucidate metal detoxification pathways. Phytochelatin synthase (PCS), an enzyme involved in the biosynthesis of phytochelatins (PCs), plays an important role in cadmium detoxification. The PCS and PCs are however not restricted to plants, but are also present in some lower metazoans. The model nematode Caenorhabditis elegans, for example, contains a fully functional phytochelatin synthase and phytochelatin pathway. By means of a transgenic nematode strain expressing a pcs-1 promoter-tagged GFP (pcs-1::GFP) and a pcs-1 specific qPCR assay, further evidence is presented that the expression of the C. elegans phytochelatin synthase gene (pcs-1) is transcriptionally non-responsive to a chronic (48 h) insult of high levels of zinc (500 μM) or acute (3 h) exposures to high levels of cadmium (300 μM). However, the accumulation of cadmium, but not zinc, is dependent on the pcs-1 status of the nematode. Synchrotron based X-ray fluorescence imaging uncovered that the cadmium body burden increased significantly in the pcs-1(tm1748) knockout allele. Taken together, this suggests that whilst the transcription of pcs-1 may not be mediated by an exposure zinc or cadmium, it is nevertheless an integral part of the cadmium detoxification pathway in C. elegans. PMID:26907254

  16. Characterization of the Phytochelatin Synthase from the Human Parasitic Nematode Ancylostoma ceylanicum

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    Rigouin, Coraline; Vermeire, Jon J.; Nylin, Elyse; Williams, David L.

    2013-01-01

    Hookworm disease is a debilitating worm infection that affects hundreds of millions of people. Despite the existence of anthelmintic drugs, reports have testified of a decrease in efficacy of these drugs. Therefore, it is imperative to find new drugs and drug targets for hookworm disease treatment. In this study we identify the gene encoding the phytochelatin synthase in the human hookworm, Ancylostoma ceylanicum (AcePCS). Phytochelatin synthase catalyzes the production of metal chelating peptides, the phytochelatins, from glutathione (GSH). In plants, algae, and fungi phytochelatin production is important for metal tolerance and detoxification. Phytochelatin synthase proteins also function in the elimination of xenobiotics by processing GSH S-conjugates. We found that in vitro AcePCS could both synthesize phytochelatins and hydrolyze a GSH S-conjugate. Interestingly, the enzyme works through a thiol-dependant and, notably, metal-independent mechanism for both transpeptidase (phytochelatin synthesis) and peptidase (hydrolysis of GSH S-conjugates) activities. AcePCS mRNAs are expressed in vivo throughout the life cycle of A. ceylanicum. Mature adult male hookworms isolated from the small intestines of their hosts displayed significantly enhanced expression of AcePCS with transcript levels 5-fold greater than other developmental forms. Although the role of AcePCS in A. ceylanicum biology has yet to be fully investigated the results reported here provide encouraging evidence of the potential that this enzyme holds as a target for new chemotherapeutic intervention. PMID:23916800

  17. Characterization of the phytochelatin synthase from the human parasitic nematode Ancylostoma ceylanicum.

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    Rigouin, Coraline; Vermeire, Jon J; Nylin, Elyse; Williams, David L

    2013-09-01

    Hookworm disease is a debilitating worm infection that affects hundreds of millions of people. Despite the existence of anthelmintic drugs, reports have testified of a decrease in efficacy of these drugs. Therefore, it is imperative to find new drugs and drug targets for hookworm disease treatment. In this study we identify the gene encoding the phytochelatin synthase in the human hookworm, Ancylostoma ceylanicum (AcePCS). Phytochelatin synthase catalyzes the production of metal chelating peptides, the phytochelatins, from glutathione (GSH). In plants, algae, and fungi phytochelatin production is important for metal tolerance and detoxification. Phytochelatin synthase proteins also function in the elimination of xenobiotics by processing GSH S-conjugates. We found that in vitro AcePCS could both synthesize phytochelatins and hydrolyze a GSH S-conjugate. Interestingly, the enzyme works through a thiol-dependent and, notably, metal-independent mechanism for both transpeptidase (phytochelatin synthesis) and peptidase (hydrolysis of GSH S-conjugates) activities. AcePCS mRNAs are expressed in vivo throughout the life cycle of A. ceylanicum. Mature adult male hookworms isolated from the small intestines of their hosts displayed significantly enhanced expression of AcePCS with transcript levels 5-fold greater than other developmental forms. Although the role of AcePCS in A. ceylanicum biology has yet to be fully investigated the results reported here provide encouraging evidence of the potential that this enzyme holds as a target for new chemotherapeutic intervention.

  18. Towards an Understanding of the Function of the Phytochelatin Synthase of Schistosoma mansoni

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    Rigouin, Coraline; Nylin, Elyse; Cogswell, Alexis A.; Schaumlöffel, Dirk; Dobritzsch, Dirk; Williams, David L.

    2013-01-01

    Phytochelatin synthase (PCS) is a protease-like enzyme that catalyzes the production of metal chelating peptides, the phytochelatins, from glutathione (GSH). In plants, algae, and fungi phytochelatin production is important for metal tolerance and detoxification. PCS proteins also function in xenobiotic metabolism by processing GSH S-conjugates. The aim of the present study is to elucidate the role of PCS in the parasitic worm Schistosoma mansoni. Recombinant S. mansoni PCS proteins expressed in bacteria could both synthesize phytochelatins and hydrolyze various GSH S-conjugates. We found that both the N-truncated protein and the N- and C-terminal truncated form of the enzyme (corresponding to only the catalytic domain) work through a thiol-dependant and, notably, metal-independent mechanism for both transpeptidase (phytochelatin synthesis) and peptidase (hydrolysis of GSH S-conjugates) activities. PCS transcript abundance was increased by metals and xenobiotics in cultured adult worms. In addition, these treatments were found to increase transcript abundance of other enzymes involved in GSH metabolism. Highest levels of PCS transcripts were identified in the esophageal gland of adult worms. Taken together, these results suggest that S. mansoni PCS participates in both metal homoeostasis and xenobiotic metabolism rather than metal detoxification as previously suggested and that the enzyme may be part of a global stress response in the worm. Because humans do not have PCS, this enzyme is of particular interest as a drug target for schistosomiasis. PMID:23383357

  19. Towards an understanding of the function of the phytochelatin synthase of Schistosoma mansoni.

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    Coraline Rigouin

    Full Text Available Phytochelatin synthase (PCS is a protease-like enzyme that catalyzes the production of metal chelating peptides, the phytochelatins, from glutathione (GSH. In plants, algae, and fungi phytochelatin production is important for metal tolerance and detoxification. PCS proteins also function in xenobiotic metabolism by processing GSH S-conjugates. The aim of the present study is to elucidate the role of PCS in the parasitic worm Schistosoma mansoni. Recombinant S. mansoni PCS proteins expressed in bacteria could both synthesize phytochelatins and hydrolyze various GSH S-conjugates. We found that both the N-truncated protein and the N- and C-terminal truncated form of the enzyme (corresponding to only the catalytic domain work through a thiol-dependant and, notably, metal-independent mechanism for both transpeptidase (phytochelatin synthesis and peptidase (hydrolysis of GSH S-conjugates activities. PCS transcript abundance was increased by metals and xenobiotics in cultured adult worms. In addition, these treatments were found to increase transcript abundance of other enzymes involved in GSH metabolism. Highest levels of PCS transcripts were identified in the esophageal gland of adult worms. Taken together, these results suggest that S. mansoni PCS participates in both metal homoeostasis and xenobiotic metabolism rather than metal detoxification as previously suggested and that the enzyme may be part of a global stress response in the worm. Because humans do not have PCS, this enzyme is of particular interest as a drug target for schistosomiasis.

  20. Towards an understanding of the function of the phytochelatin synthase of Schistosoma mansoni.

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    Rigouin, Coraline; Nylin, Elyse; Cogswell, Alexis A; Schaumlöffel, Dirk; Dobritzsch, Dirk; Williams, David L

    2013-01-01

    Phytochelatin synthase (PCS) is a protease-like enzyme that catalyzes the production of metal chelating peptides, the phytochelatins, from glutathione (GSH). In plants, algae, and fungi phytochelatin production is important for metal tolerance and detoxification. PCS proteins also function in xenobiotic metabolism by processing GSH S-conjugates. The aim of the present study is to elucidate the role of PCS in the parasitic worm Schistosoma mansoni. Recombinant S. mansoni PCS proteins expressed in bacteria could both synthesize phytochelatins and hydrolyze various GSH S-conjugates. We found that both the N-truncated protein and the N- and C-terminal truncated form of the enzyme (corresponding to only the catalytic domain) work through a thiol-dependant and, notably, metal-independent mechanism for both transpeptidase (phytochelatin synthesis) and peptidase (hydrolysis of GSH S-conjugates) activities. PCS transcript abundance was increased by metals and xenobiotics in cultured adult worms. In addition, these treatments were found to increase transcript abundance of other enzymes involved in GSH metabolism. Highest levels of PCS transcripts were identified in the esophageal gland of adult worms. Taken together, these results suggest that S. mansoni PCS participates in both metal homoeostasis and xenobiotic metabolism rather than metal detoxification as previously suggested and that the enzyme may be part of a global stress response in the worm. Because humans do not have PCS, this enzyme is of particular interest as a drug target for schistosomiasis.

  1. Homologous cloning, characterization and expression of a new halophyte phytochelatin synthase gene in Suaeda salsa

    Science.gov (United States)

    Cong, Ming; Zhao, Jianmin; Lü, Jiasen; Ren, Zhiming; Wu, Huifeng

    2016-09-01

    The halophyte Suaeda salsa can grow in heavy metal-polluted areas along intertidal zones having high salinity. Since phytochelatins can eff ectively chelate heavy metals, it was hypothesized that S. salsa possessed a phytochelatin synthase (PCS) gene. In the present study, the cDNA of PCS was obtained from S. salsa (designated as SsPCS) using homologous cloning and the rapid amplification of cDNA ends (RACE). A sequence analysis revealed that SsPCS consisted of 1 916 bp nucleotides, encoding a polypeptide of 492 amino acids with one phytochelatin domain and one phytochelatin C domain. A similarity analysis suggested that SsPCS shared up to a 58.6% identity with other PCS proteins and clustered with PCS proteins from eudicots. There was a new kind of metal ion sensor motif in its C-terminal domain. The SsPCS transcript was more highly expressed in elongated and fibered roots and stems ( Pcloned from a halophyte, and it might contain a diff erent metal sensing capability than the first PCS from Thellungiella halophila. This study provided a new view of halophyte PCS genes in heavy metal tolerance.

  2. Characterization of the Sesbania rostrata Phytochelatin Synthase Gene: Alternative Splicing and Function of Four Isoforms

    Directory of Open Access Journals (Sweden)

    Zeng-Fu Xu

    2009-07-01

    Full Text Available Phytochelatins (PCs play an important role in detoxification of heavy metals in plants. PCs are synthesized from glutathione by phytochelatin synthase (PCS, a dipeptidyltransferase. Sesbania rostrata is a tropical legume plant that can tolerate high concentrations of Cd and Zn. In this study, the S. rostrata PCS gene (SrPCS and cDNAs were isolated and characterized. Southern blot and sequence analysis revealed that a single copy of the SrPCS gene occurs in the S. rostrata genome, and produces four different SrPCS mRNAs and proteins, SrPCS1-SrPCS4, by alternative splicing of the SrPCS pre-mRNA. The SrPCS1 and SrPCS3 proteins conferred Cd tolerance when expressed in yeast cells, whereas the SrPCS2 and SrPCS4 proteins, which lack the catalytic triad and the N-terminal domains, did not. These results suggested that SrPCS1 and SrPCS3 have potential applications in genetic engineering of plants for enhancing heavy metal tolerance and phytoremediation of contaminated soils.

  3. Phytochelatin synthase: of a protease a peptide polymerase made.

    Science.gov (United States)

    Rea, Philip A

    2012-05-01

    Of the mechanisms known to protect vascular plants and some algae, fungi and invertebrates from the toxic effects of non-essential heavy metals such as As, Cd or Hg, one of the most sophisticated is the enzyme-catalyzed synthesis of phytochelatins (PCs). PCs, (γ-Glu-Cys)(n) Gly polymers, which serve as high-affinity, thiol-rich cellular chelators and contribute to the detoxification of heavy metal ions, are derived from glutathione (GSH; γ-Glu-Cys-Gly) and related thiols in a reaction catalyzed by phytochelatin synthases (PC synthases, EC 2.3.2.15). Using the enzyme from Arabidopsis thaliana (AtPCS1) as a model, the reasoning and experiments behind the conclusion that PC synthases are novel papain-like Cys protease superfamily members are presented. The status of S-substituted GSH derivatives as generic PC synthase substrates and the sufficiency of the N-terminal domain of the enzyme from eukaryotic and its half-size equivalents from prokaryotic sources, for net PC synthesis and deglycylation of GSH and its derivatives, respectively, are emphasized. The question of the common need or needs met by PC synthases and their homologs is discussed. Of the schemes proposed to account for the combined protease and peptide polymerase capabilities of the eukaryotic enzymes vs the limited protease capabilities of the prokaryotic enzymes, two that will be considered are the storage and homeostasis of essential heavy metals in eukaryotes and the metabolism of S-substituted GSH derivatives in both eukaryotes and prokaryotes.

  4. Enhanced arsenic accumulation by engineered yeast cells expressing Arabidopsis thaliana phytochelatin synthase.

    Science.gov (United States)

    Singh, Shailendra; Lee, Wonkyu; Dasilva, Nancy A; Mulchandani, Ashok; Chen, Wilfred

    2008-02-01

    Phytochelatins (PCs) are naturally occurring peptides with high-binding capabilities for a wide range of heavy metals including arsenic (As). PCs are enzymatically synthesized by phytochelatin synthases and contain a (gamma-Glu-Cys)(n) moiety terminated by a Gly residue that makes them relatively proteolysis resistant. In this study, PCs were introduced by expressing Arabidopsis thaliana Phytochelatin Synthase (AtPCS) in the yeast Saccharomyces cerevisiae for enhanced As accumulation and removal. PCs production in yeast resulted in six times higher As accumulation as compared to the control strain under a wide range of As concentrations. For the high-arsenic concentration, PCs production led to a substantial decrease in levels of PC precursors such as glutathione (GSH) and gamma-glutamyl cysteine (gamma-EC). The levels of As(III) accumulation were found to be similar between AtPCS-expressing wild type strain and AtPCS-expressing acr3Delta strain lacking the arsenic efflux system, suggesting that the arsenic uptake may become limiting. This is further supported by the roughly 1:3 stoichiometric ratio between arsenic and PC2 (n = 2) level (comparing with a theoretical value of 1:2), indicating an excess availability of PCs inside the cells. However, at lower As(III) concentration, PC production became limiting and an additive effect on arsenic accumulation was observed for strain lacking the efflux system. More importantly, even resting cells expressing AtPCS pre-cultured in Zn(2+) enriched media showed PCs production and two times higher arsenic removal than the control strain. These results open up the possibility of using cells expressing AtPCS as an inexpensive sorbent for the removal of toxic arsenic.

  5. Chloroplast targeting of phytochelatin synthase in Arabidopsis: effects on heavy metal tolerance and accumulation.

    Science.gov (United States)

    Picault, N; Cazalé, A C; Beyly, A; Cuiné, S; Carrier, P; Luu, D T; Forestier, C; Peltier, G

    2006-11-01

    The enzymatically synthesized thiol peptide phytochelatin (PC) plays a central role in heavy metal tolerance and detoxification in plants. In response to heavy metal exposure, the constitutively expressed phytochelatin synthase enzyme (PCS) is activated leading to synthesis of PCs in the cytosol. Recent attempts to increase plant metal accumulation and tolerance reported that PCS over-expression in transgenic plants paradoxically induced cadmium hypersensitivity. In the present paper, we investigate the possibility of synthesizing PCs in plastids by over-expressing a plastid targeted phytochelatin synthase (PCS). Plastids represent a relatively important cellular volume and offer the advantage of containing glutathione, the precursor of PC synthesis. Using a constitutive CaMV 35S promoter and a RbcS transit peptide, we successfully addressed AtPCS1 to chloroplasts, significant PCS activity being measured in this compartment in two independent transgenic lines. A substantial increase in the PC content and a decrease in the glutathione pool were observed in response to cadmium exposure, when compared to wild-type plants. While over-expressing AtPCS1 in the cytosol importantly decreased cadmium tolerance, both cadmium tolerance and accumulation of plants expressing plastidial AtPCS1 were not significantly affected compared to wild-type. Interestingly, targeting AtPCS1 to chloroplasts induced a marked sensitivity to arsenic while plants over-expressing AtPCS1 in the cytoplasm were more tolerant to this metalloid. These results are discussed in relation to heavy metal trafficking pathways in higher plants and to the interest of using plastid expression of PCS for biotechnological applications.

  6. Enhanced Cadmium Accumulation in Transgenic Tobacco Expressing the Phytochelatin Synthase Gene of Cynodon dactylon L.

    Institute of Scientific and Technical Information of China (English)

    Jiangchuan Li; Jiangbo Guo; Wenzhong Xu; Mi Ma

    2006-01-01

    Bermudagrass (Cynodon dactylon L. cv. Goldensun) is highly resistant to and accumulates large amounts of cadmium (Cd). A phytochelatin synthase (PCS) cDNA (CdPCS1) was isolated from this grass by rapid amplification of cDNA ends. The putative CdPCS1 protein shared a high homology with PCS from other plants, with 79% homology at the N-terminal and 47% homology at the C-terminal. However, 16 Cys residues were found at the C-terminal of CdPCS1, and among these residues, three positions were different from other PCS proteins. Semiquantitative reverse transcription-polymerase chain reaction analysis showed that Cd stress induced CdPCS1 expression in both roots and leaves in Bermudagrass. We verified that CdPCS1 plays an important role in Cd tolerance in yeast cells by expressing the gene in ABDE1, a Cd-sensitive mutant. CdPCS1 was then introduced into tobacco plants. The phytochelatin level in some transgenic tobacco lines increased 3.88-fold more than in wild type plants and Cd accumulation in these transgenic plants was enhanced 3.21-fold accordingly. The results suggested that CdPCS1 could be used as a gene element for phytoremediation in the future.

  7. Overexpression of phytochelatin synthase in Arabidopsis leads to enhanced arsenic tolerance and cadmium hypersensitivity.

    Science.gov (United States)

    Li, Yujing; Dhankher, Om Parkash; Carreira, Laura; Lee, David; Chen, Alice; Schroeder, Julian I; Balish, Rebecca S; Meagher, Richard B

    2004-12-01

    Phytochelatin synthase (PCS) catalyzes the final step in the biosynthesis of phytochelatins, which are a family of cysteine-rich thiol-reactive peptides believed to play important roles in processing many thiol-reactive toxicants. A modified Arabidopsis thaliana PCS sequence (AtPCS1) was active in Escherichia coli. When AtPCS1 was overexpressed in Arabidopsis from a strong constitutive Arabidopsis actin regulatory sequence (A2), the A2::AtPCS1 plants were highly resistant to arsenic, accumulating 20-100 times more biomass on 250 and 300 microM arsenate than wild type (WT); however, they were hypersensitive to Cd(II). After exposure to cadmium and arsenic, the overall accumulation of thiol-peptides increased to 10-fold higher levels in the A2::AtPCS1 plants compared with WT, as determined by fluorescent HPLC. Whereas cadmium induced greater increases in traditional PCs (PC2, PC3, PC4), arsenic exposure resulted in the expression of many unknown thiol products. Unexpectedly, after arsenate or cadmium exposure, levels of the dipeptide substrate for PC synthesis, gamma-glutamyl cysteine (gamma-EC), were also dramatically increased. Despite these high thiol-peptide concentrations, there were no significant increases in concentrations of arsenic and cadmium in above-ground tissues in the AtPCS1 plants relative to WT plants. The potential for AtPCS1 overexpression to be useful in strategies for phytoremediating arsenic and to compound the negative effects of cadmium are discussed.

  8. 植物螯合肽合成酶的研究进展%Research Progress on Phytochelatin Synthase

    Institute of Scientific and Technical Information of China (English)

    李安明; 李德华; 邓青云; 汪宜宇

    2011-01-01

    植物螯合肽(phytochelatins,PCs)在植物解除重金属的毒性方面具有重要作用,其结构为(γ-Glu-Cys)n-Gly(n=2~11),它不是基因的编码产物,而是在植物螯合肽合成酶(phytochelatin synthase,PCS)的催化下以谷胱甘肽(glutathione,GSH)为底物合成的.PCS能够被金属离子激活,高度保守的N-端是催化结构域,而其C-端则是多变的.本文就PCS的结构,功能与催化机制以及PCS的最新研究进行了介绍.%Phytochelatins (PCs), with a general structure (γ-Glu-Cys)n-Gly (n=2-11), play an important role in heavy metal detoxification in plants. PCs are post-translationally synthesized directly from glutathione (GSH) by the enzyme phytochelatin synthase (PCS), of which the activity is dependent on the presence of heavy metals.The conserved N-terminal core domain of PCS functions as the catalytic domain, whereas the C-terminal region is highly variable. The review focused on the structure, functions and catalytic mechanisms of PCS, prospects were also included.

  9. Co-expression of Arabidopsis thaliana phytochelatin synthase and Treponema denticola cysteine desulfhydrase for enhanced arsenic accumulation.

    Science.gov (United States)

    Tsai, Shen-Long; Singh, Shailendra; Dasilva, Nancy A; Chen, Wilfred

    2012-02-01

    Arsenic is one of the most hazardous pollutants found in aqueous environments and has been shown to be a carcinogen. Phytochelatins (PCs), which are cysteine-rich and thio-reactive peptides, have high binding affinities for various metals including arsenic. Previously, we demonstrated that genetically engineered Saccharomyces cerevisiae strains expressing phytochelatin synthase (AtPCS) produced PCs and accumulated arsenic. In an effort to further improve the overall accumulation of arsenic, cysteine desulfhydrase, an aminotransferase that converts cysteine into hydrogen sulfide under aerobic condition, was co-expressed in order to promote the formation of larger AsS complexes. Yeast cells producing both AtPCS and cysteine desulfhydrase showed a higher level of arsenic accumulation than a simple cumulative effect of expressing both enzymes, confirming the coordinated action of hydrogen sulfide and PCs in the overall bioaccumulation of arsenic.

  10. Isolation and characterization of Arabidopsis halleri and Thlaspi caerulescens phytochelatin synthases.

    Science.gov (United States)

    Meyer, Claire-Lise; Peisker, Daniel; Courbot, Mikael; Craciun, Adrian Radu; Cazalé, Anne-Claire; Desgain, Denis; Schat, Henk; Clemens, Stephan; Verbruggen, Nathalie

    2011-07-01

    The synthesis of phytochelatins (PC) represents a major metal and metalloid detoxification mechanism in various species. PC most likely play a role in the distribution and accumulation of Cd and possibly other metals. However, to date, no studies have investigated the phytochelatin synthase (PCS) genes and their expression in the Cd-hyperaccumulating species. We used functional screens in two yeast species to identify genes expressed by two Cd hyperaccumulators (Arabidopsis halleri and Thlaspi caerulescens) and involved in cellular Cd tolerance. As a result of these screens, PCS genes were identified for both species. PCS1 was in each case the dominating cDNA isolated. The deduced sequences of AhPCS1 and TcPCS1 are very similar to AtPCS1 and their identity is particularly high in the proposed catalytic N-terminal domain. We also identified in A. halleri and T. caerulescens orthologues of AtPCS2 that encode functional PCS. As compared to A. halleri and A. thaliana, T. caerulescens showed the lowest PCS expression. Furthermore, concentrations of PC in Cd-treated roots were the highest in A. thaliana, intermediate in A. halleri and the lowest in T. caerulescens. This mirrors the known capacity of these species to translocate Cd to the shoot, with T. caerulescens being the best translocator. Very low or undetectable concentrations of PC were measured in A. halleri and T. caerulescens shoots, contrary to A. thaliana. These results suggest that extremely efficient alternative Cd sequestration pathways in leaves of Cd hyperaccumulators prevent activation of PC synthase by Cd²⁺ ions.

  11. RNA Interference-mediated Silencing of Phytochelatin Synthase Gene Reduce Cadmium Accumulation in Rice Seeds

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Phytochelatins (PCs) play an important role in heavy metal resistance and accumulation. To reduce the accumulation of cadmium (Cd) in rice seeds, the expression of phytochelatin synthase (PCS) gene OsPCS1 was suppressed by RNA interference (RNAi). A hairpin construct of a PCS fragment was designed in the pRNAi-OsPCS1 under the control of ZMM1, a seed-specific promoter from maize. The construct was introduced into rice (japonica) through Agrobacterium tumefaciens. The RNAi rice plantlets were selected and cultivated in pots exposured to 10 mg/kg Cd. The transcriptional level of OsPCS1 declined in seeds of some RNAi rice compared to the wild type. As a result Cd accumulation was reduced by about half in the seeds of RNAi rice. As expected, no apparent difference of growth appeared between RNAi and wild-type plants. The results suggest that this new approach can be used to control heavy metal accumulation in crops.

  12. Tentative identification of the second substrate binding site in Arabidopsis phytochelatin synthase.

    Directory of Open Access Journals (Sweden)

    Ju-Chen Chia

    Full Text Available Phytochelatin synthase (PCS uses the substrates glutathione (GSH, γGlu-Cys-Gly and a cadmium (Cd-bound GSH (Cd∙GS2 to produce the shortest phytochelatin product (PC2, (γGlu-Cys2-Gly through a ping-pong mechanism. The binding of the 2 substrates to the active site, particularly the second substrate binding site, is not well-understood. In this study, we generated a structural model of the catalytic domain of Arabidopsis AtPCS1 (residues 12-218 by using the crystal structure of the γGlu-Cys acyl-enzyme complex of the PCS of the cyanobacterium Nostoc (NsPCS as a template. The modeled AtPCS1 revealed a cavity in proximity to the first substrate binding site, consisting of 3 loops containing several conserved amino acids including Arg152, Lys185, and Tyr55. Substitutions of these amino acids (R152K, K185R, or double mutation resulted in the abrogation of enzyme activity, indicating that the arrangement of these 2 positive charges is crucial for the binding of the second substrate. Recombinant AtPCS1s with mutations at Tyr55 showed lower catalytic activities because of reduced affinity (3-fold for Y55W for the Cd∙GS2, further suggesting the role of the cation-π interaction in recognition of the second substrate. Our study results indicate the mechanism for second substrate recognition in PCS. The integrated catalytic mechanism of PCS is further discussed.

  13. Characterization and transcription studies of a phytochelatin synthase gene from the solitary tunicate Ciona intestinalis exposed to cadmium

    Energy Technology Data Exchange (ETDEWEB)

    Franchi, Nicola [Department of Biology, University of Padova, Padova (Italy); Department of Biological, Chemical, Pharmaceutical Science and Technology, University of Palermo, Palermo (Italy); Piccinni, Ester [Department of Biology, University of Padova, Padova (Italy); Ferro, Diana [Department of Biology, University of Padova, Padova (Italy); Institute for Evolution and Biodiversity, Westfälische Wilhelms-Universität, Münster (Germany); Basso, Giuseppe [Department of Woman and Child Health, University of Padova, Padova (Italy); Spolaore, Barbara [CRIBI Biotechnology Centre, University of Padova, Padova (Italy); Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova (Italy); Santovito, Gianfranco, E-mail: gianfranco.santovito@unipd.it [Department of Biology, University of Padova, Padova (Italy); Ballarin, Loriano [Department of Biology, University of Padova, Padova (Italy)

    2014-07-01

    Highlights: • Ciona intestinalis have a functional phytochelatin synthase (PCS) gene (cipcs). • CiPCS amino acid sequence is phylogentically related to other metazoan PCSs. • CiPCS catalyze the synthesis of PC2. • cipcs are mostly transcribed in circulating hemocytes, in both tunic and blood lacunae. • Cadmium exposure results in a significant increase of cipcs and cipcna transcription. - Abstract: The major thiol-containing molecules involved in controlling the level of intracellular ROS in eukaryotes, acting as a nonenzymatic detoxification system, are metallothioneins (MTs), glutathione (GSH) and phytochelatins (PCs). Both MTs and GSH are well-known in the animal kingdom. PC was considered a prerogative of the plant kingdom but, in 2001, a phytochelatin synthase (PCS) gene was described in the nematode Caenorhabditis elegans; additional genes encoding this enzyme were later described in the earthworm Eisenia fetida and in the parasitic nematode Schistosoma mansoni but scanty data are available, up to now, for Deuterostomes. Here, we describe the molecular characteristics and transcription pattern, in the presence of Cd, of a PCS gene from the invertebrate chordate Ciona intestinalis, a ubiquitous solitary tunicate and demonstrate the presence of PCs in tissue extracts. We also studied mRNA localization by in situ hybridization. In addition, we analyzed the behavior of hemocytes and tunic cells consequent to Cd exposure as well as the transcription pattern of the Ciona orthologous for proliferating cell nuclear antigen (PCNA), usually considered a proliferation marker, and observed that cell proliferation occurs after 96 h of Cd treatment. This matches the hypothesis of Cd-induced cell proliferation, as already suggested by previous data on the expression of a metallothionein gene in the same animal.

  14. 植物络合素及其合酶在重金属抗性中的功能研究进展%RESEARCH ADVANCE OF PHYTOCHELATINS AND PHYTOCHELATIN SYNTHASE ON HEAVY METAL TOLERANCE

    Institute of Scientific and Technical Information of China (English)

    冯保民; 麻密

    2003-01-01

    Under heavy metal stress, higher plants initiate a set of defense responses, among which biosynthesis of phytochelatins (PCs) is important. PCs are rich in cystein and biosynthesized by phytochelatin synthase. The chemical structure of PCs and their ability to form complexes with a large range of metal ions is clear. Up to now, these peptides are known to play an important role in both endogenous metal ion homeostasis and heavy metal ion detoxification. The mechanism of cadmium tolerance is illustrated in detail. A model of this mechanism suggested that the detoxification process of cadmium include such steps as PCs induction, transport of cadmium into the tonoplast, formation of the HMW-Cd-PCs complexes and sequestration in vacuole. At the same time, PCs also have other functions, such as detoxification of arsenic, protecting enzyme from metal ion inhibition and supplying metal ion as a cofactor to the enzyme potentially. However, a lot of questions about its biological function remain to be answered. In 1999, three independent labs isolated the genes encoding the PCs synthase. This breakthrough of plant heavy metal tolerance research gave us a chance to further study the heavy metal tolerance mechanism. All the results from the reserch of PCs have a ffreat application potential in phytoremidation. Fig 1, Ref 25

  15. Coordinated responses of phytochelatin synthase and metallothionein genes in black mangrove, Avicennia germinans, exposed to cadmium and copper

    Energy Technology Data Exchange (ETDEWEB)

    Gonzalez-Mendoza, Daniel [Departamento de Recursos del Mar, Cinvestav-Unidad Merida, Merida, Yucatan (Mexico); Moreno, Adriana Quiroz [Unidad de biotecnologia, CICY, Merida, Yucatan (Mexico); Zapata-Perez, Omar [Departamento de Recursos del Mar, Cinvestav-Unidad Merida, Merida, Yucatan (Mexico)]. E-mail: ozapata@mda.cinvestav.mx

    2007-08-01

    To evaluate the role of phytochelatins and metallothioneins in heavy metal tolerance of black mangrove Avicennia germinans, 3-month-old seedlings were exposed to cadmium or copper for 30 h, under hydroponic conditions. Degenerate Mt2 and PCS primers were synthesized based on amino acid and nucleotide alignment sequences reported for Mt2 and PCS in other plant species found in GenBank. Total RNA was isolated from A. germinans leaves and two partial fragments of metallothionein and phytochelatin synthase genes were isolated. Gene expression was evaluated with reverse transcripatase-polymerase chain reaction (RT-PCR) amplification technique. Temporal analysis showed that low Cd{sup 2+} and Cu{sup 2+} concentrations caused a slight (but not significant) increase in AvMt2 expression after a 16 h exposure time, while AvPCS expression showed a significant increase under the same conditions but only after 4 h. Results strongly suggest that the rapid increase in AvPCS expression may contribute to Cd{sup 2+} and Cu{sup 2+} detoxification. Moreover, we found that A. germinans has the capacity to over-express both genes (AvMt2 and AvPCS), which may constitute a coordinated detoxification response mechanism targeting non-essential metals. Nonetheless, our results confirm that AvPCS was the most active gene involved in the regulation of essential metals (e.g., Cu{sup 2+}) in A. germinans leaves.

  16. The capability to synthesize phytochelatins and the presence of constitutive and functional phytochelatin synthases are ancestral (plesiomorphic) characters for basal land plants.

    Science.gov (United States)

    Petraglia, Alessandro; De Benedictis, Maria; Degola, Francesca; Pastore, Giovanni; Calcagno, Margherita; Ruotolo, Roberta; Mengoni, Alessio; Sanità di Toppi, Luigi

    2014-03-01

    Bryophytes, a paraphyletic group which includes liverworts, mosses, and hornworts, have been stated as land plants that under metal stress (particularly cadmium) do not synthesize metal-binding peptides such as phytochelatins. Moreover, very little information is available to date regarding phytochelatin synthesis in charophytes, postulated to be the direct ancestors of land plants, or in lycophytes, namely very basal tracheophytes. In this study, it was hypothesized that basal land plants and charophytes have the capability to produce phytochelatins and possess constitutive and functional phytochelatin synthases. To verify this hypothesis, twelve bryophyte species (six liverworts, four mosses, and two hornworts), three charophytes, and two lycophyte species were exposed to 0-36 μM cadmium for 72 h, and then assayed for: (i) glutathione and phytochelatin quali-quantitative content by HPLC and mass spectrometry; (ii) the presence of putative phytochelatin synthases by western blotting; and (iii) in vitro activity of phytochelatin synthases. Of all the species tested, ten produced phytochelatins in vivo, while the other seven did not. The presence of a constitutively expressed and functional phytochelatin synthase was demonstrated in all the bryophyte lineages and in the lycophyte Selaginella denticulata, but not in the charophytes. Hence, current knowledge according to phytochelatins have been stated as being absent in bryophytes was therefore confuted by this work. It is argued that the capability to synthesize phytochelatins, as well as the presence of active phytochelatin synthases, are ancestral (plesiomorphic) characters for basal land plants.

  17. Metalloproteins and phytochelatin synthase may confer protection against zinc oxide nanoparticle induced toxicity in Caenorhabditis elegans.

    Science.gov (United States)

    Polak, Natasa; Read, Daniel S; Jurkschat, Kerstin; Matzke, Marianne; Kelly, Frank J; Spurgeon, David J; Stürzenbaum, Stephen R

    2014-03-01

    Zinc oxide nanoparticles (ZnONPs) are used in large quantities by the cosmetic, food and textile industries. Here we exposed Caenorhabditis elegans wild-type and a metal sensitive triple knockout mutant (mtl-1;mtl-2;pcs-1) to ZnONPs (0-50mg/L) to study strain and exposure specific effects on transcription, reactive oxygen species generation, the biomolecular phenotype (measured by Raman microspectroscopy) and key endpoints of the nematode life cycle (growth, reproduction and lifespan). A significant dissolution effect was observed, where dissolved ZnO constituted over 50% of total Zn within a two day exposure to the test medium, suggesting that the nominal exposure to pure ZnONPs represents in vivo, at best, a mixture exposure of ionic zinc and nanoparticles. Nevertheless, the analyses provided evidence that the metallothioneins (mtl-1 and mtl-2), the phytochelatin synthase (pcs-1) and an apoptotic marker (cep-1) were transcriptionally activated. In addition, the DCFH-DA assay provided in vitro evidence of the oxidative potential of ZnONPs in the metal exposure sensitive triple mutant. Raman spectroscopy highlighted that the biomolecular phenotype changes significantly in the mtl-1;mtl-2;pcs-1 triple knockout worm upon ZnONP exposure, suggesting that these metalloproteins are instrumental in the protection against cytotoxic damage. Finally, ZnONP exposure was shown to decrease growth and development, reproductive capacity and lifespan, effects which were amplified in the triple knockout. By combining diverse toxicological strategies, we identified that individuals (genotypes) housing mutations in key metalloproteins and phytochelatin synthase are more susceptible to ZnONP exposure, which underlines their importance to minimize ZnONP induced toxicity.

  18. A cyanobacterial protein with similarity to phytochelatin synthases catalyzes the conversion of glutathione to gamma-glutamylcysteine and lacks phytochelatin synthase activity.

    Science.gov (United States)

    Harada, Emiko; von Roepenack-Lahaye, Edda; Clemens, Stephan

    2004-12-01

    Phytochelatins are glutathione-derived, non-translationally synthesized peptides essential for cadmium and arsenic detoxification in plant, fungal and nematode model systems. Recent sequencing programs have revealed the existence of phytochelatin synthase-related genes in a wide range of organisms that have not been reported yet to produce phytochelatins. Among those are several cyanobacteria. We have studied one of the encoded proteins (alr0975 from Nostoc sp. strain PCC 7120) and demonstrate here that it does not possess phytochelatin synthase activity. Instead, this protein catalyzes the conversion of glutathione to gamma-glutamylcysteine. The thiol spectrum of yeast cells expressing alr0975 shows the disappearance of glutathione and the formation of a compound that by LC-MSMS analysis was unequivocally identified as gamma-glutamylcysteine. Purified recombinant protein catalyzes the respective reaction. Unlike phytochelatin synthesis, the conversion of glutathione to gamma-glutamylcysteine is not dependent on activation by metal cations. No evidence was found for the accumulation of phytochelatins in cyanobacteria even after prolonged exposure to toxic Cd2+ concentrations. Expression of alr0975 was detected in Nostoc sp. cells with an antiserum raised against the protein. No indication for a responsiveness of expression to toxic metal exposure was found. Taken together, these data provide further evidence for possible additional functions of phytochelatin synthase-related proteins in glutathione metabolism and provide a lead as to the evolutionary history of phytochelatin synthesis.

  19. Zn-bis-glutathionate is the best co-substrate of the monomeric phytochelatin synthase from the photosynthetic heavy metal-hyperaccumulator Euglena gracilis.

    Science.gov (United States)

    García-García, Jorge D; Girard, Lourdes; Hernández, Georgina; Saavedra, Emma; Pardo, Juan P; Rodríguez-Zavala, José S; Encalada, Rusely; Reyes-Prieto, Adrián; Mendoza-Cózatl, David G; Moreno-Sánchez, Rafael

    2014-03-01

    The phytochelatin synthase from photosynthetic Euglena gracilis (EgPCS) was analyzed at the transcriptional, kinetic, functional, and phylogenetic levels. Recombinant EgPCS was a monomeric enzyme able to synthesize, in the presence of Zn(2+) or Cd(2+), phytochelatin2-phytochelatin4 (PC2-PC4) using GSH or S-methyl-GS (S-methyl-glutathione), but not γ-glutamylcysteine or PC2 as a substrate. Kinetic analysis of EgPCS firmly established a two-substrate reaction mechanism for PC2 synthesis with Km values of 14-22 mM for GSH and 1.6-2.5 μM for metal-bis-glutathionate (Me-GS2). EgPCS showed the highest Vmax and catalytic efficiency with Zn-(GS)2, and was inactivated by peroxides. The EgPCS N-terminal domain showed high similarity to that of other PCSases, in which the typical catalytic core (Cys-70, His-179 and Asp-197) was identified. In contrast, the C-terminal domain showed no similarity to other PCSases. An EgPCS mutant comprising only the N-terminal 235 amino acid residues was inactive, suggesting that the C-terminal domain is essential for activity/stability. EgPCS transcription in Euglena cells was not modified by Cd(2+), whereas its heterologous expression in ycf-1 yeast cells provided resistance to Cd(2+) stress. Phylogenetic analysis of the N-terminal domain showed that EgPCS is distant from plants and other photosynthetic organisms, suggesting that it evolved independently. Although EgPCS showed typical features of PCSases (constitutive expression; conserved N-terminal domain; kinetic mechanism), it also exhibited distinct characteristics such as preference for Zn-(GS)2 over Cd-(GS)2 as a co-substrate, a monomeric structure, and ability to solely synthesize short-chain PCs, which may be involved in conferring enhanced heavy-metal resistance.

  20. A new pathway for heavy metal detoxification in animals. Phytochelatin synthase is required for cadmium tolerance in Caenorhabditis elegans.

    Science.gov (United States)

    Vatamaniuk, O K; Bucher, E A; Ward, J T; Rea, P A

    2001-06-15

    Increasing emissions of heavy metals such as cadmium, mercury, and arsenic into the environment pose an acute problem for all organisms. Considerations of the biochemical basis of heavy metal detoxification in animals have focused exclusively on two classes of peptides, the thiol tripeptide, glutathione (GSH, gamma-Glu-Cys-Gly), and a diverse family of cysteine-rich low molecular weight proteins, the metallothioneins. Plants and some fungi, however, not only deploy GSH and metallothioneins for metal detoxification but also synthesize another class of heavy metal binding peptides termed phytochelatins (PCs) from GSH. Here we show that PC-mediated heavy metal detoxification is not restricted to plants and some fungi but extends to animals by demonstrating that the ce-pcs-1 gene of the nematode worm Caenorhabditis elegans encodes a functional PC synthase whose activity is critical for heavy metal tolerance in the intact organism.

  1. Enhanced tolerance and accumulation of heavy metal ions by engineered Escherichia coli expressing Pyrus calleryana phytochelatin synthase.

    Science.gov (United States)

    Li, Hui; Cong, Yu; Lin, Jing; Chang, Youhong

    2015-03-01

    Contamination by heavy metals is a major environmental problem worldwide and microbial bioremediation is an efficient method for removing this type of pollution. The plant enzymephytochelatin synthase (PCS, also known as glutathione g-glutamylcysteinyltransferase, EC2.3.2.15) involved in the synthesis of phytochelatins (PCs), which are metal-binding cysteine-rich peptides, has a major role in the detoxification of heavy metals in plants. Expression of the PcPCS1 gene from the bean pear (Pyrus calleryana Dcne.) was induced after cadmium and copper treatments. However, functional analysis of this gene in vivo has not been reported. And it is or not suitable for bioremediation also needs to be assessed. In this study, we found Escherichia coli with over-expressed PcPCS1 had enhanced tolerance to cadmium, copper, sodium, and mercury. E. colicells transformed with pPcPCS1 was found to survive in solid M9 medium containing 2.0 mM Cd(2+), 4.0 mM Cu(2+). 4.5% (w/v) Na+, or 200 μ MHg(2+). Moreover, the growth curve showed 1.5 mM Cd(2+), 2.5 mM Cu(2+), 3.5% (w/v) Naþ, and 100 μ MHg(2+) had no effect on the growth of the E. coli cells transformed with pPcPCS1. Also, we found the contents of PCs and the accumulation of cadmium,copper, sodium, and mercury ions were enhanced in the recombinant E. coli strain Rosetta(TM) (DE3).These results suggested the PcPCS1 gene might be a candidate for heavy metal bioremediation via recombinant bacteria.

  2. [Effects of lower concentrations of Cd on micronutrients uptake and production of phytochelatins (PCs) in Ceratophyllum demersum].

    Science.gov (United States)

    Wang, Chao; Wang, Li-Ya; Sun, Qin

    2009-04-15

    A solution experiment with a range of lower cadmium concentrations (0.01-0.64 micromol/L) was conducted to study micronutrients uptake and production of phytochelatins (PCs) in a submerged aquatic plant Ceratophyllum demersum after exposure for 7 d, 14 d, 21 d. The results showed the uptake of Cu, Zn in Ceratophyllum demersum was promoted when exposed to lower concentrations of cadmium, whereas Mn uptake was inhibited. Unlike Cu, Zn and Mn, the content of Fe was unaffected by Cd uptake. Our data showed that Ceratophyllum demersum had stronger Cd tolerance and obvious absorption and cumulative effects to external lower concentrations of Cd exposure. The inhibition of growth was observed after 21 d in 0.08 micromol/L Cd. The production of PCs was significantly induced by 0.02-0.64 micromol/L Cd for 7 d (p < 0.05), while declined after exposure for 14 d, 21 d. The good dose-related response of PCs was observed in manner of linear for 7 d and parabola for 14 d and 21 d. Regressive analysis showed that there was a significantly positive correlation between PC contents and Cd toxicity in Ceratophyllum demersum. The results suggested that PCs could be a sensitive biomarker for estimating Cd phytotoxicity and subsequently a qualitative tool for studies on Cd contamination.

  3. Effects of arsenate (AS5+) on growth and production of glutathione (GSH) and phytochelatins (PCS) in Chlorella vulgaris.

    Science.gov (United States)

    Jiang, Ying; Purchase, Diane; Jones, Huw; Garelick, Hemda

    2011-09-01

    The effect of arsenate (As5+) on growth and chlorophyll a production in Chlorella vulgaris, its removal by C. vulgaris and the role of glutathione (GSH) and phytochelatins (PCs) were investigated. C. vulgaris was tolerant to As5+ at up to 200 mg/L and was capable of consistently removing around 70% of the As5+ present in growth media over a wide range of exposure concentrations. Spectral analysis revealed that PCs and their arsenic-combined complexes were absent, indicating that the high bioaccumulation and tolerance to arsenic observed was not due to intracellular chelation. In contrast, GSH was found in all samples ranging from 0.8 mg/L in the control to 6.5 mg/L in media containing 200 mg/L As5+ suggesting that GSH plays a more prominent role in the detoxification of As5+ in C. vulgaris than PC. At concentrations below 100 mg/L cell surface binding and other mechanisms may play the primary role in As5+ detoxification, whereas above this concentration As5+ begins to accumulate inside the algal cells and activates a number of intracellular cell defense mechanisms, such as increased production of GSH. The overall findings complement field studies which suggest C. vulgaris as an increasingly promising low cost As phytoremediation method for developing countries.

  4. A Cd/Fe/Zn-responsive phytochelatin synthase is constitutively present in the ancient liverwort Lunularia cruciata (L.) dumort.

    Science.gov (United States)

    Degola, Francesca; De Benedictis, Maria; Petraglia, Alessandro; Massimi, Alberto; Fattorini, Laura; Sorbo, Sergio; Basile, Adriana; Sanità di Toppi, Luigi

    2014-11-01

    Lunularia cruciata occupies a very basal position in the phylogenetic tree of liverworts, which in turn have been recognized as a very early clade of land plants. It would therefore seem appropriate to take L. cruciata as the startingpoint for investigating character evolution in plants' metal(loid) response. One of the strongest evolutionary pressures for land colonization by plants has come from potential access to much greater amounts of nutritive ions from surface rocks, compared to water. This might have resulted in the need to precisely regulate trace element homeostasis and to minimize the risk of exposure to toxic concentrations of certain metals, prompting the evolution of a number of response mechanisms, such as synthesis of phytochelatins, metal(loid)-binding thiol-peptides. Accordingly, if the ability to synthesize phytochelatins and the occurrence of an active phytochelatin synthase are traits present in a basal liverwort species, and have been even reinforced in 'modern' tracheophytes, e.g. Arabidopsis thaliana, then such traits would presumably have played an essential role in plant fitness over time. Hence, we demonstrated here that: (i) L. cruciata compartmentalizes cadmium in the vacuoles of the phototosynthetic parenchyma by means of a phytochelatin-mediated detoxification strategy, and possesses a phytochelatin synthase that is activated by cadmium and homeostatic concentrations of iron(II) and zinc; and (ii) A. thaliana phytochelatin synthase displays a higher and broader response to several metal(loid)s [namely: cadmium, iron(II), zinc, copper, mercury, lead, arsenic(III)] than L. cruciata phytochelatin synthase.

  5. Metabolites and metals in Metazoa--what role do phytochelatins play in animals?

    Science.gov (United States)

    Bundy, J G; Kille, P

    2014-09-01

    Phytochelatins are sulfur-rich metal-binding peptides, and phytochelatin synthesis is one of the key mechanisms by which plants protect themselves against toxic soft metal ions such as cadmium. It has been known for a while now that some invertebrates also possess functional phytochelatin synthase (PCS) enzymes, and that at least one species, the nematode Caenorhabditis elegans, produces phytochelatins to help detoxify cadmium, and probably also other metal and metalloid ions including arsenic, zinc, selenium, silver, and copper. Here, we review recent studies on the occurrence, utilization, and regulation of phytochelatin synthesis in invertebrates. The phytochelatin synthase gene has a wide phylogenetic distribution, and can be found in species that cover almost all of the animal tree of life. The evidence to date, though, suggests that the occurrence is patchy, and even though some members of particular taxonomic groups may contain PCS genes, there are also many species without these genes. For animal species that do possess PCS genes, some of them (e.g. earthworms) do synthesize phytochelatins in response to potentially toxic elements, whereas others (e.g. Schistosoma mansoni, a parasitic helminth) do not appear to do so. Just how (and if) phytochelatins in invertebrates complement the function of metallothioneins remains to be elucidated, and the temporal, spatial, and metal specificity of the two systems is still unknown.

  6. Phytochelatins are synthesized by two vacuolar serine carboxypeptidases in Saccharomyces cerevisiae.

    Science.gov (United States)

    Wünschmann, Jana; Beck, Andreas; Meyer, Laurent; Letzel, Thomas; Grill, Erwin; Lendzian, Klaus J

    2007-04-17

    Phytochelatins (PCs) are cysteine-rich peptides that chelate heavy metal ions, thereby mediating heavy metal tolerance in plants, fission yeast, and Caenorhabditis elegans. They are synthesized from glutathione by PC synthase, a specific dipeptidyltransferase. While Saccharomyces cerevisiae synthesizes PCs upon exposure to heavy metal ions, the S. cerevisiae genome does not encode a PC synthase homologue. How PCs are synthesized in yeast is unclear. This study shows that the vacuolar serine carboxypeptidases CPY and CPC are responsible for PC synthesis in yeast. The finding of a PCS-like activity of these enzymes in vivo discloses another route for PC biosynthesis in eukaryotes.

  7. Mutagenic definition of a papain-like catalytic triad, sufficiency of the N-terminal domain for single-site core catalytic enzyme acylation, and C-terminal domain for augmentative metal activation of a eukaryotic phytochelatin synthase.

    Science.gov (United States)

    Romanyuk, Nataliya D; Rigden, Daniel J; Vatamaniuk, Olena K; Lang, Albert; Cahoon, Rebecca E; Jez, Joseph M; Rea, Philip A

    2006-07-01

    Phytochelatin (PC) synthases are gamma-glutamylcysteine (gamma-Glu-Cys) dipeptidyl transpeptidases that catalyze the synthesis of heavy metal-binding PCs, (gamma-Glu-Cys)nGly polymers, from glutathione (GSH) and/or shorter chain PCs. Here it is shown through investigations of the enzyme from Arabidopsis (Arabidopsis thaliana; AtPCS1) that, although the N-terminal half of the protein, alone, is sufficient for core catalysis through the formation of a single-site enzyme acyl intermediate, it is not sufficient for acylation at a second site and augmentative stimulation by free Cd2+. A purified N-terminally hexahistidinyl-tagged AtPCS1 truncate containing only the first 221 N-terminal amino acid residues of the enzyme (HIS-AtPCS1_221tr) is competent in the synthesis of PCs from GSH in media containing Cd2+ or the synthesis of S-methyl-PCs from S-methylglutathione in media devoid of heavy metal ions. However, whereas its full-length hexahistidinyl-tagged equivalent, HIS-AtPCS1, undergoes gamma-Glu-Cys acylation at two sites during the Cd2+-dependent synthesis of PCs from GSH and is stimulated by free Cd2+ when synthesizing S-methyl-PCs from S-methylglutathione, HIS-AtPCS1_221tr undergoes gamma-Glu-Cys acylation at only one site when GSH is the substrate and is not directly stimulated, but instead inhibited, by free Cd2+ when S-methylglutathione is the substrate. Through the application of sequence search algorithms capable of detecting distant homologies, work we reported briefly before but not in its entirety, it has been determined that the N-terminal half of AtPCS1 and its equivalents from other sources have the hallmarks of a papain-like, Clan CA Cys protease. Whereas the fold assignment deduced from these analyses, which substantiates and is substantiated by the recent determination of the crystal structure of a distant prokaryotic PC synthase homolog from the cyanobacterium Nostoc, is capable of explaining the strict requirement for a conserved Cys residue, Cys-56

  8. Phytochelatins: peptides involved in heavy metal detoxification.

    Science.gov (United States)

    Pal, Rama; Rai, J P N

    2010-03-01

    Phytochelatins (PCs) are enzymatically synthesized peptides known to involve in heavy metal detoxification and accumulation, which have been measured in plants grown at high heavy metal concentrations, but few studies have examined the response of plants even at lower environmentally relevant metal concentrations. Recently, genes encoding the enzyme PC synthase have been identified in plants and other species enabling molecular biological studies to untangle the mechanisms underlying PC synthesis and its regulation. The present paper embodies review on recent advances in structure of PCs, their biosynthetic regulation, roles in heavy metal detoxification and/or accumulation, and PC synthase gene expression for better understanding of mechanism involved and to improve phytoremediation efficiency of plants for wider application.

  9. The metabolomic responses of Caenorhabditis elegans to cadmium are largely independent of metallothionein status, but dominated by changes in cystathionine and phytochelatins.

    Science.gov (United States)

    Hughes, Samantha L; Bundy, Jacob G; Want, Elizabeth J; Kille, Peter; Stürzenbaum, Stephen R

    2009-07-01

    Cadmium is a widely distributed toxic environmental pollutant. Using proton NMR spectroscopy and UPLC-MS, we obtained metabolic profiles from the model organism Caenorhabditis elegans exposed to sublethal concentrations of cadmium. Neither in the presence nor absence of cadmium did the metallothionein status (single or double mtl knockouts) markedly modulate the metabolic profile. However, independent of strain, cadmium exposure resulted in a decrease in cystathionine concentrations and an increase in the nonribosomally synthesized peptides phytochelatin-2 and phytochelatin-3. This suggests that a primary response to low levels of cadmium is the differential regulation of the C. elegans trans-sulfuration pathway, which channels the flux from methionine through cysteine into phytochelatin synthesis. These results were backed up by the finding that phytochelatin synthase mutants (pcs-1) were at least an order of magnitude more sensitive to cadmium than single or double metallothionein mutants. However, an additive sensitivity toward cadmium was observed in the mtl-1; mtl-2; pcs-1 triple mutant.

  10. Metalloid tolerance based on phytochelatins is not functionally equivalent to the arsenite transporter Acr3p.

    Science.gov (United States)

    Wysocki, Robert; Clemens, Stephan; Augustyniak, Daria; Golik, Pawel; Maciaszczyk, Ewa; Tamás, Markus J; Dziadkowiec, Dorota

    2003-05-01

    Active transport of metalloids by Acr3p and Ycf1p in Saccharomyces cerevisiae and chelation by phytochelatins in Schizosaccharomyces pombe, nematodes, and plants represent distinct strategies of metalloid detoxification. In this report, we present results of functional comparison of both resistance mechanisms. The S. pombe and wheat phytochelatin synthase (PCS) genes, when expressed in S. cerevisiae, mediate only modest resistance to arsenite and thus cannot functionally compensate for Acr3p. On the other hand, we show for the first time that phytochelatins also contribute to antimony tolerance as PCS fully complement antimonite sensitivity of ycf1Delta mutant. Remarkably, heterologous expression of PCS sensitizes S. cerevisiae to arsenate, while ACR3 confers much higher arsenic resistance in pcsDelta than in wild-type S. pombe. The analysis of PCS and ACR3 homologues distribution in various organisms and our experimental data suggest that separation of ACR3 and PCS genes may lead to the optimal tolerance status of the cell.

  11. Adaptive Engineering of Phytochelatin-based Heavy Metal Tolerance.

    Science.gov (United States)

    Cahoon, Rebecca E; Lutke, W Kevin; Cameron, Jeffrey C; Chen, Sixue; Lee, Soon Goo; Rivard, Rebecca S; Rea, Philip A; Jez, Joseph M

    2015-07-10

    Metabolic engineering approaches are increasingly employed for environmental applications. Because phytochelatins (PC) protect plants from heavy metal toxicity, strategies directed at manipulating the biosynthesis of these peptides hold promise for the remediation of soils and groundwaters contaminated with heavy metals. Directed evolution of Arabidopsis thaliana phytochelatin synthase (AtPCS1) yields mutants that confer levels of cadmium tolerance and accumulation greater than expression of the wild-type enzyme in Saccharomyces cerevisiae, Arabidopsis, or Brassica juncea. Surprisingly, the AtPCS1 mutants that enhance cadmium tolerance and accumulation are catalytically less efficient than wild-type enzyme. Metabolite analyses indicate that transformation with AtPCS1, but not with the mutant variants, decreases the levels of the PC precursors, glutathione and γ-glutamylcysteine, upon exposure to cadmium. Selection of AtPCS1 variants with diminished catalytic activity alleviates depletion of these metabolites, which maintains redox homeostasis while supporting PC synthesis during cadmium exposure. These results emphasize the importance of metabolic context for pathway engineering and broaden the range of tools available for environmental remediation.

  12. Long-distance root-to-shoot transport of phytochelatins and cadmium in Arabidopsis.

    Science.gov (United States)

    Gong, Ji-Ming; Lee, David A; Schroeder, Julian I

    2003-08-19

    Phytochelatin synthases (PCS) mediate cellular heavy-metal resistance in plants, fungi, and worms. However, phytochelatins (PCs) are generally considered to function as intracellular heavy-metal detoxification mechanisms, and whether long-distance transport of PCs occurs during heavy-metal detoxification remains unknown. Here, wheat TaPCS1 cDNA expression was either targeted to Arabidopsis roots with the Arabidopsis alcohol dehydrogenase (Adh) promoter (Adh::TaPCS1/cad1-3) or ectopically expressed with the cauliflower mosaic virus 35S promoter (35S::TaPCS1/cad1-3) in the PC-deficient mutant cad1-3. Adh::TaPCS1/cad1-3 and 35S::TaPCS1/cad1-3 complemented the cadmium, mercury, and arsenic sensitivities of the cad1-3 mutant. Northern blot, RT-PCR, and Western blot analyses showed Adh promoter-driven TaPCS1 expression only in roots and thus demonstrated lack of long-distance TaPCS1 mRNA and protein transport in plants. Fluorescence HPLC analyses showed that under Cd2+ stress, no PCs were detectable in cad1-3. However, in Adh::TaPCS1/cad1-3 plants, PCs were detected in roots and in rosette leaves and stems. Inductively coupled plasma atomic emission spectrometer analyses showed that either root-specific or ectopic expression of TaPCS1 significantly enhanced long-distance Cd2+ transport into stems and rosette leaves. Unexpectedly, transgenic expression of TaPCS1 reduced Cd2+ accumulation in roots compared with cad1-3. The reduced Cd2+ accumulation in roots and enhanced root-to-shoot Cd2+ transport in transgenic plants were abrogated by l-buthionine sulfoximine. The presented findings show that (i) transgenic expression of TaPCS1 suppresses the heavy-metal sensitivity of cad1-3, (ii) PCs can be transported from roots to shoots, and (iii) transgenic expression of the TaPCS1 gene increases long-distance root-to-shoot Cd2+ transport and reduces Cd2+ accumulation in roots.

  13. Arsenic response of AtPCS1- and CePCS-expressing plants - effects of external As(V) concentration on As-accumulation pattern and NPT metabolism.

    Science.gov (United States)

    Wojas, Sylwia; Clemens, Stephan; Skłodowska, Aleksandra; Maria Antosiewicz, Danuta

    2010-02-15

    Phytochelatins (PCs) are small, cysteine-rich peptides, known to play a major role in detoxification of both cadmium and arsenic. The aim of this study was to determine whether overexpression of either of two PC synthase (PCS) genes, AtPCS1 and CePCS in Nicotiana tabacum (previously shown to cause decrease and increase, respectively, of cadmium tolerance of tobacco - Wojas et al., 2008) also contributes to such contrasting phenotypes with respect to arsenic (As) tolerance and accumulation, and how observed responses relate to non-protein thiol (NPT) metabolism. The expression of both genes resulted in an increase of As-tolerance, with CePCS plants most tolerant. We showed for the first time that the response of PCS overexpressing plants to As qualitatively depends on the external As(V) concentration. At the less toxic 50muM As(V), AtPCS1 and CePCS transformants accumulated more As in roots and leaves than WT. An increase in PC production and the level of PC2 species was detected in leaves of AtPCS1 and CePCS plants, which might explain their enhanced As-accumulation and tolerance. In contrast, at the highly toxic 200muM As(V), several disturbances in thiol metabolism of PCS overexpressing plants were found, surprisingly, including decrease of PC levels both in roots and leaves of transgenic plants relative to WT. The increase in As-tolerance and accumulation due to AtPCS1 and CePCS overexpression, observed at the As(V) concentrations similar to those found in As-contaminated soils, makes these genes promising candidates for plant engineering for phytoremediation.

  14. Effects of arsenic speciation on the phytochelatins (PCs) synthesis by Hydrilla verticillata%不同形态砷对黑藻植物络合素合成的影响

    Institute of Scientific and Technical Information of China (English)

    李红梅; 王宏镔; 王海娟; 宋雁辉; 钟正燕

    2012-01-01

    采用反相高效液相色谱(RP-HPLC)法,研究了水培条件下3种形态砷[三价砷As(Ⅲ)、五价砷As(Ⅴ)和二甲基砷(DMA)]的5个处理浓度(0、0.3、1.0、3.0和5.0 mg·L-1)对黑藻(Hydrilla venicillata(L.f.)Royle)砷吸收和植物络合素(PCs)合成的影响.结果表明:黑藻对As表现出明显的吸收和富集效应,低浓度As能促进黑藻生长,对As (Ⅲ)的吸收显著高于As(Ⅴ)和DMA;As(Ⅲ)和As(Ⅴ)处理显著诱导了谷胱甘肽(GSH)、PC2和PC4的合成,且与黑藻体内As含量呈显著正相关(P<0.05);PCs对DMA处理不敏感,只有在0.3 mg·L-1下能合成PCs;在As( Ⅲ)处理下,GSH和PC2的合成随As(Ⅲ)浓度的增加呈先增加后减少的趋势,而PC4的合成却随As(Ⅲ)浓度的增加而增加;在As(Ⅴ)处理下,GSH的合成随As(Ⅴ)浓度的增加呈先增加后减少的趋势,而PG2和PC4的合成则随As(Ⅴ)浓度的增加而增加;在3种形态As的不同浓度处理中PC3的合成很少,只在0.3 mg·L-1下有少量合成.研究结果表明,PCs对As( Ⅲ)和As(Ⅴ)的胁迫较敏感,可选择性地作为这2种形态砷胁迫下黑藻的生物标记物.%A hydroponie experiment was conducted to study the effects of different arsenic species [As(III), As(V), and DMA] at five concentrations (0, 0.3, 1.0, 3.0, and 5.0 mg· L-1) on the arsenic uptake and phytochelatins (PCs) synthesis by Hydrilla verticillaia. The PCs contents were identified by reversed phase-high performance liquid chromatography ( RP-HPLC). H. vertkillata had an obvious arsenic uptake and accumulation. Low concentrations arsenic promoted the growth of H. veniciilata, and the uptake of As( III) by H. vertkillata was significantly higher than that of As(V) or DMA. As(III) and As(V) promoted the synthesis of GSH, PC2, and PC4 significantly, and the synthesis was significantly positively correlated with the arsenic concentration in H. venkillala (P<0.05). DMA had little effects on the synthesis of PCs, with a detectable synthesis

  15. Earthworms produce phytochelatins in response to arsenic.

    Directory of Open Access Journals (Sweden)

    Manuel Liebeke

    Full Text Available Phytochelatins are small cysteine-rich non-ribosomal peptides that chelate soft metal and metalloid ions, such as cadmium and arsenic. They are widely produced by plants and microbes; phytochelatin synthase genes are also present in animal species from several different phyla, but there is still little known about whether these genes are functional in animals, and if so, whether they are metal-responsive. We analysed phytochelatin production by direct chemical analysis in Lumbricus rubellus earthworms exposed to arsenic for a 28 day period, and found that arsenic clearly induced phytochelatin production in a dose-dependent manner. It was necessary to measure the phytochelatin metabolite concentrations directly, as there was no upregulation of phytochelatin synthase gene expression after 28 days: phytochelatin synthesis appears not to be transcriptionally regulated in animals. A further untargetted metabolomic analysis also found changes in metabolites associated with the transsulfuration pathway, which channels sulfur flux from methionine for phytochelatin synthesis. There was no evidence of biological transformation of arsenic (e.g. into methylated species as a result of laboratory arsenic exposure. Finally, we compared wild populations of earthworms sampled from the field, and found that both arsenic-contaminated and cadmium-contaminated mine site worms had elevated phytochelatin concentrations.

  16. Earthworms produce phytochelatins in response to arsenic.

    Science.gov (United States)

    Liebeke, Manuel; Garcia-Perez, Isabel; Anderson, Craig J; Lawlor, Alan J; Bennett, Mark H; Morris, Ceri A; Kille, Peter; Svendsen, Claus; Spurgeon, David J; Bundy, Jacob G

    2013-01-01

    Phytochelatins are small cysteine-rich non-ribosomal peptides that chelate soft metal and metalloid ions, such as cadmium and arsenic. They are widely produced by plants and microbes; phytochelatin synthase genes are also present in animal species from several different phyla, but there is still little known about whether these genes are functional in animals, and if so, whether they are metal-responsive. We analysed phytochelatin production by direct chemical analysis in Lumbricus rubellus earthworms exposed to arsenic for a 28 day period, and found that arsenic clearly induced phytochelatin production in a dose-dependent manner. It was necessary to measure the phytochelatin metabolite concentrations directly, as there was no upregulation of phytochelatin synthase gene expression after 28 days: phytochelatin synthesis appears not to be transcriptionally regulated in animals. A further untargetted metabolomic analysis also found changes in metabolites associated with the transsulfuration pathway, which channels sulfur flux from methionine for phytochelatin synthesis. There was no evidence of biological transformation of arsenic (e.g. into methylated species) as a result of laboratory arsenic exposure. Finally, we compared wild populations of earthworms sampled from the field, and found that both arsenic-contaminated and cadmium-contaminated mine site worms had elevated phytochelatin concentrations.

  17. Biosynthetic regulation of phytochelatins, heavy metal-binding peptides.

    Science.gov (United States)

    Hirata, Kazumasa; Tsuji, Naoki; Miyamoto, Kazuhisa

    2005-12-01

    Phytochelatins (PCs) are heavy metal-binding peptides that play important roles in the detoxification of toxic heavy metals and the regulation of intracellular concentrations of essential metals in eukaryotes, including higher plants, fungi, and microalgae. Recently, PC synthase genes in higher plants and fission yeast have been identified and characterized, enabling molecular biological studies to unravel the mechanisms underlying PC synthesis. Moreover, recent routine database searches have unexpectedly identified genes that are similar to plant PC synthase genes in the genomes of worms and some prokaryotes. In this review, we introduce these recent advances in our understanding of the molecular mechanisms for PC biosynthesis and functions in order to supply basic information about the unique and attractive peptides applicable to various fields.

  18. Enzyme mediated synthesis of phytochelatin-capped CdS nanocrystals

    Science.gov (United States)

    Liu, Fang; Kang, Seung Hyun; Lee, Young-In; Choa, Yong-ho; Mulchandani, Ashok; Myung, Nosang V.; Chen, Wilfred

    2010-09-01

    We reported the enzyme mediated synthesis of CdS nanocrystals by immobilized phytochelatin synthase, which converts glutathione into the metal-binding peptide phytochelatin (PC). Formation of CdS nanocrystals were observed upon the addition of CdCl2 and Na2S with PC as the capping agent. By varying the reaction times, different compositions of PCs (form PC2 to PC3) can be synthesized, resulting in the formation of highly stable nanocrystals with tunable sizes (from 2.0 to 1.6 nm diameter). This approach may be generalized to guide the in vitro self assembly of a wide range of nanocrystals with different compositions and sizes.

  19. Phytochelatins inhibit the metal-induced aggregation of alpha-crystallin.

    Science.gov (United States)

    Hori, Yasuhisa; Yoshikawa, Tomoaki; Tsuji, Naoki; Bamba, Takeshi; Aso, Yoshikazu; Kudou, Motonori; Uchida, Yoshiki; Takagi, Masahiro; Harada, Kazuo; Hirata, Kazumasa

    2009-02-01

    Phytochelatins (PCs) are heavy-metal-binding peptides found in some eukaryotes. This study investigates the use of plant-derived PCs for the inhibition of metal-induced protein aggregation. The results of this study show that PCs inhibit zinc-induced alpha-crystallin aggregation, and suggest that PCs might be useful as anti-cataract agents.

  20. Response differences between Ectocarpus siliculosus populations to copper stress involve cellular exclusion and induction of the phytochelatin biosynthetic pathway.

    Science.gov (United States)

    Roncarati, Francesca; Sáez, Claudio A; Greco, Maria; Gledhill, Martha; Bitonti, Maria B; Brown, Murray T

    2015-02-01

    Some populations of brown seaweed species inhabit metal-polluted environments and can develop tolerance to metal stress, but the mechanisms by which this is accomplished are still to be elucidated. To address this, the responses of two strains of the model brown alga Ectocarpus siliculosus isolated from sites with different histories of metal contamination exposed to total copper (CuT) concentrations ranging between 0 and 2.4 μM for 10 days were investigated. The synthesis of the metal-chelator phytochelatin (PCs) and relative levels of transcripts encoding the enzymes γ-glutamylcysteine synthetase (γ-GCS), glutathione synthase (GS) and phytochelatin synthase (PCS) that participate in the PC biosynthetic pathway were measured, along with the effects on growth, and adsorption and uptake of Cu. Growth of strain LIA, from a pristine site in Scotland, was inhibited to a greater extent, and at lower concentrations, than that of Es524, isolated from a Cu-contaminated site in Chile. Concentrations of intra-cellular Cu were higher and the exchangeable fraction was lower in LIA than Es524, especially at the highest exposure levels. Total glutathione concentrations increased in both strains with Cu exposure, whereas total PCs levels were higher in Es524 than LIA; PC2 and PC3 were detected in Es524 but PC2 only was found in LIA. The greater production and levels of polymerisation of PCs in Es524 can be explained by the up-regulation of genes encoding for key enzymes involved in the synthesis of PCs. In Es524 there was an increase in the transcripts of γ-GCS, GS and PCS, particularly under high Cu exposure, whereas in LIA4 transcripts of γ-GCS1 increased only slightly, γ-GCS2 and GS decreased and PCS did not change. The consequences of higher intra-cellular concentrations of Cu, lower production of PCs, and lower expression of enzymes involved in GSH-PCs synthesis may be contributing to an induced oxidative stress condition in LIA, which explains, at least in part, the

  1. Systematic engineering of phytochelatin synthesis and arsenic transport for enhanced arsenic accumulation in E. coli.

    Science.gov (United States)

    Singh, Shailendra; Kang, Seung Hyun; Lee, Wonkyu; Mulchandani, Ashok; Chen, Wilfred

    2010-03-01

    Phytochelatin (PC) is a naturally occurring peptide with high affinity towards arsenic (As). In this article, we demonstrated the systematic engineering of PC-producing E. coli for As accumulation by addressing different bottlenecks in PC synthesis as well as As transport. Phytochelatin synthase from Schizosaccharomyces pombe (SpPCS) was expressed in E. coli resulting in 18 times higher As accumulation. PC production was further increased by co-expressing a feedback desensitized gamma-glutamylcysteine synthetase (GshI*), resulting in 30-fold higher PC levels and additional 2-fold higher As accumulation. The significantly increased PC levels were exploited further by co-expressing an arsenic transporter GlpF, leading to an additional 1.5-fold higher As accumulation. These engineering steps were finally combined in an arsenic efflux deletion E. coli strain to achieve an arsenic accumulation level of 16.8 micromol/g DCW, a 80-fold improvement when compared to a control strain not producing phytochelatins.

  2. An improved grafting technique for mature Arabidopsis plants demonstrates long-distance shoot-to-root transport of phytochelatins in Arabidopsis.

    Science.gov (United States)

    Chen, Alice; Komives, Elizabeth A; Schroeder, Julian I

    2006-05-01

    Phytochelatins (PCs) are peptides that function in heavy-metal chelation and detoxification in plants and fungi. A recent study showed that PCs have the ability to undergo long-distance transport in a root-to-shoot direction in transgenic Arabidopsis (Arabidopsis thaliana). To determine whether long-distance transport of PCs can occur in the opposite direction, from shoots to roots, the wheat (Triticum aestivum) PC synthase (TaPCS1) gene was expressed under the control of a shoot-specific promoter (CAB2) in an Arabidopsis PC-deficient mutant, cad1-3 (CAB2TaPCS1/cad1-3). Analyses demonstrated that TaPCS1 is expressed only in shoots and that CAB2TaPCS1/cad1-3 lines complement the cadmium (Cd) and arsenic metal sensitivity of cad1-3 shoots. CAB2TaPCS1/cad1-3 plants exhibited higher Cd accumulation in roots and lower Cd accumulation in shoots compared to wild type. Fluorescence HPLC coupled to mass spectrometry analyses directly detected PC2 in the roots of CAB2:TaPCS1/cad1-3 but not in cad1-3 controls, suggesting that PC2 is transported over long distances in the shoot-to-root direction. In addition, wild-type shoot tissues were grafted onto PC synthase cad1-3 atpcs2-1 double loss-of-function mutant root tissues. An Arabidopsis grafting technique for mature plants was modified to obtain an 84% success rate, significantly greater than a previous rate of approximately 11%. Fluorescence HPLC-mass spectrometry showed the presence of PC2, PC3, and PC4 in the root tissue of grafts between wild-type shoots and cad1-3 atpcs2-1 double-mutant roots, demonstrating that PCs are transported over long distances from shoots to roots in Arabidopsis.

  3. Arsenic accumulation and tolerance in rootless macrophyte Najas indica are mediated through antioxidants, amino acids and phytochelatins.

    Science.gov (United States)

    Tripathi, Rudra Deo; Singh, Ragini; Tripathi, Preeti; Dwivedi, Sanjay; Chauhan, Reshu; Adhikari, Bijan; Trivedi, Prabodh Kumar

    2014-12-01

    Arsenic (As) accumulation and tolerance response of a submerged rootless macrophyte Najas indica were evaluated during arsenate (As(V); 10-250 μM) and arsenite (As(III); 1-50 μM) exposure. Higher As accumulation at As(III) exposure and more tolerance upon As(V) exposure resulted in more toxicity during As(III) stress than As(V), which was evident through measurement of growth parameters and oxidative stress related parameters viz., lipid peroxidation (MDA content), electrical conductivity (EC) and hydrogen peroxide (H2O2) levels. Antioxidant enzymes and various amino acids were more prominent during moderate exposure of As(V), suggesting their possible role in As tolerance and detoxification. Various non-enzymatic antioxidant metabolites viz., ascorbic acid (ASC), glutathione (GSH), non-protein thiols (NPTs) and phytochelatins (PCs) biosynthesis involving phytochelatin synthase (PCS) activity increased more significantly during As(III) stress. However, PCs content seems inadequate in response to As accumulation leading to lower PC-SH:As molar ratio and higher As phytotoxicity during As(III) stress. N. indica may prove useful plant species for phytoremediation purpose in moderately As contaminated water bodies due to high As accumulation and tolerance potential.

  4. An HPLC-ICP-MS technique for determination of cadmium-phytochelatins in genetically modified Arabidopsis thaliana.

    Science.gov (United States)

    Sadi, Baki B M; Vonderheide, Anne P; Gong, Ji-Ming; Schroeder, Julian I; Shann, Jodi R; Caruso, Joseph A

    2008-01-01

    A reversed-phase high-performance liquid chromatographic technique was developed to separate cadmium-phytochelatin complexes (Cd-PC2, Cd-PC3, and Cd-PC4) of interest in the plant Arapidopsis thaliana. High-performance liquid chromatography (HPLC) was coupled to an inductively coupled plasma mass spectrometric (ICP-MS) system with some modification to the interface. This was done in order to sustain the plasma with optimum sensitivity for cadmium detection in the presence of the high methanol loads used in the gradient elution of the reversed-phase separation. The detection limits were found to be 91.8 ngl(-1), 77.2 ngl(-1) and 49.2 ngl(-1) for Cd-PC2, Cd-PC3, and Cd-PC4 respectively. The regression coefficients (r2) for Cd-PC2 to Cd-PC4 detection ranged from 0.998 to 0.999. The method was then used to investigate the occurrence and effect of cadmium-phytochelatin complexes in wild-type Arabidopsis and a phytochelatin-deficient mutant cad1-3 that had been genetically modified to ectopically express the wheat TaPCS1 phytochelatin synthase enzyme. The primary complex found in both wild-type and transgenic plants was Cd-PC2. In both lines, higher levels of Cd-PC2 were found in shoots than in roots, showing that phytochelatin synthases contribute to the accumulation of cadmium in shoots, in the Cd-PC2 form. Genetic modification did, however, impact the overall accumulation of Cd. Transgenic plants contained almost two times more cadmium in the form of Cd-PC2 in their roots than did the corresponding wild-type plants. Similarly, the shoot samples of the modified species also contained more (by 1.6 times) cadmium in the form of Cd-PC2 than the wild type. The enhanced role of PC2 in the transgenic Arabidopsis correlates with data showing long-distance transport of Cd in transgenic plants. Targeted transgenic expression of non-native phytochelatin synthases may contribute to improving the efficiency of plants for phytoremediation.

  5. Detoxification of arsenic by phytochelatins in plants.

    Science.gov (United States)

    Schmöger, M E; Oven, M; Grill, E

    2000-03-01

    As is a ubiquitous element present in the atmosphere as well as in the aquatic and terrestrial environments. Arsenite and arsenate are the major forms of As intoxication, and these anions are readily taken up by plants. Both anions efficiently induce the biosynthesis of phytochelatins (PCs) ([gamma-glutamate-cysteine](n)-glycine) in vivo and in vitro. The rapid induction of the metal-binding PCs has been observed in cell suspension cultures of Rauvolfia serpentina, in seedlings of Arabidopsis, and in enzyme preparations of Silene vulgaris upon challenge to arsenicals. The rate of PC formation in enzyme preparations was lower compared with Cd-induced biosynthesis, but was accompanied by a prolonged induction phase that resulted finally in higher peptide levels. An approximately 3:1 ratio of the sulfhydryl groups from PCs to As is compatible with reported As-glutathione complexes. The identity of the As-induced PCs and of reconstituted metal-peptide complexes has unequivocally been demonstrated by electrospray ionization mass spectroscopy. Gel filtration experiments and inhibitor studies also indicate a complexation and detoxification of As by the induced PCs.

  6. Copper-induced synthesis of ascorbate, glutathione and phytochelatins in the marine alga Ulva compressa (Chlorophyta).

    Science.gov (United States)

    Mellado, Macarena; Contreras, Rodrigo A; González, Alberto; Dennett, Geraldine; Moenne, Alejandra

    2012-02-01

    In order to analyze the synthesis of antioxidant and heavy metal-chelating compounds in response to copper stress, the marine alga Ulva compressa (Chlorophyta) was exposed to 10 μM copper for 7 days and treated with inhibitors of ASC synthesis, lycorine, and GSH synthesis, buthionine sulfoximine (BSO). The levels of ascorbate, in its reduced (ASC) and oxidized (DHA) forms, glutathione, in its reduced (GSH) and oxidized (GSSG) forms, and phytochelatins (PCs) were determined as well as activities of enzymes involved in ASC synthesis, L-galactose dehydrogenase (GDH) and L-galactono 1,4 lactone dehydrogenase (GLDH), and in GSH synthesis, γ-glutamylcysteine synthase (γ-GCS) and glutathione synthase (GS). The level of ASC rapidly decreased to reach a minimum at day 1 that remained low until day 7, DHA decreased until day 1 but slowly increased up to day 7 and its accumulation was inhibited by lycorine. In addition, GSH level increased to reach a maximal level at day 5 and GSSG increased up to day 7 and their accumulation was inhibited by BSO. Activities of GDH and GLDH increased until day 7 and GLDH was inhibited by lycorine. Moreover, activities of γ-GCS and GS increased until day 7 and γ-GCS was inhibited by BSO. Furthermore, PC2, PC3 and PC4, increased until day 7 and their accumulation was inhibited by BSO. Thus, copper induced the synthesis of ascorbate, glutathione and PCs in U. compressa suggesting that these compounds are involved in copper tolerance. Interestingly, U. compressa is, until now, the only ulvophyte showing ASC, GSH and PCs synthesis in response to copper excess.

  7. First report of phytochelatins in a mushroom: induction of phytochelatins by metal exposure in Boletus edulis.

    Science.gov (United States)

    Collin-Hansen, Christian; Pedersen, Sindre A; Andersen, Rolf A; Steinnes, Eiliv

    2007-01-01

    Some species of macromycetes (mushrooms) consistently are found to contain high concentrations of toxic metals such as cadmium (Cd) and mercury (Hg), and consumption of wild-growing mushrooms is acknowledged as a significant source for Cd and Hg in humans. Yet little is known about the speciation of Cd and Hg in mushroom tissues. Here we present the first evidence of peptides of the phytochelatin family being responsible for binding a large fraction of Cd in caps of the macromycete Boletus edulis exposed to excess metals. Concentrations of Cd, Zn, Cu and Hg, as well as cytosolic Cd-binding capacity (CCBC), glutathione (GSH) and free proline (Pro) were quantified in fruiting bodies of B. edulis differentially exposed to a wide range of metals. Metal distribution among cytosolic compounds were investigated by size exclusion chromatography (SEC), followed by metal determinations with atomic absorption chromatography (AAS) and HR-ICP-MS. Cd-binding compounds in SEC elutates were investigated further by high performance liquid chromatography-mass spectrometry (HPLC-MS). CCBC was >90 times higher in the exposed group relative to the reference group (Mann-Whitney's P phytochelatins (PCs), a family of cystein-rich oligopeptides, was confirmed in Cd-containing SEC fractions by HPLC-MS. The appearance of more complex PCs was coupled to declining concentrations of GSH. To our knowledge this is the first report demonstrating the presence of PCs in a macromycete.

  8. [Effects of environmental factors on cd biotoxicity and phytochelatins production in Triticum aestivum].

    Science.gov (United States)

    Sun, Qin; Yuan, Xinfang; Wang, Xiaorong

    2005-07-01

    In this paper, a solution culture experiment was conducted to study the effects of environmental factors on Cd biotoxicity and phytochelatins (PCs) production in wheat. The results showed that Cd stress had significant inhibitory effects on wheat growth and PCs overproduction. The Cd biotoxicity and Cd uptake by wheat were affected in varying degrees by soil pH, Ca and S, and the levels of PCs production in root were consistent with the changes of Cd biotoxicity. Furthermore, the Cd biotoxicity was decreased with increasing P supply, coinciding with the decrease of PCs level in root. Mg had no obvious effect on both Cd biotoxicity and PCs level in root. The present results further confirmed that the induced PCs production level by Cd was related to Cd biotoxicity in plant, suggesting that PCs could be a promising biomarker for estimating Cd phytotoxicity.

  9. PCs in the factory

    CERN Document Server

    2013-01-01

    Please note this is a short discount publication.PCs have become as essential to the factory environment as they are to the office environment. This in-depth report examines how specially adapted PCs and peripherals are being established in Factory Process Control and Reporting. The report covers: * Hardware and Software* Typical Applications* Implementation Issues* Case Studies and Real Applications

  10. Synthetic phytochelatins complement a phytochelatin-deficient Arabidopsis mutant and enhance the accumulation of heavy metal(loid)s.

    Science.gov (United States)

    Shukla, Devesh; Tiwari, Manish; Tripathi, Rudra D; Nath, Pravendra; Trivedi, Prabodh Kumar

    2013-05-10

    Phytochelatins (PCs) are naturally occurring thiol-rich peptides containing gamma (γ) peptide bonds and are well known for their metal-binding and detoxification capabilities. Whether synthetic phytochelatins (ECs) can be used as an alternative approach for enhancing the metal-binding capacity of plants has been investigated in this study. The metal-binding potential of ECs has been demonstrated in bacteria; however, no report has investigated the expression of ECs in plants. We have expressed three synthetic genes encoding ECs of different lengths in wild type (WT) Arabidopsis (Col-0 background) and a phytochelatin-deficient Arabidopsis mutant (cad1-3). After exposure to different heavy metals, the transgenic plants were examined for phenotypic changes, and metal accumulation was evaluated. The expression of EC genes rescued the sensitive phenotype of the cad1-3 mutant under heavy metal(loid) stress. Transgenic Arabidopsis plants expressing EC genes accumulated a significantly enhanced level of heavy metal(loid)s in comparison with the WT plant. The mutant complementation and enhanced heavy metal(loid) accumulation in the transgenic Arabidopsis plants suggest that ECs work in a manner similar to that of PCs in plants and that ECs could be used as an alternative for phytoremediation of heavy metal(loid) exposure.

  11. PCs The Missing Manual

    CERN Document Server

    Karp, David

    2005-01-01

    Your vacuum comes with one. Even your blender comes with one. But your PC--something that costs a whole lot more and is likely to be used daily and for tasks of far greater importance and complexity--doesn't come with a printed manual. Thankfully, that's not a problem any longer: PCs: The Missing Manual explains everything you need to know about PCs, both inside and out, and how to keep them running smoothly and working the way you want them to work. A complete PC manual for both beginners and power users, PCs: The Missing Manual has something for everyone. PC novices will appreciate the una

  12. Fission yeast HMT1 lowers seed cadmium through phytochelatin-dependent vacuolar sequestration in Arabidopsis.

    Science.gov (United States)

    Huang, Jing; Zhang, Yu; Peng, Jia-Shi; Zhong, Chen; Yi, Hong-Ying; Ow, David W; Gong, Ji-Ming

    2012-04-01

    Much of our dietary uptake of heavy metals is through the consumption of plants. A long-sought strategy to reduce chronic exposure to heavy metals is to develop plant varieties with reduced accumulation in edible tissues. Here, we describe that the fission yeast (Schizosaccharomyces pombe) phytochelatin (PC)-cadmium (Cd) transporter SpHMT1 produced in Arabidopsis (Arabidopsis thaliana) was localized to tonoplast, and enhanced tolerance to and accumulation of Cd2+, copper, arsenic, and zinc. The action of SpHMT1 requires PC substrates, and failed to confer Cd2+ tolerance and accumulation when glutathione and PC synthesis was blocked by L-buthionine sulfoximine, or only PC synthesis is blocked in the cad1-3 mutant, which is deficient in PC synthase. SpHMT1 expression enhanced vacuolar Cd2+ accumulation in wild-type Columbia-0, but not in cad1-3, where only approximately 35% of the Cd2+ in protoplasts was localized in vacuoles, in contrast to the near 100% found in wild-type vacuoles and approximately 25% in those of cad2-1 that synthesizes very low amounts of glutathione and PCs. Interestingly, constitutive SpHMT1 expression delayed root-to-shoot metal transport, and root-targeted expression confirmed that roots can serve as a sink to reduce metal contents in shoots and seeds. These findings suggest that SpHMT1 function requires PCs in Arabidopsis, and it is feasible to promote food safety by engineering plants using SpHMT1 to decrease metal accumulation in edible tissues.

  13. HPLC-HRMS method for fast phytochelatins determination in plants. Application to analysis of Clinopodium vulgare L.

    Science.gov (United States)

    Bardarov, Krum; Naydenov, Mladen; Djingova, Rumyana

    2015-09-01

    An optimized analytical method based on C8 core-shell reverse phase chromatographic separation and high resolution mass spectral (HRMS) detection is developed for a fast analysis of unbound phytochelatins (PCs) in plants. Its application to analysis of Clinopodium vulgare L. is demonstrated where proper PCs liberating and preservation conditions were employed using dithiotreitol in the extraction step. A baseline separation of glutathione (GSH) and phytochelatins from 2 to 5 (PC2-PC5) for 3 min was achieved at conventional HPLC backpressure, with detection limits from 3 ppt (for GSH) to 2.5 ppb (for PC5). It is shown, that the use of HRMS with tandem mass spectral (MS/MS) capabilities permits additional wide range screening ability for iso-phytochelatins and PC similar compounds, based on exact mass and fragment spectra in a post acquisition manner.

  14. Phytochelatin synthesis is essential for the detoxification of excess zinc and contributes significantly to the accumulation of zinc.

    Science.gov (United States)

    Tennstedt, Pierre; Peisker, Daniel; Böttcher, Christoph; Trampczynska, Aleksandra; Clemens, Stephan

    2009-02-01

    The synthesis of phytochelatins (PCs) is essential for the detoxification of nonessential metals and metalloids such as cadmium and arsenic in plants and a variety of other organisms. To our knowledge, no direct evidence for a role of PCs in essential metal homeostasis has been reported to date. Prompted by observations in Schizosaccharomyces pombe and Saccharomyces cerevisiae indicating a contribution of PC synthase expression to Zn2+ sequestration, we investigated a known PC-deficient Arabidopsis (Arabidopsis thaliana) mutant, cad1-3, and a newly isolated second strong allele, cad1-6, with respect to zinc (Zn) homeostasis. We found that in a medium with low cation content PC-deficient mutants show pronounced Zn2+ hypersensitivity. This phenotype is of comparable strength to the well-documented Cd2+ hypersensitivity of cad1 mutants. PC deficiency also results in significant reduction in root Zn accumulation. To be able to sensitively measure PC accumulation, we established an assay using capillary liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry of derivatized extracts. Plants grown under control conditions consistently showed PC2 accumulation. Analysis of plants treated with same-effect concentrations revealed that Zn2+-elicited PC2 accumulation in roots reached about 30% of the level of Cd2+-elicited PC2 accumulation. We conclude from these data that PC formation is essential for Zn2+ tolerance and provides driving force for the accumulation of Zn. This function might also help explain the mysterious occurrence of PC synthase genes throughout the plant kingdom and in a wide range of other organisms.

  15. Determination of Phytochelatins in Rice by Stable Isotope Labeling Coupled with Liquid Chromatography-Mass Spectrometry.

    Science.gov (United States)

    Liu, Ping; Cai, Wen-Jing; Yu, Lei; Yuan, Bi-Feng; Feng, Yu-Qi

    2015-07-01

    A highly sensitive method was developed for the detection of phytochelatins (PCs) in rice by stable isotope labeling coupled with liquid chromatography-electrospray ionization-tandem mass spectrometry (IL-LC-ESI-MS/MS) analysis. A pair of isotope-labeling reagents [ω-bromoacetonylquinolinium bromide (BQB) and BQB-d(7)] were used to label PCs in plant sample and standard PCs, respectively, and then combined prior to LC/MS analysis. The heavy labeled standards were used as the internal standards for quantitation to minimize the matrix and ion suppression effects in MS analysis. In addition, the ionization efficiency of PCs was greatly enhanced through the introduction of a permanent charged moiety of quaternary ammonium of BQB into PCs. The detection sensitivities of PCs upon BQB labeling improved by 14-750-fold, and therefore, PCs can be quantitated using only 5 mg of plant tissue. Furthermore, under cadmium (Cd) stress, we found that the contents of PCs in rice dramatically increased with the increased concentrations and treatment time of Cd. It was worth noting that PC5 was first identified and quantitated in rice tissues under Cd stress in the current study. Taken together, this IL-LC-ESI-MS/MS method demonstrated to be a promising strategy in detection of PCs in plants with high sensitivity and reliability.

  16. Phytochelatin induction by selenate in Chlorella vulgaris, and regulation of effect by sulfate levels.

    Science.gov (United States)

    Simmons, Denina B D; Emery, R J Neil

    2011-02-01

    Phytochelatins (PCs) are short metal detoxification peptides made from the sulfur-rich molecule glutathione. The production of PCs by algae caused by Se exposure has never been studied, although many algae accumulate Se, forming Se-rich proteins and peptides, and higher plants have demonstrated PC production when treated with Se; therefore, a goal of the current study was to examine whether Se induces PC production in algae. Furthermore, selenate is thought to compete with sulfate in the S assimilation pathway, and sulfate therefore may have a protective effect against the toxic effects of high doses of Se in algae. Hence, the interaction of selenate and sulfate was investigated with respect to the induction of PCs. Chlorella vulgaris was cultured in media with either low (31.2 µM) or high (312 µM) concentrations of sulfate. These cultures were exposed to selenate in doses of 7, 35, and 70 nM for 48 h. In a separate treatment, Cd (890 nM) was added as a positive PC-inducing control, and one no-metal negative control was used. Total Se and Se speciation were determined, and glutathione, phytochelatin-2, and phytochelatin-3 were quantified in each of cell digests, cell medium, and cell lysates. We found that PCs and their precursor glutathione were induced by selenate as well as by a Cd control. The high concentration of sulfate was able to counter selenate-induced production of PCs and glutathione. These data support two possible mechanisms: a negative feedback system in the S assimilation pathway that affects PC production when sulfate is abundant, and competition for uptake at the ion transport level between selenate and sulfate.

  17. Bioremediation of cadmium contaminated soil using symbiosis between leguminous plant and recombinant rhizobia with the MTL4 and the PCS genes.

    Science.gov (United States)

    Ike, Akiko; Sriprang, Rutchadaporn; Ono, Hisayo; Murooka, Yoshikatsu; Yamashita, Mitsuo

    2007-01-01

    Cadmium contamination in rice grains is one of the important issues in Asian countries. We have developed a novel bio-remediation system based on the symbiosis between leguminous plant and genetically engineered rhizobia. We designed two types of recombinant rhizobia, carrying two genes, synthetic tetrameric metallothionein (MTL4) and cDNA encoding phytochelatin synthase from Arabidopsis thaliana (AtPCS). The MTL4 and AtPCS genes were transferred to Mesorhizobium huakuii subsp. rengei B3, which can infect and form nodules on Chinese milk vetch, Astragalus sinicus. The two genes were fused to the nolB or nifH promoter, which generated nodule specific expression of these genes in strain B3. The two recombinant strains, B3(pMPnolBMTL4nifHPCS) and B3::nifHMTL4(pMPnifHPCS), showed 25 and 12-fold increase in Cd concentration, in the free-living cells, respectively. When these recombinant strains established the symbiotic relationship with A. sinicus, the symbionts increased Cd accumulation in nodules by two-fold in hydroponic culture. The expression of the both MTL4 and AtPCS genes showed additive effect on cadmium accumulation in nodules. We also applied these recombinant bacteria to rice paddy soil polluted with Cd (1mgkg(-1) dry weight soil). The accumulation of Cd increased not only in nodules but also in the roots of A. sinicus infected by the recombinant rhizobia. The accumulation of Cd in the plant roots infected by B3(pMPnolBMTL4nifHPCS) achieved three-fold than that by the wild-type B3. After two months of cultivation of the symbiont, a maximum of 9% of Cd in paddy soil was removed. Thus, the symbiosis will be useful in phytoremediation for heavy metals.

  18. Characterization of lead induced metal-phytochelatin complexes in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Scheidegger, Christian; Sigg, Laura; Behra, Renata

    2011-11-01

    Accumulation of Pb and induction of phytochelatin synthesis were observed in Chlamydomonas reinhardtii upon Pb(II) exposure. Our aim was to examine whether Pb(II) is bound by phytochelatins (PCs) in C. reinhardtii and to examine formed complexes for their stoichiometry and composition. Metal-phytochelatin (Me-PC) complexes induced by Pb were isolated by size-exclusion chromatography in 13 collected fractions, which were analyzed for their PC and metal content by high-performance liquid chromatography and inductively coupled plasma mass spectrometry. A recovery of more than 90% of Pb from standard Pb-PC₂ complexes within the total volume of the size-exclusion column indicated the adequacy of the method for Pb-PC(n) complex separation and characterization. Phytochelatins were detected mainly in a molecular weight ranging from 1,000 to 5,300 daltons (Da), indicating the formation of complexes with various stoichiometries. Approximately 72% of total PC₂ eluted in the range from 1,000 to 1,600 Da, and 80% of total PC₃ eluted in the molecular weight range from 1,600 to 2,300 Da. The distribution of Cu, Zn, and Pb showed that more than 70% of these metals were associated with the high-molecular-weight fractions. Copper, zinc, and lead were also observed in PC-containing fractions, suggesting the formation of various Me-PC complexes. The results of the present study indicate that the role of PCs in Pb detoxification is minor, because only 13% of total Pb was associated with PCs.

  19. Contribution of phytochelatins to cadmium tolerance in peanut plants.

    Science.gov (United States)

    Bianucci, Eliana; Sobrino-Plata, Juan; Carpena-Ruiz, Ramón O; Del Carmen Tordable, María; Fabra, Adriana; Hernández, Luis E; Castro, Stella

    2012-10-01

    Cadmium (Cd) is a well known heavy metal considered as one of the most toxic metals on Earth, affecting all viable cells that are exposed even at low concentration. It is introduced to agricultural soils mainly by phosphate fertilizers and causes many toxic symptoms in cells. Phytochelatins (PCs) are non-protein thiols which are involved in oxidative stress protection and are strongly induced by Cd. In this work, we analyzed metal toxicity as well as PCs implication on protection of peanut plants exposed to Cd. Results showed that Cd exposure induced a reduction of peanut growth and produced changes in the histological structure with a deposit of unknown material on the epidermal and endodermal cells. When plants were exposed to 10 μM Cd, no modification of chlorophyll, lipid peroxides, carbonyl groups, or hydrogen peroxide (H₂O₂) content was observed. At this concentration, peanut leaves and roots glutathione (GSH) content decreased. However, peanut roots were able to synthesize different types of PCs (PC2, PC3, PC4). In conclusion, PC synthesis could prevent metal disturbance on cellular redox balance, avoiding oxidative damage to macromolecules.

  20. Tablet PCs: The Write Approach

    Science.gov (United States)

    Milner, Jacob

    2006-01-01

    This article discusses the transforming effects of tablet PCs in the classroom. As 1-to-1 computing becomes the goal on K-12 campuses, school districts are turning to this newer, pen-based technology. Saint Mary's School's new Lenovo ThinkPad X41 tablet PCs had transformed the way Saint Mary's teachers did their jobs. Teachers created outlines for…

  1. Quantification of phytochelatins in Chlamydomonas reinhardtii using ferrocene-based derivatization.

    Science.gov (United States)

    Bräutigam, Anja; Bomke, Susanne; Pfeifer, Thorben; Karst, Uwe; Krauss, Gerd-Joachim; Wesenberg, Dirk

    2010-08-01

    A method for the identification and quantification of canonic and isoforms of phytochelatins (PCs) from Chlamydomonas reinhardtii was developed. After disulfide reduction with tris(2-carboxyethyl)phosphine (TCEP) PCs were derivatized with ferrocenecarboxylic acid (2-maleimidoyl)ethylamide (FMEA) in order to avoid oxidation of the free thiol functions during analysis. Liquid chromatography (LC) coupled to electrospray mass spectrometry (ESI-MS) and inductively coupled plasma-mass spectrometry (ICP-MS) was used for rapid and quantitative analysis of the precolumn derivatized PCs. PC(2-4), CysGSH, CysPC(2-4), CysPC(2)desGly, CysPC(2)Glu and CysPC(2)Ala were determined in the algal samples depending on the exposure of the cells to cadmium ions.

  2. Detection of phytochelatins in the hyperaccumulator Sedum alfredii exposed to cadmium and lead.

    Science.gov (United States)

    Zhang, Zhongchun; Gao, Xiang; Qiu, Baosheng

    2008-02-01

    Phytochelatins (PCs) are known to play an essential role in the heavy metal detoxification of some higher plants and fungi by chelating heavy metals. However, three recent papers reported that no PCs could be detected in the hyperaccumulator Sedum alfredii Hance upon cadmium, lead or zinc treatment, respectively. In this paper, PC synthesis was assayed again in the mine population of S. alfredii with the help of reversed phase high-performance liquid chromatography (HPLC), HPLC-mass spectrometry, and HPLC-tandem mass spectrometry. Our data showed that PC formation could be induced in the leaf, stem and root tissues of S. alfredii upon exposure to 400 microM cadmium, and only in the stem and root when exposed to 700 microM lead. However, no PCs were found in any part of S. alfredii when it was subjected to exposure to 1600 microM zinc.

  3. Quantitative Relationship between Cadmium Uptake and the Kinetics of Phytochelatin Induction by Cadmium in a Marine Diatom

    Science.gov (United States)

    Wu, Yun; Guo, Zhiqiang; Zhang, Wei; Tan, Qiaoguo; Zhang, Li; Ge, Xinlei; Chen, Mindong

    2016-01-01

    Heavy metals activate the synthesis of phytochelatins (PCs), while the induced PCs might affect metal uptake via chelating intracellular free metals. However, the relationship of PCs to metal uptake is poorly understood. In this study, we examined the kinetics of cadmium (Cd) accumulation and the synthesis of PCs in a marine diatom, Thalassiosira weissflogii, under different irradiance levels. Irradiance alone could not change the concentrations of PCs in the Cd-free treatments, while higher irradiance accelerated the induction of intracellular PCs at the same [Cd2+] level. PC-SH (2 × PC2 + 3 × PC3 + 4 × PC4) was bound with Cd at a stoichiometric ratio of 2 to 49 in our short-term uptake experiments, indicating that PC induction is sufficient to serve as the first line of defense against Cd stress. A positive linear correlation between the induction rate of PCs and the Cd uptake rate was observed, while the ratio of the PC content to intracellular Cd varied greatly when the irradiance was increased several fold. Because metal uptake has been successfully used in predicting acute metal toxicity, our findings are helpful for understanding the role of PCs in metal detoxification and developing PCs as biomarkers for metal sensitivity. PMID:27779209

  4. Cadmium uptake and interaction with phytochelatins in wheat protoplasts.

    Science.gov (United States)

    Lindberg, Sylvia; Landberg, Tommy; Greger, Maria

    2007-01-01

    In order to investigate the role of phytochelatins in short-time uptake of Cd(2+) into the cytosol of wheat protoplasts, a new method was applied, using fluorescence microscopy and the heavy metal-specific fluorescent dye, 5-nitrobenzothiazole coumarin, BTC-5N. The uptake of Cd(2+) into protoplasts from 5- to 7-day-old wheat seedlings (Triticum aestivum, L. cv. Kadett) was lower in protoplasts from seedlings raised in the presence of 1 microM CdCl(2), than in the absence. Presence of CdCl(2) in the cultivation medium increased the content of phytochelatins (PCs) in the protoplasts. When seedlings were raised in the presence of both Cd(2+) and buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, only little PC was found in the protoplasts. Pre-treatment with BSO alone did not affect the content of PC, but inhibited that of GSH. The inhibition of GSH was independent of pre-treatment with Cd(2+). Unidirectional flux analyses, using (109)Cd(2+), showed approximately the same uptake pattern of Cd(2+) as did the fluorescence experiments showing the cytosolic uptake of Cd(2+). Thus, the diminished uptake of Cd(2+) into protoplasts from cadmium-pre-treated plants was not depending on PCs. Instead, it is likely that pre-treatment with Cd(2+) causes a down-regulation of the short-term Cd(2+) uptake, or an up-regulation of the Cd(2+) extrusion. Moreover, since addition of Cd(2+) to protoplasts from control plants caused a cytosol acidification, it is likely that a Cd(2+/)H(+)-antiport mechanism is involved in the extrusion of Cd(2+) from these protoplasts.

  5. Neutron proton crystallography station (PCS)

    Energy Technology Data Exchange (ETDEWEB)

    Fisher, Zoe [Los Alamos National Laboratory; Kovalevsky, Andrey [Los Alamos National Laboratory; Johnson, Hannah [Los Alamos National Laboratory; Mustyakimov, Marat [Los Alamos National Laboratory

    2009-01-01

    The PCS (Protein Crystallography Station) at Los Alamos Neutron Science Center (LANSCE) is a unique facility in the USA that is designed and optimized for detecting and collecting neutron diffraction data from macromolecular crystals. PCS utilizes the 20 Hz spallation neutron source at LANSCE to enable time-of-flight measurements using 0.6-7.0 {angstrom} neutrons. This increases the neutron flux on the sample by using a wavelength range that is optimal for studying macromolecular crystal structures. The diagram below show a schematic of PCS and photos of the detector and instrument cave.

  6. Phytoremediation capacity of aquatic plants is associated with the degree of phytochelatin polymerization.

    Science.gov (United States)

    Török, Anamaria; Gulyás, Zsolt; Szalai, Gabriella; Kocsy, Gábor; Majdik, Cornelia

    2015-12-15

    Phytochelatins (PCs) play important role in phytoremediation as heavy metal binding peptides. In the present study, the association between heavy metal removal capacity and phytochelatin synthesis was compared through the examination of three aquatic plants: Elodea canadensis, Salvinia natans and Lemna minor. In case of a Cd treatment, or a Cd treatment combined with Cu and Zn, the highest removal capacity was observed in L. minor. At the same time, E. canadensis showed the lowest removal capacity except for Zn. The heavy metal-induced (Cu+Zn+Cd) oxidative stress generated the highest ascorbate level in L. minor. Cd in itself or combined with the other two metals induced a 10-15-fold increase in the amount of ɣ-glutamylcysteine in L. minor while no or smaller changes were observed in the other two species. Correspondingly, the total PC content was 6-8-fold greater in L. minor. In addition, PCs with higher degree of polymerization were only observed in L. minor (PC4, PC6 and PC7) while PC2 and PC3 occurred in E. canadensis and S. natans only. The correlation analysis indicated that the higher phytoremediation capacity of L. minor was associated with the synthesis of PCs and their higher degree of polymerization.

  7. Effects of Interaction Between Cadmium and Plumbum on Phytochelatins and Glutathione Production in Wheat (Triticum aestivum L.)

    Institute of Scientific and Technical Information of China (English)

    Qin SUN; Xiao-Rong WANG; Shi-Ming DING; Xin-Fang YUAN

    2005-01-01

    Phytochelatins (PCs) may function as a potential biomarker for metal toxicity. However, less attention has been paid to the effects of metal interactions on the production of PCs and glutathione (GSH),the most prominent cellular thiol. In the present study, the effects of interactions between cadmium (Cd) and plumbum (Pb) on the production of PCs and GSH were monitored over a period of 14 d in wheat (Triticum aestivum L.) tissues. The results showed that combination of Cd and Pb led to synergistic growth inhibition in wheat. Exposure to Cd or Pb increased levels of PCs in a concentration-, tissue-, and time-dependent manner. Cadmium was more effective that Pb in increasing PCs production. Compared with the effects of Cd or Pb alone on the production of PCs, the combination of Cd and Pb acted synergistically, resulting in an enhanced production of PCs. Cadmium also stimulated GSH production in a concentration-, tissue-, and time-dependent manner. However, Pb had no obvious effects on GSH levels. The combination of Pb and Cd antagonized GSH production over the course of the growth period. The results of the present study suggest that metal interactions should be considered in the application of PCs and GSH as potential biomarkers for the evaluation of metal toxicity.

  8. Biosynthesis of Cd-bound phytochelatins by Phaeodactylum tricornutum and their speciation by size-exclusion chromatography and ion-pair chromatography coupled to ICP-MS.

    Science.gov (United States)

    Loreti, Valeria; Toncelli, Daniel; Morelli, Elisabetta; Scarano, Gioacchino; Bettmer, Jörg

    2005-10-01

    Cd-bound phytochelatins (Cd-PCs) have been synthesised by incubation of Phaeodactylum tricornutum cell cultures with Cd and purified by size-exclusion chromatography-UV-Vis. These complexes, which were identified in previous work, have now been used as model substances to develop and optimise ion-pair chromatography (IPC) coupled to inductively coupled plasma-mass spectrometry (ICP-MS) for analysis of Cd-PCs. Subsequent analysis of samples taken from Silene vulgaris plants cultivated under heavy metal stress conditions revealed Cd signals but no Cd-PC signals. By use of isotopically enriched (116)Cd-PCs the sample preparation steps were verified to determine the stability of the analytes. We observed species transformation between Cd-PCs and other unidentified Cd complexes. Consequently, the kinetic and thermodynamic lability of Cd-PCs are decisive factors in their detection.

  9. Characterization of Hg-phytochelatins complexes in vines (Vitis vinifera cv Malbec) as defense mechanism against metal stress.

    Science.gov (United States)

    Spisso, Adrian A; Cerutti, Soledad; Silva, Fernanda; Pacheco, Pablo H; Martinez, Luis D

    2014-06-01

    An approach to understand vines (Vitis vinifera) defense mechanism against heavy metal stress by isolation and determination of Hg-phytochelatins (PCs) complexes was performed. PCs are important molecules involved in the control of metal concentration in plants. PCs complex toxic metals through -SH groups and stores them inside cells vacuole avoiding any toxic effect of free metals in the cytosol. The Hg-PCs identification was achieved by determination of Hg and S as hetero-tagged atoms. A method involving two-dimensional chromatographic analysis coupled to atomic spectrometry and confirmation by tandem mass spectrometry is proposed. An approach involving size exclusion chromatography coupled to inductively coupled plasma mass spectrometry on roots, stems, and leaves extracts describing Hg distribution according to molecular weight and sulfur associations is proposed for the first time. Medium-low molecular weight Hg-S associations of 29-100 kDa were found, suggesting PCs presence. A second approach employing reversed-phase chromatography coupled to atomic fluorescence spectrometry analysis allowed the determination of Hg-PCs complexes within the mentioned fractions. Chromatograms showed Hg-PC2, Hg-PC3 and Hg-PC4 presence only in roots. Hg-PCs presence in roots was confirmed by ESI-MS/MS analysis.

  10. Comparative analysis of the contribution of phytochelatins to cadmium and arsenic tolerance in soybean and white lupin.

    Science.gov (United States)

    Vázquez, Saúl; Goldsbrough, Peter; Carpena, Ramón O

    2009-01-01

    The biosynthesis of phytochelatins (PCs) plays a crucial role in the detoxification and homeostasis of heavy metals and metalloids in plants. However, in an increasing number of plant species metal(loid) tolerance is not well correlated with the accumulation of PCs: tolerant ecotypes frequently contain lower levels of PCs than non-tolerant ecotypes. In this study we have compared the responses of soybean (Glycine max L. cv. Resnik) and white lupin (Lupinus albus L. cv. Marta) to cadmium and arsenate in order to assess the role of homophytochelatins (hPCs) in the tolerance of soybean to these toxic elements. Soybean plants treated with Cd and As showed a high contribution of homo-glutathione (hGSH) to the pool of thiols in shoots in comparison to white lupin. Higher levels of hPCs in Cd-treated soybeans compared to PCs in lupins did not prevent growth inhibition. In contrast, the role of hPCs in the detoxification mechanism to arsenate in soybean seems to be clearer, showing higher thiol concentrations and lower growth reductions than those present in lupin plants.

  11. Fly-ash induced synthesis of phytochelatins in chickpea (Cicer arietinum L.) plants.

    Science.gov (United States)

    Gupta, D K; Rai, U N; Tripathi, R D; Sinha, S; Rai, P; Inouhe, M

    2005-07-01

    Phytochelatins and related metabolites (cysteine and GSH) were found to be induced in the shoots of two varieties of Cicer arietinum viz., CSG-8962 and C-235 grown under different amendments of fly-ash with garden soil and press mud. Cysteine, GSH, PCs and its speciation were found in higher concentrations in amended fly-ash than in the control 100% soil. Two species of metal binding peptides i.e., PC2 and PC4 were found in both varieties and in amendments, however, their concentration varied depending upon the fly-ash concentrations in both amendments. Further, var. CSG-8962 was found more tolerant than var. C-235 because of higher concentrations of PCs and related metabolites.

  12. Induction of phytochelatins in hydrilla verticillata (l.f.) Royle under cadmium stress

    Energy Technology Data Exchange (ETDEWEB)

    Tripathi, R.D.; Rai, U.N.; Gupta, M. [National Botanical Research Institute, Lucknow (India)] [and others

    1996-03-01

    Plants tolerate Cd by sequestering them through synthesizing phytochelatins with the general structure (t-Glu-cys)n-gly where n= 2-11 depending upon the species from which these peptides are isolated. Recent biochemical evidence suggests that these peptides are synthesized via posttranslationally activated, metal-dependent enzymatic pathways from the precursor glutathione. However, most of these studies are confined to terrestrial species and only a few studies have been made on higher aquatic plants. Recently H. verticillata and other aquatic higher plants have been reported to be hyperaccumulators of Cd and have demonstrated the ability to remove many toxic metals, including Cd, from wastewater. It is hypothesized that cadmium hyperaccumulating ability of the macrophyte is associated with induction of the metal chelating peptides, the phytochelatins (PCs), to copeup with high cellular Cd levels. In view of this, it was considered worthwhile to examine the induction of phytochelatins and changes in levels of glutathione and related metabolites in H. verticillata under Cd stress.

  13. Phytochelatin synthesis in response to elevated CO2 under cadmium stress in Lolium perenne L.

    Science.gov (United States)

    Jia, Yan; Ju, Xuehai; Liao, Shangqiang; Song, Zhengguo; Li, Zhongyang

    2011-10-15

    The increasing atmospheric CO(2) and heavy metal contamination in soil are two of the major environmental problems. Knowledge of the Cd stress coping mechanisms is needed to understand the regulation of the plants' metabolism under the increasing atmospheric CO(2) levels. Lolium perenne L. was grown hydroponically under two concentrations of atmospheric CO(2) (360 and 1000μLL(-1)) and six concentrations of cadmium (0-160μmolL(-1)) to investigate Cd uptake, Cd transportation, and variations in phytochelatin (PC) concentration. Cd concentrations in roots and shoots were decreased, but transport index (Ti) was increased under elevated CO(2) compared to ambient CO(2). Regardless of CO(2) concentrations, Cd and PC concentrations, especially the concentrations of high molecular weight PCs (PC(4), PC(5), PC(6)) were higher with increasing Cd concentration in growth media and longer Cd exposure time. Under the elevated CO(2), more high molecular weight PCs (PC(4), PC(5), PC(6)) in shoots and roots were synthesized compared to ambient CO(2), with higher SH:Cd ratio in roots as well. These results indicate that under elevated CO(2), L. perenne may be better protected against Cd stress with higher biomass, lower Cd concentration and better detoxification by phytochelatins.

  14. Reduced translocation of cadmium from roots is associated with increased production of phytochelatins and their precursors.

    Science.gov (United States)

    Akhter, Fardausi; McGarvey, Brian; Macfie, Sheila M

    2012-12-15

    Cadmium (Cd) is a non-essential trace element and its environmental concentrations are approaching toxic levels, especially in some agricultural soils. Understanding how and where Cd is stored in plants is important for ensuring food safety. In this study, we examined two plant species that differ in the distribution of Cd among roots and leaves. Lettuce and barley were grown in nutrient solution under two conditions: chronic (4 weeks) exposure to a low, environmentally relevant concentration (1.0 μM) of Cd and acute (1 h) exposure to a high concentration (5.0 mM) of Cd. Seedlings grown in solution containing 1.0 μM CdCl₂ did not show symptoms of toxicity and, at this concentration, 77% of the total Cd was translocated to leaves of lettuce, whereas only 24% of the total Cd was translocated to barley leaves. We tested the hypothesis that differential accumulation of Cd in roots and leaves is related to differential concentrations of phytochelatins (PCs), and its precursor peptides. The amounts of PCs and their precursor peptides in the roots and shoots were measured using HPLC. Each of PC₂₋₄ was synthesized in the barley root upon chronic exposure to Cd and did not increase further upon acute exposure. In the case of lettuce, no PCs were detected in the root given either Cd treatment. The high amounts of PCs produced in barley root could have contributed to preferential retention of Cd in barley roots.

  15. Phytochelatin concentrations in the equatorial Pacific

    Science.gov (United States)

    Ahner, Beth A.; Lee, Jennifer G.; Price, Neil M.; Morel, François M. M.

    1998-11-01

    Phytochelatin, an intracellular metal-binding polypeptide synthesized in eucaryotic algae in response to metals such as Cd and Cu, was measured in particulate samples collected from the equatorial Pacific. The concentrations in these samples (normalized to total particulate chl a) were unexpectedly high compared to laboratory culture data and were on average slightly more than in coastal areas where the metal concentrations are typically much greater. In part, the high field concentrations can be explained by the low cellular concentrations of chlorophyll a resulting from very low ambient Fe, but laboratory experiments provide a possible explanation for the rest of the difference. At low concentrations of inorganic Cd (Cd'=3 pM), increasing amounts of phytochelatin were induced by decreasing Zn concentrations in the culture medium of two diatoms: Thalassiosira weissflogii, a coastal species, and T. parthenaia, an isolate from the equatorial Pacific. In all previous studies, phytochelatin production has been directly correlated with increasing metal concentrations. Decreasing Co also resulted in higher phytochelatin concentrations in T. weissflogii and Emiliania huxleyi. Replicating the field concentrations of Zn, Co, and Cd in the laboratory results in cellular concentrations (amol -1 cell) that are very similar to those estimated for the field. Contrary to the expectation that high metal concentrations in the equatorial upwelling would cause elevated phytochelatin concentrations, there was no increase in phytochelatin concentrations from 20° S to 10° N—near surface samples were roughly the same at all stations. Also, most of the depth profiles had a distinct subsurface maximum. Neither of these features is readily explained by the available Zn and Cd data. Incubations with additions of Cd and Cu performed on water sampled at four separate stations induced significantly higher concentrations of phytochelatins than those in controls in a majority of the samples

  16. Analysis of plant Pb tolerance at realistic submicromolar concentrations demonstrates the role of phytochelatin synthesis for Pb detoxification.

    Science.gov (United States)

    Fischer, Sina; Kühnlenz, Tanja; Thieme, Michael; Schmidt, Holger; Clemens, Stephan

    2014-07-01

    Lead (Pb) ranks first among metals with respect to tonnage produced and released into the environment. It is highly toxic and therefore an important pollutant of worldwide concern. Plant Pb uptake, accumulation, and detoxification mobilize Pb into food webs. Still, knowledge about the underlying mechanisms is very limited. This is largely due to serious experimental challenges with respect to Pb availability. In most studies, Pb(II) concentrations in the millimolar range have been used even though the toxicity threshold is in the nanomolar range. We therefore developed a low-phosphate, low-pH assay system that is more realistic with respect to soil solution conditions. In this system the growth of Arabidopsis thaliana seedlings was significantly affected by the addition of only 0.1 μM Pb(NO3)2. Involvement of phytochelatins in the detoxification of Pb(II) could be demonstrated by investigating phytochelatin synthase mutants. They showed a stronger inhibition of root growth and a lack of Pb-activated phytochelatin synthesis. In contrast, other putative Pb hypersensitive mutants were unaffected under these conditions, further supporting the essential role of phytochelatins for Pb detoxification. Our findings demonstrate the need to monitor plant Pb responses at realistic concentrations under controlled conditions and provide a strategy to achieve this.

  17. Identification and quantification of phytochelatins in roots of rice to long-term exposure: evidence of individual role on arsenic accumulation and translocation.

    Science.gov (United States)

    Batista, Bruno Lemos; Nigar, Meher; Mestrot, Adrien; Rocha, Bruno Alves; Barbosa Júnior, Fernando; Price, Adam H; Raab, Andrea; Feldmann, Jörg

    2014-04-01

    Rice has the predilection to take up arsenic in the form of methylated arsenic (o-As) and inorganic arsenic species (i-As). Plants defend themselves using i-As efflux systems and the production of phytochelatins (PCs) to complex i-As. Our study focused on the identification and quantification of phytochelatins by HPLC-ICP-MS/ESI-MS, relating them to the several variables linked to As exposure. GSH, 11 PCs, and As-PC complexes from the roots of six rice cultivars (Italica Carolina, Dom Sofid, 9524, Kitrana 508, YRL-1, and Lemont) exposed to low and high levels of i-As were compared with total, i-As, and o-As in roots, shoots, and grains. Only Dom Sofid, Kitrana 508, and 9524 were found to produce higher levels of PCs even when exposed to low levels of As. PCs were only correlated to i-As in the roots (r=0.884, P <0.001). However, significant negative correlations to As transfer factors (TF) roots-grains (r= -0.739, P <0.05) and shoots-grains (r= -0.541, P <0.05), suggested that these peptides help in trapping i-As but not o-As in the roots, reducing grains' i-As. Italica Carolina reduced i-As in grains after high exposure, where some specific PCs had a special role in this reduction. In Lemont, exposure to elevated levels of i-As did not result in higher i-As levels in the grains and there were no significant increases in PCs or thiols. Finally, the high production of PCs in Kitrana 508 and Dom Sofid in response to high As treatment did not relate to a reduction of i-As in grains, suggesting that other mechanisms such as As-PC release and transport seems to be important in determining grain As in these cultivars.

  18. Efficiency of cadmium chelation by phytochelatins in Nitzschia palea (Kützing) W. Smith.

    Science.gov (United States)

    Figueira, Etelvina; Freitas, Rosa; Guasch, Helena; Almeida, Salomé F P

    2014-03-01

    Phytochelatins (PCs) are thiol-rich peptides, enzymatically synthesized by plants and algae under exposure to certain metals (Cd, Pb, Zn, Ag, As, Cu). Due to their ability to bind metal ions, they play an important role in the cellular detoxification, forming stable metal-PC complexes that minimize the intracellular deleterious effects of metals. The aim of the present work was to evaluate the efficiency of PC-Cd chelation in the freshwater diatom Nitzschia palea under 0, 0.1 and 0.2 mg Cd L(-1), which induced different levels of oxidative stress. This objective was accomplished by the isolation of PC-Cd complexes through size exclusion chromatography. Two peaks were identified, corresponding to high molecular weight (HMW) and low molecular weight (LMW) complexes. In each of the complexes, Cd was quantified by inductively coupled plasma-mass spectrometry, thiol composition was determined by HPLC analysis and the efficiency of Cd chelation calculated by -SH/Cd ratios in HMW and LMW complexes at both Cd concentrations. Results showed that the majority of intracellular Cd was complexed with PCs (75.2-91.2 %). PCs-binding efficiency in this diatom species was higher at HMW than at LMW complexes and enhanced with the increase of Cd concentration exposure. Our work evidenced the important role of PCs as the main intracellular tolerance mechanism in this species. The efficiency increase of Cd-PC binding is related to the increment of PCs synthesis and to the number of Cd ions coordinately bonded to -SH groups in LMW and HMW complexes.

  19. Phytochelatins play a key role in arsenic accumulation and tolerance in the aquatic macrophyte Wolffia globosa.

    Science.gov (United States)

    Zhang, Xin; Uroic, M Kalle; Xie, Wan-Ying; Zhu, Yong-Guan; Chen, Bao-Dong; McGrath, Steve P; Feldmann, Jörg; Zhao, Fang-Jie

    2012-06-01

    The rootless duckweed Wolffia globosa can accumulate and tolerate relatively large amounts of arsenic (As); however, the underlying mechanisms were unknown. W. globosa was exposed to different concentrations of arsenate with or without l-buthionine sulphoximine (BSO), a specific inhibitor of γ-glutamylcysteine synthetase. Free thiol compounds and As(III)-thiol complexes were identified and quantified using HPLC - high resolution ICP-MS - accurate mass ESI-MS. Without BSO, 74% of the As accumulated in the duckweed was complexed with phytochelatins (PCs), with As(III)-PC(4) and As(III)-PC(3) being the main species. BSO was taken up by the duckweed and partly deaminated. The BSO treatment completely suppressed the synthesis of PCs and the formation of As(III)-PC complexes, and also inhibited the reduction of arsenate to arsenite. BSO markedly decreased both As accumulation and As tolerance in W. globosa. The results demonstrate an important role of PCs in detoxifying As and enabling As accumulation in W. globosa.

  20. An electrophoretic profiling method for thiol-rich phytochelatins and metallothioneins.

    Science.gov (United States)

    Fan, Teresa W M; Lane, Andrew N; Higashi, Richard M

    2004-01-01

    Thiol-rich peptides such as phytochelatins (PCs) and metallothioneins (MTs) are important cellular chelating agents which function in metal detoxification and/or homeostasis. The variations in molecular sizes and lack of chromophores of these peptides make their analysis difficult. This paper reports an electrophoresis-based method for a broad screen of thiol-rich peptides and proteins. The method uses the thiol-selective fluorescent tag, monobromobimane, coupled with Tricine--sodium dodecyl sulphate--urea polyacrylamide gel electrophoresis for a sensitive determination of both PCs and MTs. Results for PCs were confirmed by two-dimensional NMR and HPLC-tandem MS analyses. Sample throughput is substantially improved over chromatography-based methods through parallel sample analysis in 1 h of electrophoretic separation. The method is versatile in that peptides ranging from glutathione to large proteins can be analysed by simple modification(s) of the extraction and electrophoretic conditions, and the nature of the method supports serendipitous detection of unexpected or novel thiol metabolites.

  1. Complexation of Hg with phytochelatins is important for plant Hg tolerance.

    Science.gov (United States)

    Carrasco-Gil, Sandra; Alvarez-Fernández, Ana; Sobrino-Plata, Juan; Millán, Rocío; Carpena-Ruiz, Ramón O; Leduc, Danika L; Andrews, Joy C; Abadía, Javier; Hernández, Luís E

    2011-05-01

    Three-week-old alfalfa (Medicago sativa), barley (Hordeum vulgare) and maize (Zea mays) were exposed for 7 d to 30 µm of mercury (HgCl(2) ) to characterize the Hg speciation in root, with no symptoms of being poisoned. The largest pool (99%) was associated with the particulate fraction, whereas the soluble fraction (SF) accounted for a minor proportion (phytochelatins (PCs) in root SF, which was particularly varied in alfalfa (eight ligands and five stoichiometries), a species that also accumulated homophytochelatins. Spatial localization of Hg in alfalfa roots by microprobe synchrotron X-ray fluorescence spectroscopy showed that most of the Hg co-localized with sulphur in the vascular cylinder. Extended X-ray Absorption Fine Structure (EXAFS) fingerprint fitting revealed that Hg was bound in vivo to organic-S compounds, i.e. biomolecules containing cysteine. Albeit a minor proportion of total Hg, Hg-PCs complexes in the SF might be important for tolerance to Hg, as was found with Arabidopsis thaliana mutants cad2-1 (with low glutathione content) and cad1-3 (unable to synthesize PCs) in comparison with wild type plants. Interestingly, high-performance liquid chromatography-electrospray ionization-time of flight analysis showed that none of these mutants accumulated Hg-biothiol complexes.

  2. Phytochelatin-metal(loid) transport into vacuoles shows different substrate preferences in barley and Arabidopsis.

    Science.gov (United States)

    Song, Won-Yong; Mendoza-Cózatl, David G; Lee, Youngsook; Schroeder, Julian I; Ahn, Sang-Nag; Lee, Hyun-Sook; Wicker, Thomas; Martinoia, Enrico

    2014-05-01

    Cadmium (Cd) and arsenic (As) are toxic to all living organisms, including plants and humans. In plants, Cd and As are detoxified by phytochelatins (PCs) and metal(loid)-chelating peptides and by sequestering PC-metal(loid) complexes in vacuoles. Consistent differences have been observed between As and Cd detoxification. Whereas chelation of Cd by PCs is largely sufficient to detoxify Cd, As-PC complexes must be sequestered into vacuoles to be fully detoxified. It is not clear whether this difference in detoxification pathways is ubiquitous among plants or varies across species. Here, we have conducted a PC transport study using vacuoles isolated from Arabidopsis and barley. Arabidopsis vacuoles accumulated low levels of PC2 -Cd, and vesicles from yeast cells expressing either AtABCC1 or AtABCC2 exhibited negligible PC2 -Cd transport activity compared with PC2 -As. In contrast, barley vacuoles readily accumulated comparable levels of PC2 -Cd and PC2 -As. PC transport in barley vacuoles was inhibited by vanadate, but not by ammonium, suggesting the involvement of ABC-type transporters. Interestingly, barley vacuoles exhibited enhanced PC2 transport activity when essential metal ions, such as Zn(II), Cu(II) and Mn(II), were added to the transport assay, suggesting that PCs might contribute to the homeostasis of essential metals and detoxification of non-essential toxic metal(loid)s.

  3. Synergistic defensive mechanism of phytochelatins and antioxidative enzymes in Brassica chinensis L. against Cd stress

    Institute of Scientific and Technical Information of China (English)

    CHEN LiQin; GUO YiFei; YANG LiMin; WANG QiuQuan

    2008-01-01

    Brassica chinensis L. was chosen and exposed to different concentrations of Cd exposure to evaluate its Cd-accumulating capacity and its potential cellular defensive mechanisms. Cd accumulation in the shoots and roots of B. chinensis was up to 1348.3±461.8 and 3761.0±795.0 mg per killogram of dry weight, respectively, under 200 μmol/L of Cd exposure. Increasing Cd accumulation in the plant was accompanied by rapid accumulation of phytochelatins (PCs), and the sequestration of Cd by PCs pro-vided a primary cellular mechanism for Cd detoxification and tolerance of B. chinensis. Furthermore, malondialdehyde formation, hydrogen peroxide content and antioxidative enzyme activities such as superoxide dismutase, catalase, guaiacol peroxidase and ascorbate peroxidase were observed in the shoots of Cd-stressed B. chinensis. Increasing enzyme activities in response to concentrations of 5 to 50 μmol/L Cd showed an efficient defense against oxidative stress, suggesting that the antioxidative system was a secondary defensive mechanism. These resulted in reduced free Cd damage and en-hanced Cd accumulation and tolerance. Glutathione plays a pivotal role in these two detoxification pathways. In general, these results suggested that PCs and the antioxidative system are synergistic in combatting Cd-induced oxidative stress and that they play important roles in Cd detoxification of B. chinensis, and also give a deep understanding of the natural defensive mechanisms in plants under heavy metal stress.

  4. Chemometrics applied to the analysis of induced phytochelatins in Hordeum vulgare plants stressed with various toxic non-essential metals and metalloids.

    Science.gov (United States)

    Dago, Àngela; González, Inmaculada; Ariño, Cristina; Díaz-Cruz, José Manuel; Esteban, Miquel

    2014-01-01

    Hordeum vulgare plants were stressed with Hg, Cd and As and their phytotoxicity was evaluated in terms of growth inhibition and total metal uptake by the plant. The synthesised phytochelatins ((γ-Glu-Cys)n-Gly, n=2-5; PCs) were determined by HPLC with amperometric detection at a glassy carbon electrode. The results indicate that H. vulgare is a good phytostabilisation plant due to its capacity to accumulate heavy metals in roots. Cd and Hg are the most uptake toxic elements, being Cd the most potent inducer of PCs. The data obtained on the different PCs and related peptides induced by each heavy metal were used to perform a Principal Component Analysis (PCA) of the results as a function of the contaminating toxic element or its concentration level. The nature of the stressor element could be predicted from the pattern of PCs and related peptides identified by PCA. PCs were the most strongly induced peptides under Cd and Hg stress, whereas As only tended to synthesise small thiols such as glutathione and γ-glutamylcysteine, both precursors of PCs synthesis. This finding indicates that PCs are induced at different rates depending on the metal stressor used.

  5. Effects of exogenous organic chelators on phytochelatins production and its relationship with cadmium toxicity in wheat (Triticum aestivum L.) under cadmium stress.

    Science.gov (United States)

    Sun, Q; Wang, X R; Ding, S M; Yuan, X F

    2005-06-01

    Phytochelatins (PCs) have been proposed as a potential biomarker for metal toxicity. In this study, cadmium (Cd) toxicity, PCs production and their relationship in wheat under Cd stress were examined using various exogenous organic chelator-buffered nutrient solutions. Single Cd stress produced strong toxic effects, as indicated by decreases of growth parameters, high level of lipid peroxidation in leaf and overproduction of PCs in root. Exogenous organic chelators with proper dose more or less reduced Cd toxicity by increasing growth parameters and decreasing lipid peroxidation in leaves. Of organic chelators (EDTA, DTPA, citric acid, malic acid and oxalic acid), EDTA was the most effective in decreasing Cd toxicity in plants, followed by DTPA and citric acid. Simultaneously, the concentrations of Cd-induced PCs in roots decreased, and the greatest decrease was caused by application of EDTA and DTPA. Linearly positive relationships were observed between Cd toxicity and root PCs concentrations under the influences of organic chelators, particularly EDTA, DTPA and citric acid. Furthermore, present results provide stronger evidence that PCs synthesis in plant cells was related to free Cd ion concentrations, not total Cd, and demonstrate that the levels of PCs production in plants correlated well with toxic effects caused by the bioavailable Cd levels.

  6. The detoxification of lead in Sedum alfredii H. is not related to phytochelatins but the glutathione.

    Science.gov (United States)

    Gupta, D K; Huang, H G; Yang, X E; Razafindrabe, B H N; Inouhe, M

    2010-05-15

    Two ecotypes of S. alfredii [Pb accumulating (AE) and Pb non-accumulating (NAE)] differing in their ability in accumulating Pb were exposed to different Pb levels to evaluate the effects on plant length, photosynthetic pigments, antioxidant enzymes (SOD and APX), cysteine, non-protein thiols (NP-SH), phytochelatins (PCs) and glutathione (GSH) vis-à-vis Pb accumulation. Both ecotypes showed significant Pb accumulation in roots, however only the AE showed significant Pb accumulation in shoots. We found that both AE and NAE of S. alfredii-induced biosynthesis of GSH rather than phytochelatins in their tissue upon addition of even high Pb levels (200 microM). Root and shoot length were mostly affected in both ecotypes after addition of higher Pb concentrations and on longer durations, however photosynthetic pigments did not alter upon addition of any Pb treatment. Both superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities of AE were higher than NAE. The levels of cysteine and NP-SH were also higher in AE than in NAE. Hence, the characteristic Pb accumulation of ecotypes differed presumably in relation to their capacity for detoxification of Pb. These results suggest that enzymatic and non-enzymatic antioxidants play a key role in the detoxification of Pb-induced toxic effects in Sedum alfredii. This plant can be used as an indicator species for Pb contamination.

  7. Physiological and biochemical responses of Suaeda fruticosa to cadmium and copper stresses: growth, nutrient uptake, antioxidant enzymes, phytochelatin, and glutathione levels.

    Science.gov (United States)

    Bankaji, I; Caçador, I; Sleimi, N

    2015-09-01

    Environmental pollution by trace metal elements (TMEs) is a serious problem worldwide, increasing in parallel with the development of human technology. The present research aimed to examine the response of halophytic species Suaeda fruticosa to oxidative stress posed by combined abiotic stresses. Plants have been grown for 1 month with an irrigation solution supplemented with 200 mM NaCl and 400 μM Cd(2+) or 400 μM Cu(2+). The level of glutathione (GSH), phytochelatins (PCs), and antioxidant enzyme activities [ascorbate peroxidase (APX), guaiacol peroxidase (GPX), and catalase (CAT)] as well as lipid peroxidation was studied to see the stress exerted by the TME and the level of tolerance and detoxification strategy adopted by S. fruticosa. Relative growth rate (RGR) decreased under Cd(2+) stress in this species, whereas Cu(2+) did not have any impact on S. fruticosa performance. Cd(2+) or Cu(2+) enhanced malondialdehyde, suggesting reactive oxygen species-induced disruption of membrane integrity and oxidative stress in S. fruticosa. On the other hand, the activities of the antioxidant enzymes CAT, APX, and GPX diminished and mineral nutrition was disturbed by metal stress. S. fruticosa was able to synthesize PCs in response to TME toxicity. However, data indicate that GSH levels underwent a significant decrease in roots and leaves of S. fruticosa stressed by Cd(2+) or Cu(2+). The GSH depletion accompanied by the increase of phytochelatin concentration suggests the involvement of GSH in the synthesis of phytochelatins.

  8. Arsenic tolerance in Arabidopsis is mediated by two ABCC-type phytochelatin transporters.

    Science.gov (United States)

    Song, Won-Yong; Park, Jiyoung; Mendoza-Cózatl, David G; Suter-Grotemeyer, Marianne; Shim, Donghwan; Hörtensteiner, Stefan; Geisler, Markus; Weder, Barbara; Rea, Philip A; Rentsch, Doris; Schroeder, Julian I; Lee, Youngsook; Martinoia, Enrico

    2010-12-01

    Arsenic is an extremely toxic metalloid causing serious health problems. In Southeast Asia, aquifers providing drinking and agricultural water for tens of millions of people are contaminated with arsenic. To reduce nutritional arsenic intake through the consumption of contaminated plants, identification of the mechanisms for arsenic accumulation and detoxification in plants is a prerequisite. Phytochelatins (PCs) are glutathione-derived peptides that chelate heavy metals and metalloids such as arsenic, thereby functioning as the first step in their detoxification. Plant vacuoles act as final detoxification stores for heavy metals and arsenic. The essential PC-metal(loid) transporters that sequester toxic metal(loid)s in plant vacuoles have long been sought but remain unidentified in plants. Here we show that in the absence of two ABCC-type transporters, AtABCC1 and AtABCC2, Arabidopsis thaliana is extremely sensitive to arsenic and arsenic-based herbicides. Heterologous expression of these ABCC transporters in phytochelatin-producing Saccharomyces cerevisiae enhanced arsenic tolerance and accumulation. Furthermore, membrane vesicles isolated from these yeasts exhibited a pronounced arsenite [As(III)]-PC(2) transport activity. Vacuoles isolated from atabcc1 atabcc2 double knockout plants exhibited a very low residual As(III)-PC(2) transport activity, and interestingly, less PC was produced in mutant plants when exposed to arsenic. Overexpression of AtPCS1 and AtABCC1 resulted in plants exhibiting increased arsenic tolerance. Our findings demonstrate that AtABCC1 and AtABCC2 are the long-sought and major vacuolar PC transporters. Modulation of vacuolar PC transporters in other plants may allow engineering of plants suited either for phytoremediation or reduced accumulation of arsenic in edible organs.

  9. Electrochemical monitoring of phytochelatin accumulation in Nicotiana tabacum cells exposed to sub-cytotoxic and cytotoxic levels of cadmium

    Energy Technology Data Exchange (ETDEWEB)

    Fojta, Miroslav [Laboratory of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65 Brno (Czech Republic)]. E-mail: fojta@ibp.cz; Fojtova, Miloslava [Laboratory of Molecular Epigenetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65 Brno (Czech Republic); Havran, Ludek [Laboratory of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65 Brno (Czech Republic); Pivonkova, Hana [Laboratory of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65 Brno (Czech Republic); Dorcak, Vlastimil [Laboratory of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65 Brno (Czech Republic); Sestakova, Ivana [J. Heyrovsky Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, Dolejskova 3, 182 23 Prague 8 (Czech Republic)

    2006-02-03

    Cadmium belongs to the most dangerous environmental pollutants among the toxic heavy metals seriously affecting vital functions in both animal and plant cells. It has been previously shown that cadmium ions at 50-100 {mu}M concentrations caused tobacco BY-2 (TBY-2) cells to enter apoptosis within several days of exposure. Phytochelatins (PCs), the 'plant metallothioneins', are cysteine-rich peptides involved in detoxification of heavy metals in plants. The PCs are synthesized in response to the heavy metal exposure. In this paper, we utilized electrochemical analysis to monitor accumulation of PCs in the TBY-2 cells exposed to cadmium ions. Measurements of a characteristic PC signal at mercury electrode in the presence of cobalt ions made it possible to detect changes in the cellular PC levels during the time of cultivation, starting from 30 min after exposure. Upon TBY-2 cultivation in the presence of cytotoxic cadmium concentrations, the PC levels remarkably increased during the pre-apoptotic phase and reached a limiting value at cultivation times coinciding with apoptosis trigger. The PC level observed for a sub-cytotoxic cadmium concentration (10 {mu}M) was about three-times lower than that observed for the 50 or 100 {mu}M cadmium ions after 5 days of exposure. We show that using a simple electrochemical analysis, synthesis of PCs in plant cells can be easily followed in parallel with other tests of the cellular response to the toxic heavy metal stress.

  10. Phytochelatin synthesis in response to Hg uptake in aquatic plants near a chlor-alkali factory.

    Science.gov (United States)

    Turull, Marta; Grmanova, Gabriela; Dago, Àngela; Ariño, Cristina; Díez, Sergi; Díaz-Cruz, José Manuel; Esteban, Miquel

    2017-06-01

    The effects of mercury (Hg) released from a chlor-alkali factory in aquatic plants along the Ebro River basin (NE Spain) were analysed considering the phytochelatins (PCn) and their isoforms content in these plants. These compounds were analyzed using HPLC with amperometric detection, and the macrophytes species Ceratophyllum demersum and Myriopyllum spicatum were collected in two sampling campaigns, autumn and spring, respectively. To correlate the PCn content in macrophytes with the Hg contamination, analysis of total Hg (THg) content in plants and suspended particulate matter, as well as the dissolved-bioavailable fraction of Hg in water measured by the diffusive gradient in thin film (DGT) technique were done. The results confirm the presence of PC2-Ala in extracts of C. demersum and PC2-desGly in M. spicatum, and the concentration of these thiol compounds depends clearly on the distance between the hot spot and the downstream sites: the higher the levels are, the closer the hot spot is. Since most of the Hg is hypothesized to be associated with SPM and transported downstream, our results of the DGT suggest that trace amounts of Hg in water can be released as free metal ions yielding a certain accumulation in plants (reaching the ppb level) that are enough for activation of induction of PCs. A few PCs species have been determined, at different seasons, indicating that they can be used as good indicators of the presence of bioavailable Hg in aquatic media throughout the year.

  11. Phytochelatins do not correlate with the level of Cd accumulation in Chlamydomonas spp.

    Science.gov (United States)

    Nishikawa, Kahoko; Onodera, Ai; Tominaga, Noriko

    2006-06-01

    Chlamydomonas acidophila KT-1 and Chlamydomonas acidophila DVB238 exhibit a strong heavy metal tolerance, but C. acidophila DVB238 can accumulate a much higher amount of Cadmium (Cd) than C. acidophila KT-1. Phytochelatins (PCs) are known to play an important role in the detoxification of several toxic heavy metals, but the relationship between PCs and Cd accumulation is not clear. PC metabolism and Cd accumulation were investigated by using three Chlamydomonas strains including Chlamydomonas reinhardtii C-9 as a standard alga. The results showed that the PC content did not correlate closely with the level of Cd accumulation, maintenance of a high GSH level seeming to be more important for Cd accumulation. The ultrastructure of C. acidophila KT-1 was extremely disrupted by a great increase in starch granules, which resulted in a moribund state, but hyper-accumulator C. acidophila DVB238 did not exhibit an increase in starch granules in its cells, in spite of Cd accumulation in its chloroplasts, cytosol and vacuoles. These results indicated that C. acidophila DVB238 probably has a developed detoxification system preventing such as destruction of the cells due to Cd toxicity.

  12. Make Projects Small Form Factor PCs

    CERN Document Server

    Wessels, Duane

    2006-01-01

    Shoebox sized and smaller, small-form-factor PCs can pack as much computing muscle as a full-sized desktop computer. They consumer less power, have few or no moving parts, and are very quiet. Whether you plan to use one as a standalone PC or want to embed it in your next hacking project, a small-form-factor PC can be a lot of fun to build. Make Projects: Small Form Factor PCs is the only book available that shows you how to build small-form-factor PCs -- from kits and from scratch -- that are more interesting and more personalized than what a full-sized PC can give you. Included in the book

  13. HPLC with electrochemical detection for studying of synthesis of phytochelatins in cadmium treated flax

    Directory of Open Access Journals (Sweden)

    Ondrej Zitka

    2010-12-01

    Full Text Available If there would be free heavy metals occurring in the organismwithout control it initiates creation of number of dangerous species (ROS. These species are disturbing of homeostasis of organism by subsequent oxidation of essential cellular structures as nucleic acids. Animal and plant organism has both one efficient defending system which includes synthesis of glutathione (GSH. But plants are limited in case of escape from metal contaminated area. Therefore plant cell has mechanism includes synthesis of phytochelatins (PCfrom GSH by phytochelatin synthase. PC´s are able to immobilize heavy metal ion via number of thiol groups. We treated the plants of flax (Linum usativum by various concentration of Cd(II ions (50, 250, and 500μM in our study. Subsequently we used our developed method for determination of oxidation stress as GSH/GSSG ratio and levels of PC-2,3,4 and 5. We observed that most synthesised PC under the highest concentration of Cd(II treatment was PC-2,4 and 5.

  14. Effect of Inoculation with Glomus versiforme on Cadmium Accumulation, Antioxidant Activities and Phytochelatins of Solanum photeinocarpum.

    Science.gov (United States)

    Tan, Shi-Yun; Jiang, Qiu-Yun; Zhuo, Feng; Liu, Hui; Wang, Yu-Tao; Li, Shao-Shan; Ye, Zhi-Hong; Jing, Yuan-Xiao

    2015-01-01

    The plant growth, phosphate acquisition, Cd translocation, phytochelatins (PCs) production and antioxidant parameters [superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), ascorbate peroxidase (APX), glutathione reductase (GR), glutathione (GSH), ascorbate (ASA) and malonaldehyde (MDA)] were investigated in Cd-hyperaccumulator Solanum photeinocarpum inoculated with Glomus versiforme BGC GD01C (Gv) in Cd-added soils (0, 5, 10, 20, 40 mg Cd kg-1 soil). Mycorrhizal colonization rates were generally high (from 77% to 94%), and hardly affected by Cd. Gv colonization significantly enhanced P acquisition, growth and total Cd uptakes in both shoots and roots of S. photeinocarpum at all Cd levels. Meanwhile, Gv symbiosis significantly increased Cd concentration in the roots, and decreased Cd concentration in the shoots at all Cd levels, which indicates that Gv could promote phytostabilization by enhancing Cd accumulation in the roots to inhibit its translocation to shoots and the "dilution effects" linked to an increase in plant dry matter yield and a reduced Cd partitioning to shoots. Moreover, the improvement of CAT, POD and APX activities in the leaves of mycorrhizal plants infers that Gv symbiosis helped S. photeinocarpum to relieve oxidative damage to biomolecules in Cd-contaminated soil. The evident decline of MDA content in the leaves of mycorrhizal plants indicates that Gv symbiosis evidently improved antioxidant activities, and the enhancement of PCs production in the leaves of mycorrhizal plants suggests that Gv-inoculated plant may be more efficient to relieve Cd phytotoxicity. Therefore, the possible mechanisms of Cd phytotoxicity alleviation by Gv can be concluded as the decline of Cd concentration in the shoots and the improvement of P acquisition, PCs production and activities of CAT, POD, APX in mycorrhizal plants.

  15. Effect of Inoculation with Glomus versiforme on Cadmium Accumulation, Antioxidant Activities and Phytochelatins of Solanum photeinocarpum.

    Directory of Open Access Journals (Sweden)

    Shi-Yun Tan

    Full Text Available The plant growth, phosphate acquisition, Cd translocation, phytochelatins (PCs production and antioxidant parameters [superoxide dismutase (SOD, catalase (CAT, guaiacol peroxidase (POD, ascorbate peroxidase (APX, glutathione reductase (GR, glutathione (GSH, ascorbate (ASA and malonaldehyde (MDA] were investigated in Cd-hyperaccumulator Solanum photeinocarpum inoculated with Glomus versiforme BGC GD01C (Gv in Cd-added soils (0, 5, 10, 20, 40 mg Cd kg-1 soil. Mycorrhizal colonization rates were generally high (from 77% to 94%, and hardly affected by Cd. Gv colonization significantly enhanced P acquisition, growth and total Cd uptakes in both shoots and roots of S. photeinocarpum at all Cd levels. Meanwhile, Gv symbiosis significantly increased Cd concentration in the roots, and decreased Cd concentration in the shoots at all Cd levels, which indicates that Gv could promote phytostabilization by enhancing Cd accumulation in the roots to inhibit its translocation to shoots and the "dilution effects" linked to an increase in plant dry matter yield and a reduced Cd partitioning to shoots. Moreover, the improvement of CAT, POD and APX activities in the leaves of mycorrhizal plants infers that Gv symbiosis helped S. photeinocarpum to relieve oxidative damage to biomolecules in Cd-contaminated soil. The evident decline of MDA content in the leaves of mycorrhizal plants indicates that Gv symbiosis evidently improved antioxidant activities, and the enhancement of PCs production in the leaves of mycorrhizal plants suggests that Gv-inoculated plant may be more efficient to relieve Cd phytotoxicity. Therefore, the possible mechanisms of Cd phytotoxicity alleviation by Gv can be concluded as the decline of Cd concentration in the shoots and the improvement of P acquisition, PCs production and activities of CAT, POD, APX in mycorrhizal plants.

  16. Arsenic accumulation in root and shoot vis-a-vis its effects on growth and level of phytochelatins in seedlings of Cicer arietinum L.

    Science.gov (United States)

    Gupta, D K; Tripathi, R D; Mishra, S; Srivastava, S; Dwivedi, S; Rai, U N; Yang, X E; Huanji, H; Inouhe, M

    2008-05-01

    Arsenic (As) contamination of water and soil has become a subject of prime interest due to its direct effect on human health through drinking water and food. In present study two varieties (CSG-8962 and C-235) of chickpea, Cicer arietinum L., which is a major supplementary food in many parts of India and a valuable source of protein, has been selected to estimate the level of arsenate in root and shoot of five day old seedlings vis-à-vis effect of arsenate on seedling growth and induction of thiols including glutathione (GSH) and phytochelatins (PCs) and their homologues. Both varieties accumulated arsenate to similar levels and most of the metalloid was confined to roots, only about 2.5% was translocated to shoot. Plant growth was also not affected significantly in both the varieties. Arsenate exposure significantly induced the levels of thiols including PCs and homophytochelatins (hPCs). The induction of thiols was much higher in roots than shoots and was greater in var C-235 between the two tested ones. Thus, both varieties tolerated and detoxified arsenic through chelation with GSH, PCs and hPCs, primarily in roots, however var C-235 performed better

  17. Cadmium hyperaccumulation leads to an increase of glutathione rather than phytochelatins in the cadmium hyperaccumulator Sedum alfredii.

    Science.gov (United States)

    Sun, Qin; Ye, Zhi Hong; Wang, Xiao Rong; Wong, Ming Hung

    2007-11-01

    Sedum alfredii has been reported to be a cadmium (Cd) hyperaccumulator. Phytochelatins (PCs) and other thiol (SH)-containing compounds have been proposed to play an important role in the detoxification and tolerance of some heavy metals, but it is not clear whether PCs are responsible for Cd hyperaccumulation and tolerance in S. alfredii. In this study, two geographically isolated populations of S. alfredii were studied: one population grew on an old Pb/Zn mine site, while the other on a non-mine site. The mine population of this species exhibited a stronger heavy metal tolerance than in the other population. Root-to-shoot transport of Cd was higher in population located at the mine site than at the non-mine site. Considerable amounts of Cd were accumulated in leaves and stems of mine plants, while most Cd was distributed in roots of non-mine plants. Non-protein SH in plant tissues of two populations were further investigated by a HPLC pre-column derivatization system. Upon exposure to Cd, no PCs were detected in all tissues of mine population, while an appreciable amount of glutathione (GSH) was observed in the descending order of stem>root>leaf. The concentrations of GSH consistently increased with the increase of exogenous Cd concentrations and time. On the contrary, Cd exposure strongly induced the production of PCs (mainly PC(2) and PC(3)) and GSH in plant tissues of non-mine population, and the concentrations of GSH showed an initial drop over the duration of 7-d exposure. The present results provided strong evidence that PCs are not involved in Cd transport, hyperaccumulation and tolerance in mine population of S. alfredii.

  18. Determination and characterization of phytochelatins by liquid chromatography coupled with on line chemical vapour generation and atomic fluorescence spectrometric detection.

    Science.gov (United States)

    Bramanti, Emilia; Toncelli, Daniel; Morelli, Elisabetta; Lampugnani, Leonardo; Zamboni, Roberto; Miller, Keith E; Zemetra, Joseph; D'Ulivo, Alessandro

    2006-11-10

    Liquid chromatography (LC) coupled on line with UV/visible diode array detector (DAD) and cold vapour generation atomic fluorescence spectrometry (CVGAFS) has been developed for the speciation, determination and characterization of phytochelatins (PCs). The method is based on a bidimensional approach, e.g. on the analysis of synthetic PC solutions (apo-PCs and Cd(2+)-complexed PCs) (i) by size exclusion chromatography coupled to UV diode array detector (SEC-DAD); (ii) by the derivatization of PC -SH groups in SEC fractions by p-hydroxymercurybenzoate (PHMB) and the indirect detection of PC-PHMB complexes by reversed phase liquid chromatography coupled to atomic fluorescence detector (RPLC-CVGAFS). MALDI-TOF/MS (matrix assisted laser desorption ionization time of flight mass spectrometry) analysis of underivatized synthetic PC samples was performed in order have a qualitative information of their composition. Quantitative analysis of synthetic PC solutions has been performed on the basis of peak area of PC-PHMB complexes of the mercury specific chromatogram and calibration curve of standard solution of glutathione (GSH) complexed to PHMB (GS-PHMB). The limit of quantitation (LOQ) in terms of GS-PHMB complex was 90 nM (CV 5%) with an injection volume of 35 microL, corresponding to 3.2 pmol (0.97 ng) of GSH. The method has been applied to analysis of extracts of cell cultures from Phaeodactylum tricornutum grown in Cd-containing nutrient solutions, analysed by SEC-DAD-CVGAFS and RPLC-DAD-CVGAFS.

  19. Dequenching of Cu(I)-bathocuproine disulfonate complexes for high-performance liquid chromatographic determination of phytochelatins, heavy-metal-binding peptides produced by the primitive red alga Cyanidioschyzon merolae.

    Science.gov (United States)

    Shirabe, Tomoo; Ito, Kyoko; Yoshimura, Etsuro

    2008-12-01

    A novel method has been devised for the determination of phytochelatins (PCs), heavy-metal-tolerant peptides produced by higher plants and algae. The method is based on the facts that fluorescence of bathocuproine disulfonate (BCS) is quenched by Cu(I) ions as a result of Cu(I)-BCS complex formation and that PCs compete with BCS for Cu(I). Detection of PCs via recovered fluorescence of BCS using the Cu(I)-BCS complex as a postcolumn reagent, following separation of peptides on an octyldecylsilane column, demonstrated a highly sensitive method for determination of PCs. PCs in the primitive red alga, Cyanidioschyzon merolae, grown in the presence or absence of added Cd(II) were successfully determined by this protocol. Unlike other methods for the determination of PCs, which rely on the SH groups in the peptides, the proposed method is unique in that detection is based on the chemical nature of PCs, which favors the formation of complexes with Cu(I). In this context, the new method yields chromatograms based on the strength of binding Cu(I) ions.

  20. Arsenite treatment induces oxidative stress, upregulates antioxidant system, and causes phytochelatin synthesis in rice seedlings.

    Science.gov (United States)

    Mishra, Shruti; Jha, A B; Dubey, R S

    2011-07-01

    The effects of arsenite treatment on generation of reactive oxygen species, induction of oxidative stress, response of antioxidative system, and synthesis of phytochelatins were investigated in two indica rice (Oryza sativa L.) cvs. Malviya-36 and Pant-12 grown in sand cultures for a period of 5-20 days. Arsenite (As(2)O(3); 25 and 50 μM) treatment resulted in increased formation of superoxide anion (O (2) (.-) ), elevated levels of H(2)O(2) and thiobarbituric acid reactive substances, showing enhanced lipid peroxidation. An enhanced level of ascorbate (AA) and glutathione (GSH) was observed irrespective of the variation in the level of dehydroascorbate (DHA) and oxidized glutathione (GSSG) which in turn influenced redox ratios AA/DHA and GSH/GSSG. With progressive arsenite treatment, synthesis of total acid soluble thiols and phytochelatins (PC) increased in the seedlings. Among antioxidative enzymes, the activities of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6), total ascorbate peroxidase (APX, EC 1.11.1.11), chloroplastic ascorbate peroxidase, guaiacol peroxidase (EC 1.11.1.7), monodehydroascorbate reductase (EC 1.6.5.4), and glutathione reductase (EC 1.6.4.2) increased in arsenite treated seedlings, while dehyroascorbate reductase (EC 1.8.5.1) activity declined initially during 5-10 days and increased thereafter. Results suggest that arsenite treatment causes oxidative stress in rice seedlings, increases the levels of many enzymatic and non-enzymatic antioxidants, and induces synthesis of thiols and PCs, which may serve as important components in mitigating arsenite-induced oxidative damage.

  1. Tablet PCs: A Physical Educator's New Clipboard

    Science.gov (United States)

    Nye, Susan B.

    2010-01-01

    Computers in education have come a long way from the abacus of 5,000 years ago to the desktop and laptop computers of today. Computers have transformed the educational environment, and with each new iteration of smaller and more powerful machines come additional advantages for teaching practices. The Tablet PC is one. Tablet PCs are fully…

  2. Modulation of exogenous glutathione in phytochelatins and photosynthetic performance against cd stress in the two rice genotypes differing in Cd tolerance.

    Science.gov (United States)

    Cai, Yue; Cao, Fangbin; Cheng, Wangda; Zhang, Guoping; Wu, Feibo

    2011-11-01

    Greenhouse hydroponic experiments were conducted using Cd-sensitive (Xiushui63) and tolerant (Bing97252) rice genotypes to evaluate genotypic differences in response of photosynthesis and phytochelatins to Cd toxicity in the presence of exogenous glutathione (GSH). Plant height, chlorophyll content, net photosynthetic rate (Pn), and biomass decreased in 5 and 50 μM Cd treatments, and Cd-sensitive genotype showed more severe reduction than the tolerant one. Cadmium stress caused decrease in maximal photochemical efficiency of PSII (Fv/Fm) and effective PSII quantum yield [Y(II)] and increase in quantum yield of regulated energy dissipation [Y(NPQ)], with changes in Cd-sensitive genotype being more evident. Cadmium-induced phytochelatins (PCs), GSH, and cysteine accumulation was observed in roots of both genotypes, with markedly higher level in PCs and GSH on day 5 in Bing97252 compared with that measured in Xiushui63. Exogenous GSH significantly alleviated growth inhibition in Xiushui63 under 5 μM Cd and in both genotypes in 50 μM Cd. External GSH significantly increased chlorophyll content, Pn, Fv/Fm, and Y(II) of plants exposed to Cd, but decreased Y(NPQ) and the coefficient of non-photochemical quenching (qN). GSH addition significantly increased root GSH content in plants under Cd exposure (except day 5 of 50 μM Cd) and induced up-regulation in PCs of 5 μM-Cd-treated Bing97252 throughout the 15-day and Xiushui63 of 5-day exposure. The results suggest that genotypic difference in the tolerance to Cd stress was positively linked to the capacity in elevation of GSH and PCs, and that alleviation of Cd toxicity by GSH is related to significant improvement in chlorophyll content, photosynthetic performance, and root GSH levels.

  3. [The radiation-increased synthesis of phytochelatins in roots of gamma-irradiated barley seedlings].

    Science.gov (United States)

    Danilin, I A; Dikarev, V G; Geras'kin, S A

    2004-01-01

    It was shown that gamma-irradiation and cadmium nitrate increased synthesis of phytochelatins in roots of barley seedlings. The stimulation of synthesis of phytochelatins in gamma-irradiated plants was shown for the first time. The results obtained indicate more important role of phytochelatins in intercellular metabolism than heavy-metal-binding.

  4. Complexation of arsenite with phytochelatins reduces arsenite efflux and translocation from roots to shoots in Arabidopsis.

    Science.gov (United States)

    Liu, Wen-Ju; Wood, B Alan; Raab, Andrea; McGrath, Steve P; Zhao, Fang-Jie; Feldmann, Jörg

    2010-04-01

    Complexation of arsenite [As(III)] with phytochelatins (PCs) is an important mechanism employed by plants to detoxify As; how this complexation affects As mobility was little known. We used high-resolution inductively coupled plasma-mass spectrometry and accurate mass electrospray ionization-mass spectrometry coupled to HPLC to identify and quantify As(III)-thiol complexes and free thiol compounds in Arabidopsis (Arabidopsis thaliana) exposed to arsenate [As(V)]. As(V) was efficiently reduced to As(III) in roots. In wild-type roots, 69% of As was complexed as As(III)-PC4, As(III)-PC3, and As(III)-(PC2)2. Both the glutathione (GSH)-deficient mutant cad2-1 and the PC-deficient mutant cad1-3 were approximately 20 times more sensitive to As(V) than the wild type. In cad1-3 roots, only 8% of As was complexed with GSH as As(III)-(GS)3 and no As(III)-PCs were detected, while in cad2-1 roots, As(III)-PCs accounted for only 25% of the total As. The two mutants had a greater As mobility, with a significantly higher accumulation of As(III) in shoots and 4.5 to 12 times higher shoot-to-root As concentration ratio than the wild type. Roots also effluxed a substantial proportion of the As(V) taken up as As(III) to the external medium, and this efflux was larger in the two mutants. Furthermore, when wild-type plants were exposed to l-buthionine sulfoximine or deprived of sulfur, both As(III) efflux and root-to-shoot translocation were enhanced. The results indicate that complexation of As(III) with PCs in Arabidopsis roots decreases its mobility for both efflux to the external medium and for root-to-shoot translocation. Enhancing PC synthesis in roots may be an effective strategy to reduce As translocation to the edible organs of food crops.

  5. Derivatization of phytochelatins from Silene vulgaris, induced upon exposure to arsenate and cadmium: comparison of derivatization with Ellman's reagent and monobromobimane.

    Science.gov (United States)

    Sneller, F E; van Heerwaarden, L M; Koevoets, P L; Vooijs, R; Schat, H; Verkleij, J A

    2000-09-01

    Phytochelatins (PCs) are a family of thiol-rich peptides, with the general structure (gamma-Glu-Cys)(n)()-Gly, with n = 2-11, induced in plants upon exposure to excessive amounts of heavy metals and some metalloids, such as arsenic. Two types of PC analyses are currently used, i.e., acid extraction and separation on HPLC with either precolumn derivatization (pH 8.2) with monobromobimane (mBBr) or postcolumn derivatization (pH 7.8) with Ellman's reagent [5, 5'-dithiobis(2-nitrobenzoic acid), DTNB]. Although both methods were satisfactory for analysis of Cd-induced PCs, formation of (RS)(3)-As complexes during extraction of As-induced PCs rendered the DTNB method useless. This paper shows that precolumn derivatization with mBBr, during which the (RS)(3)-As complexes are disrupted, provides a qualitative and quantitative analysis of both Cd- and As-induced PCs. In addition, derivatization efficiencies of both methods for the oligomers with n = 2-4 (PC(2)(-)(4)) are compared. Derivatization efficiency decreased from 71.8% and 81.4% for mBBr and DTNB derivatization, respectively, for PC(2) to 27.4% and 50.2% for PC(4). This decrease is most likely due to steric hindrance. Correction of measured thiol concentration is therefore advised for better quantification of PC concentrations in plant material.

  6. Identification of in vivo nitrosylated phytochelatins in Arabidopsis thaliana cells by liquid chromatography-direct electrospray-linear ion trap-mass spectrometry.

    Science.gov (United States)

    Elviri, L; Speroni, F; Careri, M; Mangia, A; di Toppi, L Sanità; Zottini, M

    2010-06-18

    Reversed-phase liquid chromatography (RPLC) and electrospray (ESI)-linear ion trap (LIT) mass spectrometry was applied to the direct characterization of in vivo S-nitrosylated (SNO) phytochelatins (PCs) expressed in cadmium-stressed Arabidopsis thaliana cells. Cys-nitrosylation is under discussion as in vivo redox-based post-translational modification of proteins and peptides in plants in which the -NO group is involved as signal molecule in different biological functions. The gas-phase ion chemistry of in vivo and in vitro generated SNO-PC(s) was compared with the aim of evaluating NO binding stability and improving MS knowledge about peptide nitrosation. Using RPLC separation and ESI-LIT-MS, mono-nitrosylated PCs were identified in in vivo cadmium treated A. thaliana cells without derivatization. The in vivo binding of the NO group to PC(2), PC(3) and PC(4) resulted to occur selectively on only one cystein residue. The fragmentation pathway energies of the in vitro GSNO-generated NO-PCs with respect to the in vivo NO-PCs were investigated, suggesting the presence of a different internal stability for these molecules. By carrying out MS(2) experiments on these quasi-symmetric peptides, the different stability degree of the NO group was demonstrated to be correlated with the PC chain length. In addition, the data obtained highlight a putative role of the adjacent Glu/Cys motif in the gas-phase stability of the NO-containing molecule.

  7. Identification of high levels of phytochelatins, glutathione and cadmium in the phloem sap of Brassica napus. A role for thiol-peptides in the long-distance transport of cadmium and the effect of cadmium on iron translocation.

    Science.gov (United States)

    Mendoza-Cózatl, David G; Butko, Emerald; Springer, Franziska; Torpey, Justin W; Komives, Elizabeth A; Kehr, Julia; Schroeder, Julian I

    2008-04-01

    Phytochelatins (PCs) are glutathione-derived peptides that function in heavy metal detoxification in plants and certain fungi. Recent research in Arabidopsis has shown that PCs undergo long-distance transport between roots and shoots. However, it remains unknown which tissues or vascular systems, xylem or phloem, mediate PC translocation and whether PC transport contributes to physiologically relevant long-distance transport of cadmium (Cd) between shoots and roots. To address these questions, xylem and phloem sap were obtained from Brassica napus to quantitatively analyze which thiol species are present in response to Cd exposure. High levels of PCs were identified in the phloem sap within 24 h of Cd exposure using combined mass spectrometry and fluorescence HPLC analyses. Unexpectedly, the concentration of Cd was more than four-fold higher in phloem sap compared to xylem sap. Cadmium exposure dramatically decreased iron levels in xylem and phloem sap whereas other essential heavy metals such as zinc and manganese remained unchanged. Data suggest that Cd inhibits vascular loading of iron but not nicotianamine. The high ratios [PCs]/[Cd] and [glutathione]/[Cd] in the phloem sap suggest that PCs and glutathione (GSH) can function as long-distance carriers of Cd. In contrast, only traces of PCs were detected in xylem sap. Our results suggest that, in addition to directional xylem Cd transport, the phloem is a major vascular system for long-distance source to sink transport of Cd as PC-Cd and glutathione-Cd complexes.

  8. Influence of sulfur and cadmium on antioxidants, phytochelatins and growth in Indian mustard.

    Science.gov (United States)

    Bashir, Humayra; Ibrahim, Mohamed M; Bagheri, Rita; Ahmad, Javed; Arif, Ibrahim A; Baig, M Affan; Qureshi, M Irfan

    2015-01-12

    Soils in many parts of the world are contaminated with heavy metals, leading to multiple, deleterious effects on plants and threats to world food production efficiency. Cadmium (Cd) is one such metal, being toxic at relatively low concentrations as it is readily absorbed and translocated in plants. Sulfur-rich compounds are critical to the impact of Cd toxicity, enabling plants to increase their cellular defence and/or sequester Cd into vacuoles mediated by phytochelatins (PCs). The influence of sulfur on Cd-induced stress was studied in the hyperaccumulator plant Indian mustard (Brassica juncea) using two sulfur concentrations (+S, 300 µM [Formula: see text] and S-deficient -S, [Formula: see text]) with and without the addition of Cd (100 µM CdCl2) at two different time intervals (7 and 14 days after treatment). Compared with control plants (+S/-Cd), levels of oxidative stress were higher in S-deficient (-S/-Cd) plants, and greatest in S-deficient Cd-treated (-S/+Cd) plants. However, additional S (+S/+Cd) helped plants cope with oxidative stress. Superoxide dismutase emerged as a key player against Cd stress under both -S and +S conditions. The activity of ascorbate peroxidase, glutathione reductase and catalase declined in Cd-treated and S-deficient plants, but was up-regulated in the presence of sulfur. Sulfur deficiency mediated a decrease in ascorbate and glutathione (GSH) content but changes in ascorbate (reduced : oxidized) and GSH (reduced : oxidized) ratios were alleviated by sulfur. Our data clearly indicate that a sulfur pool is needed for synthesis of GSH, non-protein thiols and PCs and is also important for growth. Sulfur-based defence mechanisms and the cellular antioxidant pathway, which are critical for tolerance and growth, collapsed as a result of a decline in the sulfur pool.

  9. Concentrations of phytochelatins and glutathione found in natural assemblages of seaweeds depend on species and metal concentrations of the habitat.

    Science.gov (United States)

    Pawlik-Skowrońska, Barbara; Pirszel, Jacek; Brown, Murray T

    2007-07-20

    The occurrence of the metal-complexing thiol peptides, phytochelatins (PC) in natural populations of brown, red and green seaweeds (marine macroalgae) was studied. Concentrations of PCs and their precursor glutathione (GSH) were measured in seaweeds collected from locations in south-west England with different levels of contamination by trace metals, to evaluate their role under natural environmental conditions. The non-protein thiols were identified and quantified in seaweed extracts by HPLC and the molecular structures of PCs were confirmed by LC-ESIMS. The capacity for production of PCs of representative seaweeds under Cd and Zn exposure was also assessed, experimentally. The concentrations of metals/metalloids (As, Cu, Cd, Pb and Zn) accumulated by the seaweeds were determined by ICP-MS. For the first time, PCs are reported in native Phaeophyceae (Fucus spp.), Rhodophyceae (Solieria chordalis) and Chlorophyceae (Rhizoclonium tortuosum) but not in thalli of Ulva spp. and Codium fragile (Chlorophyceae). The concentrations of PCs in brown and red seaweeds correlated with the contamination history of sampling sites and total metal burden of thalli. The highest concentrations of metals (5.6-7.1micromolg(-1) DW), PCs (200-240nmolSHg(-1)DW) and GSH (1,550-3,960nmolSHg(-1)DW), and the longest PC chain lengths (PC(2-4)) were found in Fucus spp. collected from the most contaminated site. A combination of PC-production and maintenance of high concentrations of GSH allows Fucus spp. and R. tortuosum (2,000nmolGSHg(-1)DW) to thrive in highly contaminated environments whereas in Ulva spp. high concentrations of GSH (1,000-1,500nmolSHg(-1)DW) together with thick cells walls and a high polysaccharide content appear to be responsible for metal-resistance. The lack of production of PCs in these green macroalgae suggests lower intracellular metal accumulation rather than an inability for synthesizing PCs. The higher concentrations of Cu (approximately 3.4micromolg(-1)DW) found in

  10. PCs All-in-One For Dummies

    CERN Document Server

    Chambers, Mark L

    2010-01-01

    One-stop shopping for everything you need to know about PCs!. If you're a PC owner, you have a pretty good idea of just how much there is to discover about your PC, whether you use it for work or play. Comprised of eight minibooks, this All-in-One guide covers essential PC topics from soup through nuts, including the latest updates to PC hardware, Windows 7, the Internet, Office 2010, digital media, upgrading and troubleshooting, social media, and home networking. This new edition features expanded coverage of using popular social media such as Twitter, Facebook, WordPress, and blogging. Plus,

  11. A simulation environment for ITER PCS development

    Energy Technology Data Exchange (ETDEWEB)

    Walker, M.L., E-mail: walker@fusion.gat.com [General Atomics, P.O. Box 85608, San Diego, CA 92186-5608 (United States); Ambrosino, G.; De Tommasi, G. [CREATE/Università di Napoli Federcico II, Napoli (Italy); Humphreys, D.A. [General Atomics, P.O. Box 85608, San Diego, CA 92186-5608 (United States); Mattei, M. [Seconda Università di Napoli, Napoli (Italy); Neu, G.; Raupp, G.; Treutterer, W. [Max-Planck-Institut fuer Plasmaphysik, EURATOM Association, 85748 Garching (Germany); Winter, A. [ITER Organization, Route de Vinon-sur-Verdon, 13115 St. Paul-lez-Durance (France)

    2014-05-15

    Highlights: • Describes task to develop simulation tool to aid development/testing of ITER PCS. • Requirements and use cases and preliminary architecture have been delivered. • Detailed design is now being developed. • Provides overview of use cases and requirements. • Provides overview of architecture and status of development. - Abstract: A simulation environment known as the Plasma Control System Simulation Platform (PCSSP), specifically designed to support development of the ITER Plasma Control System (PCS), is currently under construction by an international team encompassing a cross-section of expertise in simulation and exception handling for plasma control. The proposed design addresses the challenging requirements of supporting the PCS design. This paper provides an overview of the PCSSP project and a discussion of some of the major features of its design. Plasma control for the ITER tokamak will be significantly more challenging than for existing fusion devices. An order of magnitude greater performance (e.g. [1,2]) is needed for some types of control, which together with limited actuator authority, implies that optimized individual controllers and nonlinear saturation logic are required. At the same time, consequences of control failure are significantly more severe, which implies a conflicting requirement for robust control. It also implies a requirement for comprehensive and robust exception handling. Coordinated control of multiple competing objectives with significant interactions, together with many shared uses of actuators to control multiple variables, implies that highly integrated control logic and shared actuator management will be required. It remains a challenge for the integrated technologies to simultaneously address these multiple and often competing requirements to be demonstrated on existing fusion devices and adapted for ITER in time to support its operational schedule. We describe ways in which the PCSSP will help address

  12. The Evolution and Impact of PCS Technology.

    Science.gov (United States)

    1994-09-01

    divided into two 10 MHz blocks-one for voice services and one for data services as was revised in June 1994.45 The most important of the two PCS...CTIA), June, 1994), p. 4. 44 Communication Daily, Vol. 14, No. 137, July 18, 1994, p.3. i45 FinTech Mobile Communications, June 16, 1994. 46 FCC...a palm-sized portable answering machine 3 which can receive and replay voice messages based on Voice Now service being developed I 47 FinTech Mobile

  13. Relief of arsenate toxicity by Cd-stimulated phytochelatin synthesis in the green alga Chlamydomonas reinhardtii.

    Science.gov (United States)

    Kobayashi, Isao; Fujiwara, Shoko; Saegusa, Hirotaka; Inouhe, Masahiro; Matsumoto, Hiroko; Tsuzuki, Mikio

    2006-01-01

    In most photosynthetic organisms, inorganic arsenic taken up into the cells inhibits photosynthesis and cellular growth. In a green alga, Chlamydomonas reinhardtii, 0.5 mM arsenate inhibited photosynthesis almost completely within 30 min. However, in cells acclimated with a sublethal concentration (0.05 to 0.1 mM) of Cd, the inhibition of photosynthesis at 30 min after the addition of arsenate was relieved by more than 50%. The concentrations of arsenic incorporated into the cells were not significantly different between the Cd-acclimated and the non-acclimated cells. The Cd-acclimated cells accumulated Cd and synthesized phytochelatin (PC) peptides, which are known to play an important role in detoxification of heavy metals in plants. By the addition of an inhibitor of glutathione (an intermediate in the PC biosynthetic pathway) biosynthesis, buthionine sulfoximine, cells lost not only Cd tolerance but also arsenate tolerance. These results suggest that glutathione and/or PCs synthesized in Cd-acclimated cells are involved in mechanisms of arsenate tolerance.

  14. Effect of cadmium and calcium treatments on phytochelatin and glutathione levels in citrus plants.

    Science.gov (United States)

    López-Climent, M F; Arbona, V; Pérez-Clemente, R M; Zandalinas, S I; Gómez-Cadenas, A

    2014-01-01

    Industry residues, phosphate fertilisers and wastewater as a source of irrigation have considerably increased levels of heavy metals in the soil, mainly cadmium (Cd(2+)). To test the effects of a calcium (Ca(2+)) treatment on Cd(2+) accumulation and plant tolerance to this heavy metal, plants of two citrus genotypes, Cleopatra mandarin (CM) and Carrizo citrange (CC), were watered with increasing concentrations of Cd(2+), and phytochelatin (PC) and glutathione (GSH) content were measured. Both genotypes were able to synthesise PCs in response to heavy metal intoxication, although CM seems to be a better Cd(2+) excluder than CC. However, data indicate that CC plants had a higher capacity for regenerating GSH than CM plants. In this context, the effects of Ca(2+) treatment on Cd(2+) accumulation, plant survival and PC, GSH and oxidised glutathione (GSSG) content were assessed. Data indicate that treatment with Ca(2+) had two positive effects on citrus physiology: it reduced Cd(+2) uptake into roots and also increased GSH content (even in the absence of Cd(2+)). Overall, the data indicate that although Cd(2+) exclusion is a powerful mechanism to avoid heavy metal build-up into photosynthetic organs, the capacity to maintain optimum GSH levels to feed PC biosynthesis could also be an important factor in stress tolerance.

  15. Arsenate tolerance mechanism of Oenothera odorata from a mine population involves the induction of phytochelatins in roots.

    Science.gov (United States)

    Kim, Dae-Yeon; Park, Hyun; Lee, Sang-Hwan; Koo, Namin; Kim, Jeong-Gyu

    2009-04-01

    We investigated the arsenate tolerance mechanisms of Oenothera odorata by comparing two populations [i.e., one population from the mine site (MP) and the other population from an uncontaminated site (UP)] via the exposure of hydroponic solution containing arsenate (i.e., 0-50 microM). The MP plants were significantly more tolerant to arsenate than UP plants. The UP plants accumulated more As in their shoots and roots than did the MP plants. The UP plants translocated up to 21 microg g(-1) of As into shoots, whereas MP plants translocated less As (up to 4.5 microg g(-1)) to shoots over all treatments. The results of lipid peroxidation indicated that MP plants were less damaged by oxidative stress than were UP plants. Phytochelatin (PC) content correlated linearly with root As concentration in the MP (i.e., [PCs](root)=1.69x[As](root), r(2)=0.945) and UP (i.e., [PCs](root)=0.89x[As](root), r(2)=0.979) plants. This relationship means that increased PC to As ratio may be associated with increased tolerance. Our results suggest that PC induction in roots plays a critical role in As tolerance of O. odorata.

  16. The role of phytochelatins and antioxidants in tolerance to Cd accumulation in Brassica juncea L.

    Science.gov (United States)

    Seth, Chandra Shekhar; Kumar Chaturvedi, Pranav; Misra, Virendra

    2008-09-01

    A hydroponics experiment using Indian mustard (Brassica juncea L.) was conducted to investigate the effect of different concentrations (10-160 microM) of cadmium (Cd) and a fixed concentration (500 microM) of ethylene diamine tetra acetic acid (EDTA) on Cd accumulation and its toxicity for 14 and 28 days (d). The results showed that Cd alone and Cd+EDTA increased total dry biomass production, photosynthetic pigments and total protein content of B. juncea up to 160 microM with respect to control for 14d (hormesis effect). Further, on treatment with Cd at 160 microM for 28d, dry biomass of root and shoot, total protein content and total chlorophyll decreased up to 73%, 58%, 67% and 53% respectively, while in the case of Cd+EDTA, the decrease in the above parameters was 38%, 50%, 57% and 46% with respect to their control. It was observed that the maximum Cd accumulation after 28d in the root and shoot was 1925 and 977 mg kg(-1) dry weight (dw), respectively, while in the case of Cd+EDTA it was 1013 and 2316 mg kg(-1)dw, respectively. Levels of phytochelatins (PCs), glutathione reductase (GR; EC 1.6.4.2), non-protein thiols (NP-SH) and glutathione (GSH) were monitored as plants primary and secondary metal detoxifying responses. Glutathione reductase showed three-fold increased activity for Cd and 2.2-fold for Cd+EDTA at 160 microM after 14d followed by decreased activity after 28d with respect to control. Maximum synthesis of PCs was found at 10 microM of Cd exposure followed by a gradual decline after 28d. This may be correlated with reduced level of GSH, probably due to reduced GR activity, resulting in enhanced oxidative stress as also proved by phenotypic changes in plants such as browning of roots and yellowing of leaves. Thus, the capacity of B. juncea to accumulate and tolerate high concentrations of Cd, through enhanced level of PCs, GSH, NP-SH and GR suggests its applicability for phytoremediation.

  17. Effects of simultaneous expression of heterologous genes involved in phytochelatin biosynthesis on thiol content and cadmium accumulation in tobacco plants.

    Science.gov (United States)

    Wawrzynski, Adam; Kopera, Edyta; Wawrzynska, Anna; Kaminska, Jolanta; Bal, Wojciech; Sirko, Agnieszka

    2006-01-01

    Transgenic tobacco (Nicotiana tabacum cv. LA Burley 21) lines expressing three genes encoding enzymes thought to be critical for the efficient production of phytochelatins, (i) serine acetyltransferase (EC 2.3.1.30) involved in the production of O-acetylserine, the cysteine precursor, (ii) gamma-glutamylcysteine synthetase (EC 6.3.2.2) involved in the production of gamma-glutamylcysteine, the precursor of glutathione, and (iii) phytochelatin synthase (EC 2.3.2.15), were obtained and analysed for non-protein thiol content and cadmium accumulation. After a 3 week exposure to 15 microM CdCl2, plants expressing transgenes (either separately or in combination) had increased cadmium concentration in roots but not in shoots compared with the wild type. Nearly all transgenic lines analysed had more non-protein thiols than the wild type. The greatest effects (about 8-fold elevation of thiols) were found in one of the lines simultaneously expressing the three transgenes. Despite the fact that a multi-transgene strategy described in this work resulted in a strong increase in the levels of several classes of non-protein thiols in transgenic plants, other factors appeared to restrict cadmium accumulation in shoots.

  18. Contributions of apoplasmic cadmium accumulation, antioxidative enzymes and induction of phytochelatins in cadmium tolerance of the cadmium-accumulating cultivar of black oat (Avena strigosa Schreb.).

    Science.gov (United States)

    Uraguchi, Shimpei; Kiyono, Masako; Sakamoto, Takuya; Watanabe, Izumi; Kuno, Katsuji

    2009-07-01

    The contributions of cadmium (Cd) accumulation in cell walls, antioxidative enzymes and induction of phytochelatins (PCs) to Cd tolerance were investigated in two distinctive genotypes of black oat (Avena strigosa Schreb.). One cultivar of black oat 'New oat' accumulated Cd in the leaves at the highest concentration compared to another black oat cultivar 'Soil saver' and other major graminaceous crops. The shoot:root Cd ratio also demonstrated that 'New oat' was the high Cd-accumulating cultivar, whereas 'Soil saver' was the low Cd-accumulating cultivar. Varied levels of Cd exposure demonstrated the strong Cd tolerance of 'New oat'. By contrast, low Cd-accumulating cultivar 'Soil saver' suffered Cd toxicity such as growth defects and increased lipid peroxidation, even though it accumulated less Cd in shoots than 'New oat'. Higher activities of ascorbate peroxidase (EC 1.11.1.11) and superoxide dismutase (EC 1. 15. 1. 1) were observed in the leaves of 'New oat' than in 'Soil saver'. No advantage of 'New oat' in PCs induction was observed in comparison to Cd-sensitive cultivar 'Soil saver', although Cd exposure increased the concentration of total PCs in both cultivars. Higher and increased Cd accumulation in cell wall fraction was observed in shoots of 'New oat'. On the other hand, in 'Soil saver', apoplasmic Cd accumulation showed saturation under higher Cd exposure. Overall, the present results suggest that cell wall Cd accumulation and antioxidative activities function in the tolerance against Cd stress possibly in combination with vacuolar Cd compartmentation.

  19. Overexpressing GSH1 and AsPCS1 simultaneously increases the tolerance and accumulation of cadmium and arsenic in Arabidopsis thaliana.

    Science.gov (United States)

    Guo, Jiangbo; Dai, Xiaojing; Xu, Wenzhong; Ma, Mi

    2008-07-01

    The goal of this study was to develop transgenic plants with increased tolerance for and accumulation of heavy metals and metalloids from soil by simultaneous overexpression of AsPCS1 and GSH1 (derived from garlic and baker's yeast) in Arabidopsis thaliana. Phytochelatins (PCs) and glutathione (GSH) are the main binding peptides involved in chelating heavy metal ions in plants and other living organisms. Single-gene transgenic lines had higher tolerance to and accumulated more Cd and As than wild-type. Compared to single-gene transgenic lines, dual-gene transformants exhibited significantly higher tolerance to and accumulated more Cd and As. One of the dual-gene transgenic lines, PG1, accumulated twice the amount of Cd as single-gene transgenic lines. Simultaneous overexpression of AsPCS1 and GSH1 led to elevated total PC production in transgenic Arabidopsis. These results indicate that such a stacking of modified genes is capable of increasing Cd and As tolerance and accumulation in transgenic lines, and represents a highly promising new tool for use in phytoremediation efforts.

  20. Lead detoxification by coontail (Ceratophyllum demersum L.) involves induction of phytochelatins and antioxidant system in response to its accumulation.

    Science.gov (United States)

    Mishra, Seema; Srivastava, S; Tripathi, R D; Kumar, R; Seth, C S; Gupta, D K

    2006-11-01

    Coontail (Ceratophyllum demersum L.) plants when exposed to various concentrations of Pb (1-100microM) for 1-7days, exhibited both phytotoxic and tolerance responses. The specific responses were function of concentration and duration. Plants accumulated 1748mugPbg(-1) dw after 7d which reflected its metal accumulation ability, however most of the metal (1222microgg(-1) dw, 70%) was accumulated after 1d exposure only. The toxic effect and oxidative stress caused by Pb were evident by the reduction in biomass and photosynthetic pigments and increase in malondialddehyde (MDA) content and electrical conductivity with increase in metal concentration and exposure duration. Morphological symptoms of senescence phenomena such as chlorosis and fragmentation of leaves were observed after 7d. The metal tolerance and detoxification strategy adopted by the plant was investigated with reference to antioxidant system and synthesis of phytochelatins. Protein and antioxidant enzymes viz., superoxide dismutase (SOD, EC 1.15.1.1), guaiacol peroxidase (GPX, EC 1.11.1.7) ascorbate peroxidase (APX, EC 1.11.1.11), catalase (CAT, EC 1.11.1.6) and glutathione reductase (GR, EC 1.6.4.2) showed induction at lower concentration and duration followed by decline. All enzymes except GPX showed maximum activity after 1d. An increase in cysteine, non-protein thiols (NP-SH) and glutathione (GSH) content was observed at moderate exposure conditions followed by decline. Phytochelatins (PC(2) and PC(3)) were synthesized to significant levels at 10 and 50microM Pb with concomitant decrease in GSH levels. Thus production of PCs seems important for the detoxification of metal, however it may lead to depletion of GSH and consequently oxidative stress. Results suggest that plants responded positively to moderate Pb concentrations and accumulated high amount of metal. Due to metal accumulation coupled with detoxification potential, the plant appears to have potential for its use as phytoremediator species

  1. Public PCs: Log Out or Lose Out

    CERN Multimedia

    Computer Security Team

    2013-01-01

    Do you regularly use one of the public Windows or Linux terminals in the CERN library or in front of the Users' Office? Or do you often give presentations or run meetings, workshops or conferences? Did you recently attend a training session in the CERN Training Centre? If you answered at least once with “yes”, we have a plea for you: LOG OUT when done in order to protect your data!   You might recall that CERN considers that “Your Privacy is Paramount”. But this does not come for free. In the few past months, we have received several reports from vigilant people who have spotted open user sessions on public PCs at CERN. Those users simply forgot to log out once their work, training or meeting was over. Their session continued without them being present. Worse, with CERN using a central Single Sign-On (SSO) portal, their login credentials would allow a malicious person at CERN to use those credentials to access that user’s mailbox, DFS ...

  2. The nature of arsenic-phytochelatin complexes in Holcus lanatus and Pteris cretica.

    Science.gov (United States)

    Raab, Andrea; Feldmann, Jörg; Meharg, Andrew A

    2004-03-01

    We have developed a method to extract and separate phytochelatins (PCs)-metal(loid) complexes using parallel metal(loid)-specific (inductively coupled plasma-mass spectrometry) and organic-specific (electrospray ionization-mass spectrometry) detection systems-and use it here to ascertain the nature of arsenic (As)-PC complexes in plant extracts. This study is the first unequivocal report, to our knowledge, of PC complex coordination chemistry in plant extracts for any metal or metalloid ion. The As-tolerant grass Holcus lanatus and the As hyperaccumulator Pteris cretica were used as model plants. In an in vitro experiment using a mixture of reduced glutathione (GS), PC(2), and PC(3), As preferred the formation of the arsenite [As((III))]-PC(3) complex over GS-As((III))-PC(2), As((III))-(GS)(3), As((III))-PC(2), or As((III))-(PC(2))(2) (GS: glutathione bound to arsenic via sulphur of cysteine). In H. lanatus, the As((III))-PC(3) complex was the dominant complex, although reduced glutathione, PC(2), and PC(3) were found in the extract. P. cretica only synthesizes PC(2) and forms dominantly the GS-As((III))-PC(2) complex. This is the first evidence, to our knowledge, for the existence of mixed glutathione-PC-metal(loid) complexes in plant tissues or in vitro. In both plant species, As is dominantly in non-bound inorganic forms, with 13% being present in PC complexes for H. lanatus and 1% in P. cretica.

  3. ENHANCED BIOACCUMULATION OF HEAVY METAL BY BACTERIA CELLS DISPLAYING SYNTHETIC PHYTOCHELATINS. (R827227)

    Science.gov (United States)

    A novel strategy using synthetic phytochelatins is described for the purpose of developing microbial agents for enhanced bioaccumulation of toxic metals. Synthetic genes encoding for several metal-chelating phytochelatin analogs (Glu-Cys)nGly (EC8 (n = 8), EC11 (n = 11...

  4. Copper-induced oxidative stress and responses of antioxidants and phytochelatins in Hydrilla verticillata (L.f.) Royle.

    Science.gov (United States)

    Srivastava, Sudhakar; Mishra, Seema; Tripathi, Rudra D; Dwivedi, Sanjay; Gupta, Dharmendra K

    2006-12-30

    Copper, though essential, is potentially toxic heavy metal at supraoptimal level and has widespread contamination. The present investigation was carried out to study the responses induced by lower as well as higher doses of copper (0.1-25 microM) in an aquatic macrophyte, Hydrilla verticillata (L.f.) Royle for a period of 1-7 days. The plants accumulated copper in high amount with a maximum of 770 microg g(-1) dw on day 7 at 25 microM. Biomass and photosynthetic pigments showed less alteration up to 1 microM while at higher concentrations, significant decline occurred. Malondialdehyde (MDA) content and electrical conductivity (EC) also showed sharp increase at higher concentrations indicating oxidative stress. In response to copper exposure, plants showed significant induction of proteins and enzymes like superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), catalase (CAT) and glutathione reductase (GR), however, only up to moderate exposures. Total non-protein thiols (NP-SH) and cysteine levels increased significantly up to 5 microM copper exposure while at 25 microM, their level declined drastically. Reduced glutathione (GSH) showed decrease at all concentrations while oxidized glutathione (GSSG) simultaneously increased. Phytochelatins (PCs) were also induced significantly at studied concentrations of 1 and 5 microM on day 4 in comparison to control. However, copper chelation depicted by PC-SH to copper ratio was found to be low (6.5% at 1 microM and 2.4% at 5 microM) suggesting that PCs play only a part in integrated mechanisms of copper homeostasis and detoxification. Tolerant response of plants to moderate copper exposures and high accumulation potential warrants their suitability for remediation of moderately copper polluted water bodies.

  5. Ab initio ro-vibronic spectroscopy of the 2Π PCS radical and +1ΣPCS- anion

    Science.gov (United States)

    Finney, Brian; Mitrushchenkov, Alexander O.; Francisco, Joseph S.; Peterson, Kirk A.

    2016-12-01

    Near-equilibrium potential energy surfaces have been calculated for both the PCS radical and its anion using a composite coupled cluster approach based on explicitly correlated F12 methods in order to provide accurate structures and spectroscopic properties. These transient species are still unknown and the present study provides theoretical predictions of the radical and its anion for the first time. Since these species are strongly suggested to play an important role as intermediates in the interstellar medium, the rotational and vibrational spectroscopic parameters are presented to help aid in the identification and assignment of these spectra. The rotational constants produced will aid in ground-based observation. Both the PCS radical and the PCS- anion are linear. In the PCS- anion, which has a predicted adiabatic electron binding energy (adiabatic electron affinity of PCS) of 65.6 kcal/mol, the P-C bond is stronger than the corresponding neutral radical showing almost triple bond character, while the C-S bond is weaker, showing almost single bond character in the anion. The PCS anion shows a smaller rotational constant than that of the neutral. The ω3 stretching vibrational frequencies of PCS- are red-shifted from the radical, while the ω1 and ω2 vibrations are blue-shifted with ω1 demonstrating the largest blue shift. The ro-vibronic spectrum of the PCS radical has been accurately calculated in variational nuclear motion calculations including both Renner-Teller (RT) and spin-orbit (SO) coupling effects using the composite potential energy near-equilibrium potential energy and coupled cluster dipole moment surfaces. The spectrum is predicted to be very complicated even at low energies due to the presence of a strong Fermi resonance between the bending mode and symmetric stretch, but also due to similar values of the bending frequency, RT, and SO splittings.

  6. Synthesis of silicon carbide thin films with polycarbosilane (PCS)

    Energy Technology Data Exchange (ETDEWEB)

    Colombo, P. [Univ. di Padova (Italy). Dept. di Ingegneria Meccanica-Settore Materiali; Paulson, T.E.; Pantano, C.G. [Pennsylvania State Univ., University Park, PA (United States). Dept. of Materials Science and Engineering

    1997-09-01

    Polycarbosilane (PCS) thin films were deposited on silicon (and other) substrates and heat treated under vacuum ({approximately}10{sup {minus}6} torr) at temperatures in the range of 200--1,200 C. At temperatures in the range of 1,000--1,200 C, the initially amorphous PCS films transformed to polycrystalline {beta}-silicon carbide ({beta}-SiC). Although PCS films could be deposited at thickness up to 2 {micro}m, the films with thicknesses >1 {micro}m could not be transformed to SiC without extensive cracking. The resulting SiC coatings were characterized using Fourier transform infrared spectroscopy, glancing-angle X-ray diffractometry, secondary-ion mass spectroscopy, Raman spectroscopy, transmission electron microscopy, and scanning electron microscopy. The temperature and time dependence of the amorphous-to-crystalline transition could be associated with the evolution of free carbon, oxygen, and hydrogen in the films.

  7. Arsenate tolerance in Silene paradoxa does not rely on phytochelatin-dependent sequestration.

    Science.gov (United States)

    Arnetoli, Miluscia; Vooijs, Riet; ten Bookum, Wilma; Galardi, Francesca; Gonnelli, Cristina; Gabbrielli, Roberto; Schat, Henk; Verkleij, Jos A C

    2008-04-01

    Arsenate tolerance, As accumulation and As-induced phytochelatin accumulation were compared in populations of Silene paradoxa, one from a mine site enriched in As, Cu and Zn, the other from an uncontaminated site. The mine population was significantly more arsenate-tolerant. Arsenate uptake and root-to-shoot transport were slightly but significantly higher in the non-mine plants. The difference in uptake was quantitatively insufficient to explain the difference in tolerance between the populations. As accumulation in the roots was similar in both populations, but the mine plants accumulated much less phytochelatins than the non-mine plants. The mean phytochelatin chain length, however, was higher in the mine population, possibly due to a constitutively lower cellular glutathione level. It is argued that the mine plants must possess an arsenic detoxification mechanism other than arsenate reduction and subsequent phytochelatin-based sequestration. This alternative mechanism might explain at least some part of the superior tolerance in the mine plants.

  8. Study of phytochelatins and other related thiols as complexing biomolecules of As and Cd in wild type and genetically modified Brassica juncea plants.

    Science.gov (United States)

    Navaza, Ana Pereira; Montes-Bayón, Maria; LeDuc, Danika L; Terry, Norman; Sanz-Medel, Alfredo

    2006-03-01

    The accumulation of As and Cd in Brassica juncea plants and the formation of complexes of these elements with bioligands such as glutathione and/or phytochelatins (PCs) is studied. The genetic manipulation of these plants to induce higher As and Cd accumulation has been achieved by overexpressing the genes encoding for gamma-glutamyl cysteine synthetase (gamma-ECS) and glutathione synthetase (GS). These two enzymes are responsible for glutathione (GSH) formation in plants, which is the first step in the production of PCs. The biomass produced in both the wild type and the genetically modified plants, has been evaluated. Additionally, the total Cd and As concentration accumulated in the plant tissues was measured by inductively coupled plasma mass spectrometry (ICP-MS) after extraction. Speciation studies on the extracts were conducted using size exclusion liquid chromatography (SEC) coupled online with ICP-MS to monitor As, Cd and S. For further purification of the As fractions, reversed phase high performance liquid chromatography (RP-HPLC) was used. Structural elucidation of the PCs and other thiols, as well as their complexes with As and Cd, was performed by electrospray-quadrupole-time-of-flight (ESI-Q-TOF). In both the Cd and As exposed plants it was possible to observe the presence of oxidized PC2 ([M + H]+, m/z 538), GS-PC2(-Glu) ([M + H]+, m/z 716) as well as reduced GSH ([M + H]+, m/z 308) and oxidized glutathione (GSSG) ([M + H]+, m/z 613). However, only the GS plants exhibited the presence of As(GS)3 complex ([M + H]+, m/z 994) that was further confirmed by MS/MS. This species is reported for the first time in B. juncea plant tissues.

  9. Metal and phytochelatin content in phytoplankton from freshwater lakes with different metal concentrations

    Energy Technology Data Exchange (ETDEWEB)

    Knauer, K.; Xue, H.B.; Sigg, L. [Swiss Federal Inst. of Environmental Science and Technology EAWAG, Kastanienbaum (Switzerland); Ahner, B. [Cornell Univ., Ithaca, NY (United States). Dept. of Agricultural and Biological Engineering

    1998-12-01

    The trace metal (Cu, Zn, Cd, Mn) and phytochelatin (an intracellular chelator for metal ions) cellular content were determined in phytoplankton samples originating from four lakes. The lakes differ in their metal concentrations and in other conditions (pH, trophic state, organic matter). Total and intracellular contents of Cu and Cd were related to the experimentally determined free metal ion concentration and the total and intracellular content of Mn to the dissolved Mn. The intracellular Zn content was tightly regulated over a broad range of [Zn{sup 2+}]. Phytochelatin concentrations were measurable in phytoplankton communities from three of the lakes, in spite of low levels of free Cu, Zn, and Cd ion concentrations. Culture experiments showed that the concentration of intracellular phytochelatin in Scendesmus subspicatus and in a natural algal community increased upon addition of copper in a similar concentration range as in the lakes. Phytochelatin concentrations were below detection in the phytoplankton collected from the highly contaminated Lake Orta, perhaps suggesting that this algal community has adapted in some other way to high metal concentrations. Although the authors only sampled a few lakes, the lack of any clear relationship between phytochelatin and metal concentrations calls into question the feasibility of using phytochelatins as a bioindicator of metal exposure in lakes.

  10. Eleanor McElwee and the Formation of IEEE PCS

    Science.gov (United States)

    Malone, Edward A.

    2015-01-01

    This article examines the historical professional project that created the Institute of Radio Engineers' Professional Group on Engineering Writing an Speech (IRE PGEWS)--now called the Institute of Electrical and Electronics Engineers' Professional Communication Society (IEEE PCS)--and recounts the group's early history in detail. It also traces…

  11. Eleanor McElwee and the Formation of IEEE PCS

    Science.gov (United States)

    Malone, Edward A.

    2015-01-01

    This article examines the historical professional project that created the Institute of Radio Engineers' Professional Group on Engineering Writing an Speech (IRE PGEWS)--now called the Institute of Electrical and Electronics Engineers' Professional Communication Society (IEEE PCS)--and recounts the group's early history in detail. It also traces…

  12. Pseudouridine synthases.

    Science.gov (United States)

    Hamma, Tomoko; Ferré-D'Amaré, Adrian R

    2006-11-01

    Pseudouridine synthases are the enzymes responsible for the most abundant posttranscriptional modification of cellular RNAs. These enzymes catalyze the site-specific isomerization of uridine residues that are already part of an RNA chain, and appear to employ both sequence and structural information to achieve site specificity. Crystallographic analyses have demonstrated that all pseudouridine synthases share a common core fold and active site structure and that this core is modified by peripheral domains, accessory proteins, and guide RNAs to give rise to remarkable substrate versatility.

  13. Phytochelatins and antioxidant systems respond differentially during arsenite and arsenate stress in Hydrilla verticillata (L.f.) Royle.

    Science.gov (United States)

    Srivastava, S; Mishra, S; Tripathi, R D; Dwivedi, S; Trivedi, P K; Tandon, P K

    2007-04-15

    Serious contamination of aquatic systems by arsenic (As) in different parts of the world calls for the development of an in situ cost-effective phytoremediation technology. In the present investigation, plants of Hydrilla verticillata (L.f.) Royle were exposed to various concentrations of arsenate (As(V)) (0-250 microM) and arsenite (AsIII) (0-25 microM) and analyzed for accumulation responses vis-à-vis biochemical changes. Total As accumulation was found to be higher in plants exposed to AsIII (315 microg g(-1) dw at 25 microM) compared to As(V) (205 microg g(-1) dw at 250 microM) after 7 d of treatment. Plants tolerated low concentrations of As(III) and As(V) by detoxifying the metalloid through augmented synthesis of thiols such as phytochelatins and through increased activity of antioxidant enzymes. While As(V) predominantly stimulated antioxidant enzyme activity, As(III) primarily caused enhanced levels of thiols. The maximum amount of As chelated by PCs was found to be about 39% in plants exposed to As(III) (at 10 microM) and 35% in As(V) exposed plants (at 50 microM) after 4 d. Only the respective highest concentrations of As(III) (25 microM) and As(V) (250 microM) proved toxic for normal plant growth after prolonged treatment. Thus, H. verticillata forms a promising candidate for the phytoremediation of As contaminated water.

  14. CELL-SURFACE DISPLAY OF SYNTHETIC PHYTOCHELATINS USING ICE NUCLEATION PROTEIN FOR ENHANCED HEAVY-METAL BIOACCUMULATION. (R827227)

    Science.gov (United States)

    Synthetic phytochelatins (ECs) composed of (Glu–Cys)nGly are protein analogs of phytochelatin that exhibit improved metal-binding capacity over metallothioneins (MTs). Expression of EC20 on the surface of E. coli using the Lpp-OmpA anchor resulted in i...

  15. Arsenate tolerance in Silene paradoxa does not rely on phytochelatin-dependent sequestration

    Energy Technology Data Exchange (ETDEWEB)

    Arnetoli, Miluscia [Section of Plant Ecology and Physiology, Department of Plant Biology, University of Florence, via Micheli 1, 50121 Firenze (Italy)], E-mail: miluscia@gmail.com; Vooijs, Riet; Bookum, Wilma ten [Institute of Molecular and Cellular Biology, Faculty of Earth and Life Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam (Netherlands); Galardi, Francesca; Gonnelli, Cristina; Gabbrielli, Roberto [Section of Plant Ecology and Physiology, Department of Plant Biology, University of Florence, via Micheli 1, 50121 Firenze (Italy); Schat, Henk; Verkleij, Jos A.C. [Institute of Molecular and Cellular Biology, Faculty of Earth and Life Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam (Netherlands)

    2008-04-15

    Arsenate tolerance, As accumulation and As-induced phytochelatin accumulation were compared in populations of Silene paradoxa, one from a mine site enriched in As, Cu and Zn, the other from an uncontaminated site. The mine population was significantly more arsenate-tolerant. Arsenate uptake and root-to-shoot transport were slightly but significantly higher in the non-mine plants. The difference in uptake was quantitatively insufficient to explain the difference in tolerance between the populations. As accumulation in the roots was similar in both populations, but the mine plants accumulated much less phytochelatins than the non-mine plants. The mean phytochelatin chain length, however, was higher in the mine population, possibly due to a constitutively lower cellular glutathione level. It is argued that the mine plants must possess an arsenic detoxification mechanism other than arsenate reduction and subsequent phytochelatin-based sequestration. This alternative mechanism might explain at least some part of the superior tolerance in the mine plants. - Neither decreased uptake nor phytochelatins seem to play a role in the As tolerance in Silene paradoxa.

  16. Phytochelatins in the diatom Phaeodactylum tricornutum Bohlin: an evaluation of their use as biomarkers of metal exposure in marine waters.

    Science.gov (United States)

    Morelli, Elisabetta; Fantozzi, Laura

    2008-09-01

    The feasibility of a bioassay based on the synthesis of phytochelatins to assess metal pollution in aquatic environments was evaluated by using the marine diatom Phaeodactylum tricornutum. Short-term incubation experiments carried out in EDTA-buffered artificial seawater showed increasing cellular phytochelatin concentration with increasing free cadmium, lead or copper in the medium, indicating that phytochelatins behave as a biomarker of exposure to the bioavailable metal fraction. A linear dose-response relationship between metal exposure and phytochelatin synthesis was found in natural seawater samples enriched with known amounts of heavy metals. Phytochelatin induction tests carried out on polluted seawater samples showed an enhanced response compared to that obtained in unpolluted seawater. This finding was found to be consistent mainly with a copper contamination.

  17. PCS: The First Fluid Physics Payload on ISS

    Science.gov (United States)

    Doherty, M.; Sankaran, S.

    2002-01-01

    The Physics of Colloids in Space (PCS) experiment was accommodated within International Space Station (ISS) EXpedite the PRocessing of Experiments to Space Station (EXPRESS) Rack 2 and was remotely operated from early June 2001 until February 2002 from NASA Glenn Research Center's Telescience Support Center in Cleveland, Ohio and from a remote site at Harvard University in Cambridge, Massachusetts. PCS is an experiment conceived by Professor David A. Weitz of Harvard University (the Principal Investigator), focusing on the behavior of three different classes of colloid mixtures. The sophisticated light scattering instrumentation comprising PCS is capable of color imaging, and dynamic and static light scattering from 11 to 169 degrees, Bragg scattering over the range from 10 to 60 degrees, and laser light scattering at low angles from 0.3 to 6.0 degrees. The PCS instrumentation performed remarkably well, demonstrating a flexibility that enabled experiments to be performed that had not been envisioned prior to launch. While on-orbit, PCS accomplished 2400 hours of science operations, and was declared a resounding success. Each of the eight sample cells worked well and produced interesting and important results. Crystal nucleation and growth and the resulting structures of two binary colloidal crystal alloys were studied, with the long duration microgravity environment of the ISS facilitating extended studies on the growth and coarsening characteristics of the crystals. In another experiment run, the de-mixing of the colloid-polymer critical-point sample was studied as it phase-separates into two phases, one that resembles a gas and one that resembles a liquid. This process was studied over four decades of length scale, from 1 micron to 1 centimeter, behavior that cannot be observed in this sample on Earth because sedimentation would cause the colloids to fall to the bottom of the cell faster than the de-mixing process could occur. Similarly, the study of gelation and

  18. HPLC法测定镉、砷胁迫下水稻根系的植物螯合肽%HPLC assay of phytochelatins in rice root under cadmium or arsenic stress

    Institute of Scientific and Technical Information of China (English)

    张静; 常青晓; 孙传范; 段桂兰; 赵全志

    2011-01-01

    Phytochelatins (PCs) and other thiol-containing compounds in rice roots exposed to cadmium (Cd) and arsenic (As) were analyzed by RP-HPLC separation and fluorescence detection with monobromobimane pre-column derivatization. The results showed that GSH and PCs in the mixed standard solutions and rice roots can be separated by using CAN-O. 1% TFA binary gradient elution. PC2, PC3 and PC4 were found in the rice roots that were exposed to Cd, and the concentrations of PC3 were the highest. In response to As stress, PCs in rice roots were complicated. In addition to PC2 and PC3, there are other forms of PCn that need further identification.%利用monobromobimane衍生试剂,通过反相高效液相色谱-荧光检测体系对镉、砷胁迫下水稻根系内的植物螯合肽(PCs)等巯基化合物进行形态和含量的分析测定.结果表明,采用乙腈和0.1%三氟乙酸组成的两元梯度流动相,可以将标准样品中的谷胱甘肽(GSH)和PCs等巯基化合物很好地分离.利用上述方法,能较好地分离Cd、As处理下溶液培养的水稻幼苗根系内的PCs形态.Cd处理下,根系内合成PC2、PC3、PC4,其中以PC3的含量最大.As处理下,PCs的形态除PC2和PC3外,其它PCn的形态有待进一步定性.

  19. Benzalacetone Synthase

    Directory of Open Access Journals (Sweden)

    Ikuro eAbe

    2012-03-01

    Full Text Available Benzalacetone synthase, from the medicinal plant Rheum palmatum (Polygonaceae (RpBAS, is a plant-specific chalcone synthase (CHS superfamily of type III polyketide synthase (PKS. RpBAS catalyzes the one-step, decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce the C6-C4 benzalacetone scaffold. The X-ray crystal structures of RpBAS confirmed that the diketide-forming activity is attributable to the characteristic substitution of the conserved active-site "gatekeeper" Phe with Leu. Furthermore, the crystal structures suggested that RpBAS employs novel catalytic machinery for the thioester bond cleavage of the enzyme-bound diketide intermediate and the final decarboxylation reaction to produce benzalacetone. Finally, by exploiting the remarkable substrate tolerance and catalytic versatility of RpBAS, precursor-directed biosynthesis efficiently generated chemically and structurally divergent, unnatural novel polyketide scaffolds. These findings provided a structural basis for the functional diversity of the type III PKS enzymes.

  20. Characterization of Hg(II) binding with different length phytochelatins using liquid chromatography and amperometric detection.

    Science.gov (United States)

    Dago, Angela; González-García, Olga; Ariño, Cristina; Díaz-Cruz, José Manuel; Esteban, Miquel

    2011-06-10

    A simple and rapid methodology is optimised to analyse mixtures of different phytochelatins (PC(n), n=2-5) with Hg(II) by HPLC with amperometric detection as a first step towards the analysis of extracts of plants stressed with Hg(II). The separation was achieved in a C(18) column with a mobile phase of 0.1% trifluoroacetic acid (TFA) in water and 0.1% TFA in acetonitrile using gradient elution. Electrochemical detection with glassy carbon electrode and UV-vis detection were used in series. This methodology can clearly distinguish between the free peptides and their complexes and permits to study the evolution of the different complexes formed and predicts the possible interactions between the long chain phytochelatin complexes. ESI-MS is used as a complementary technique to find out the stoichiometries of such long chain phytochelatin complexes.

  1. Mass spectrometric detection, identification, and fragmentation of arseno-phytochelatins.

    Science.gov (United States)

    Schmied-Tobies, Maria I H; Arroyo-Abad, Uriel; Mattusch, Jürgen; Reemtsma, Thorsten

    2014-11-01

    Phytochelatins (PC) are cystein-rich oligopeptides in plants for coordination with toxic metals and metalloids via their thiol groups. The composition, structure, and mass spectrometric fragmentation of arseno-PC (As-PC) with PC of different degree of oligomerization (PC2-PC5) in solution were studied using liquid chromatography coupled in parallel to inductively coupled plasma mass spectrometry and electrospray ionization quadrupole time-of-flight mass spectrometry. As-PC were detected from As(PC2) to As(PC5) with an increasing number of isomers that differ in the position of thiol groups bound to As. Thermodynamic modeling supported the identification process in case of these isomers. Mass spectrometric fragmentation of the As-PC does not follow the established pattern of peptides but is governed by the formation of series of As-containing annular cations, which coordinate to As via S, N, or O. Structure proposals for 30 As-PC fragment ions in the range m/z 147.92 to m/z 1290.18 are elaborated. Many of these fragment ions are characteristic to several As-PC and may be suited for a screening for As-PC in plant extracts. The mass spectrometric data offer the perspective for a future more sensitive determination of As-PC by means of liquid chromatography tandem mass spectrometry with multiple reaction monitoring.

  2. PCS a code system for generating production cross section libraries

    Energy Technology Data Exchange (ETDEWEB)

    Cox, L.J.

    1997-04-01

    This document outlines the use of the PCS Code System. It summarizes the execution process for generating FORMAT2000 production cross section files from FORMAT2000 reaction cross section files. It also describes the process of assembling the ASCII versions of the high energy production files made from ENDL and Mark Chadwick`s calculations. Descriptions of the function of each code along with its input and output and use are given. {ital This document is under construction. Please submit entries, suggestions, questions, and corrections to} {bold (ljc@llnl.gov)} 3 tabs.

  3. Removal or transport of office furniture and PCs

    CERN Multimedia

    TS Department

    2008-01-01

    The TS/FM Group wishes to remind users that requests for the removal or transport of office furniture must be made using the EDH ‘Transport request’ form. Please select the "Removals" category from the drop-down menu to indicate the nature of the request. Requests for the transport of PCs from your office to Bldg. 513 or back must also be made using the EDH ‘Transport request’ form. In this case select the "PC transport" category from the drop-down menu. TS/FM Group Tel. 160239

  4. SEC ICP MS and CZE ICP MS investigation of medium and high molecular weight complexes formed by cadmium ions with phytochelatins.

    Science.gov (United States)

    Miszczak, Agata; Rosłon, Magdalena; Zbroja, Grzegorz; Brama, Katarzyna; Szalacha, Elżbieta; Gawrońska, Helena; Pawlak, Katarzyna

    2013-05-01

    Size-exclusion chromatography (SEC) and capillary zone electrophoresis (CZE) coupled with inductively coupled plasma mass spectrometry were applied to characterize low, medium, and high molecular weight cadmium complexes with glutathione and phytochelatins (PCs). The dominant stoichiometry of the complexes formed in vitro was established as 1:1 using electrospray ionization mass spectrometry. Calculated molecular masses of Cd1L1 complexes were used for calibration of the SEC and CZE methods. The results showed a lower (2 kDa) SEC column exclusion limit for cadmium complexes compared with free peptides (10 kDa), and most of the high molecular weight cadmium species were eluted in the void volume of the column. Moreover, the CZE method based on the semiempirical model of Offord to elucidate peptide migration allowed us to show a high propensity of Cd-PC complexes for polymorphism on complexation, which was also observed for extracts of Arabidopsis thaliana treated with cadmium. All the information presented is vital for understanding the mechanism of metal deactivation in plants.

  5. CRISPR-PCS: a powerful new approach to inducing multiple chromosome splitting in Saccharomyces cerevisiae

    Science.gov (United States)

    Sasano, Yu; Nagasawa, Koki; Kaboli, Saeed; Sugiyama, Minetaka; Harashima, Satoshi

    2016-01-01

    PCR-mediated chromosome splitting (PCS) was developed in the yeast Saccharomyces cerevisiae. It is based on homologous recombination and enables division of a chromosome at any point to form two derived and functional chromosomes. However, because of low homologous recombination activity, PCS is limited to a single site at a time, which makes the splitting of multiple loci laborious and time-consuming. Here we have developed a highly efficient and versatile chromosome engineering technology named CRISPR-PCS that integrates PCS with the novel genome editing CRISPR/Cas9 system. This integration allows PCS to utilize induced double strand breaks to activate homologous recombination. CRISPR-PCS enhances the efficiency of chromosome splitting approximately 200-fold and enables generation of simultaneous multiple chromosome splits. We propose that CRISPR-PCS will be a powerful tool for breeding novel yeast strains with desirable traits for specific industrial applications and for investigating genome function. PMID:27530680

  6. Effects of metal combinations on the production of phytochelatins and glutathione by the marine diatom Phaeodactylum tricornutum.

    Science.gov (United States)

    Kawakami, Silvia K; Gledhill, Martha; Achterberg, Eric P

    2006-02-01

    Copper, Cd and Zn can be found at elevated concentrations in contaminated estuarine and coastal waters and have potential toxic effects on phytoplankton species. In this study, the effects of these metals on the intracellular production of the polypeptides phytochelatin and glutathione by the marine diatom Phaeodactylum tricornutum were examined in laboratory cultures. Single additions of Cu and Cd (0.4 microM Cu2 and 0.45 microM Cd2+) to the culture medium induced the production of short-chained phytochelatins ((gamma-Glu-Cys)n-Gly where n = 2-5), whereas a single addition of Zn (2.2 microM Zn2+) did not stimulate phytochelatin production. Combination of Zn with Cu resulted in a similar phytochelatin production compared with a single Cu addition. The simultaneous exposure to Zn and Cd led to an antagonistic effect on phytochelatin production, which was probably caused by metal competition for cellular binding sites. Glutathione concentrations were affected only upon exposure to Cd (85% increase) or the combination of Cd with Zn (65% decrease), relative to the control experiment. Ratios of phytochelatins to glutathione indicated a pronounced metal stress in response to exposures to Cu or Cd combined with Zn. This study indicates that variabilities in phytochelatin and glutathione production in the field can be explained in part by metal competition for cellular binding sites.

  7. User guide to power management for PCs and monitors

    Energy Technology Data Exchange (ETDEWEB)

    Nordman, B.; Piette, M.A.; Kinney, K.; Webber, C. [Lawrence Berkeley National Lab., CA (United States). Environmental Energy Technologies Div.

    1997-01-01

    Power management of personal computers (PCs) and monitors has the potential to save significant amounts of electricity as well as deliver other economic and environmental benefits. The Environmental Protection Agency`s ENERGY STAR{reg_sign} program has transformed the PC market so that equipment capable of power management is now widely available. However, previous studies have found that many Energy Star compliant computer systems are not accomplishing energy savings. The principal reasons for this are systems not being enabled for power management or a circumstance that prevents power management from operating. This guide is intended to provide information to computer support workers to increase the portion of systems that successfully power manage. The guide introduces power management concepts and the variety of benefits that power management can bring. It then explains how the parts of a computer system work together to enter and leave power management states. Several common computer system types are addressed, as well as the complications that networks bring to power management. Detailed instructions for checking and configuring several system types are provided, along with trouble shooting advice. The guide concludes with a discussion of how to purchase Energy Star compliant systems and future directions for power management of PCs and related equipment.

  8. User guide to power management for PCs and monitors

    Energy Technology Data Exchange (ETDEWEB)

    Nordman, B.; Piette, M.A.; Kinney, K.; Webber, C. [Lawrence Berkeley National Lab., CA (United States). Environmental Energy Technologies Div.

    1997-01-01

    Power management of personal computers (PCs) and monitors has the potential to save significant amounts of electricity as well as deliver other economic and environmental benefits. The Environmental Protection Agency`s ENERGY STAR{reg_sign} program has transformed the PC market so that equipment capable of power management is now widely available. However, previous studies have found that many Energy Star compliant computer systems are not accomplishing energy savings. The principal reasons for this are systems not being enabled for power management or a circumstance that prevents power management from operating. This guide is intended to provide information to computer support workers to increase the portion of systems that successfully power manage. The guide introduces power management concepts and the variety of benefits that power management can bring. It then explains how the parts of a computer system work together to enter and leave power management states. Several common computer system types are addressed, as well as the complications that networks bring to power management. Detailed instructions for checking and configuring several system types are provided, along with trouble shooting advice. The guide concludes with a discussion of how to purchase Energy Star compliant systems and future directions for power management of PCs and related equipment.

  9. PCs all-in-one desk reference for dummies

    CERN Document Server

    Chambers, Mark L

    2013-01-01

    Need the scoop on Windows Vista? How about Office 2007? Anything you need to know about using your PC can probably be found in PCs All-In-One Desk Reference for Dummies, 4th Edition. This handy guide is made up of eight convenient minibooks, so you can find what you need in a hurry. And in case you think you've seen it before, this fourth edition is fully updated for all the newest, coolest stuff. Want a guided tour of the Vista operating system and how it differs from XP? Check out minibook #2. Have you heard about Microsoft Works but aren't sure what it's about? Find out

  10. Pricing the property claim service (PCS) catastrophe insurance options using gamma distribution

    Science.gov (United States)

    Noviyanti, Lienda; Soleh, Achmad Zanbar; Setyanto, Gatot R.

    2017-03-01

    The catastrophic events like earthquakes, hurricanes or flooding are characteristics for some areas, a properly calculated annual premium would be closely as high as the loss insured. From an actuarial perspective, such events constitute the risk that are not insurable. On the other hand people living in such areas need protection. In order to securitize the catastrophe risk, futures or options based on a loss index could be considered. Chicago Board of Trade launched a new class of catastrophe insurance options based on new indices provided by Property Claim Services (PCS). The PCS-option is based on the Property Claim Service Index (PCS-Index). The index are used to determine and payout in writing index-based insurance derivatives. The objective of this paper is to price PCS Catastrophe Insurance Option based on PCS Catastrophe index. Gamma Distribution is used to estimate PCS Catastrophe index distribution.

  11. Using Tablet PCs in Social Work Practice Education

    Directory of Open Access Journals (Sweden)

    Diane M. Hodge

    2008-06-01

    Full Text Available Within social work practice courses, video recording has been used to record and evaluate the clinical practice skills of students. This process has been limited by labor-intensive, tapebased video equipment, non-digital means of organizing and assessing specific scenes and events within the video, and paper evaluation forms. As an interdisciplinary project, professors from professional disciplines (education, social work, and counseling worked with information technology students from computer science to design and develop Table PC-based One- Note EVAs (Extended Video Application that would provide a more effective way of evaluating clinical practice skills for professional program students. This case study presents how one interdisciplinary team was able to create an EVA for use with digital recordings of clinical practice skills so that these demonstrations could be recorded, organized, and evaluated more effectively. The issues of working through communication differences, design difficulties, and the additional steps toward implementation are explored. The lessons learned from working as an interdisciplinary team and the impact of Tablet PCs in social work practice courses is also presented.

  12. Tablet PCs in Engineering Mathematics Courses at the J.B. Speed School of Engineering

    Science.gov (United States)

    Hieb, Jeffrey L.; Ralston, Patricia A. S.

    2010-01-01

    In fall 2007, J.B. Speed School of Engineering at the University of Louisville joined the ranks of universities requiring the purchase of Tablet PCs for all new entering students. This article presents a description of how the Department of Engineering Fundamentals incorporated Tablet PCs into their instruction, a review of the literature…

  13. Biomonitoring for metal contamination near two Superfund sites in Woburn, Massachusetts, using phytochelatins

    Energy Technology Data Exchange (ETDEWEB)

    Gawel, James E.; Hemond, Harold F

    2004-09-01

    Characterizing the spatial extent of groundwater metal contamination traditionally requires installing sampling wells, an expensive and time-consuming process in urban areas. Moreover, extrapolating biotic effects from metal concentrations alone is problematic, making ecological risk assessment difficult. Our study is the first to examine the use of phytochelatin measurements in tree leaves for delimiting biological metal stress in shallow, metal-contaminated groundwater systems. Three tree species (Rhamnus frangula, Acer platanoides, and Betula populifolia) growing above the shallow groundwater aquifer of the Aberjona River watershed in Woburn, Massachusetts, display a pattern of phytochelatin production consistent with known sources of metal contamination and groundwater flow direction near the Industri-Plex Superfund site. Results also suggest the existence of a second area of contaminated groundwater and elevated metal stress near the Wells G and H Superfund site downstream, in agreement with a recent EPA ecological risk assessment. Possible contamination pathways at this site are discussed.

  14. Effects of cadmium exposure on phytochelatin and the synthesis of abscisic acid in funalia trogii

    Energy Technology Data Exchange (ETDEWEB)

    Yuerekli, F.; Porgali, Z.B. [Inonu University, Arts and Science Faculty, Department of Biology, 44069 Malatya (Turkey); Uenyayar, A.; Mazmanci, M.A. [Mersin University, Engineering Faculty, Engineering Faculty, 33000 Mersin (Turkey)

    2004-08-01

    Heavy metal toxicity poses major environmental and health problems as heavy metals are more difficult to remediate than chemical contaminants, which can be degraded by microorganisms. Phytochelatins are formed in plants and some fungi upon exposure to a range of different heavy metals. Fungi show sensitivity to many environmental factors, including nutrient limitation, changes in carbon sources and heavy metal exposure. (Abstract Copyright [2004], Wiley Periodicals, Inc.)

  15. Quantification of phytochelatins and their metal(loid) complexes: critical assessment of current analytical methodology.

    Science.gov (United States)

    Wood, B Alan; Feldmann, Jörg

    2012-04-01

    Whilst there are a variety of methods available for the quantification of biothiols in sample extracts, each has their own inherent advantages and limitations. The ease with which thiols readily oxidise not only hinders their quantification but also alters the speciation profile. The challenge faced by the analyst is not only to preserve the speciation of the sample, but also to select a method which allows the retrieval of the desired information. Given that sulfur is not a chromophore and that it cannot easily be monitored by ICP-MS, a number of direct and indirect methods have been developed for this purpose. In order to assess these methods, they are compared in the context of the measurement of arsenic-phytochelatin complexes in plant extracts. The inherent instability of such complexes, along with the instabilities of reduced glutathione and phytochelatin species,necessitates a rapid and sensitive analytical protocol. Whilst being a specific example, the points raised and discussed in this review will also be applicable to the quantification of biothiols and thiol-metal(loid) species in a wide range of systems other than just the analysis of arsenic-phytochelatin species in plant extracts.

  16. Enhanced bioaccumulation of heavy metals by bacterial cells displaying synthetic phytochelatins.

    Science.gov (United States)

    Bae, W; Chen, W; Mulchandani, A; Mehra, R K

    2000-12-05

    A novel strategy using synthetic phytochelatins is described for the purpose of developing microbial agents for enhanced bioaccumulation of toxic metals. Synthetic genes encoding for several metal-chelating phytochelatin analogs (Glu-Cys)(n)Gly (EC8 (n = 8), EC11 (n = 11), and EC20 (n = 20)) were synthesized, linked to a lpp-ompA fusion gene, and displayed on the surface of E. coli. For comparison, EC20 was also expressed periplasmically as a fusion with the maltose-binding protein (MBP-EC20). Purified MBP-EC20 was shown to accumulate more Cd(2+) per peptide than typical mammalian metallothioneins with a stoichiometry of 10 Cd(2+)/peptide. Cells displaying synthetic phytochelatins exhibited chain-length dependent increase in metal accumulation. For example, 18 nmoles of Cd(2+)/mg dry cells were accumulated by cells displaying EC8, whereas cells exhibiting EC20 accumulated a maximum of 60 nmoles of Cd(2+)/mg dry cells. Moreover, cells with surface-expressed EC20 accumulated twice the amount of Cd(2+) as cells expressing EC20 periplasmically. The ability to genetically engineer ECs with precisely defined chain length could provide an attractive strategy for developing high-affinity bioadsorbents suitable for heavy metal removal. Copyright 2000 John Wiley & Sons, Inc.

  17. Cadmium-inducible expression of the ABC-type transporter AtABCC3 increases phytochelatin-mediated cadmium tolerance in Arabidopsis.

    Science.gov (United States)

    Brunetti, Patrizia; Zanella, Letizia; De Paolis, Angelo; Di Litta, Davide; Cecchetti, Valentina; Falasca, Giuseppina; Barbieri, Maurizio; Altamura, Maria Maddalena; Costantino, Paolo; Cardarelli, Maura

    2015-07-01

    The heavy metal cadmium (Cd) is a widespread environmental contaminant with harmful effects on living cells. In plants, phytochelatin (PC)-dependent Cd detoxification requires that PC-Cd complexes are transported into vacuoles. Here, it is shown that Arabidopsis thaliana seedlings defective in the ABCC transporter AtABCC3 (abcc3) have an increased sensitivity to different Cd concentrations, and that seedlings overexpressing AtABCC3 (AtABCC3ox) have an increased Cd tolerance. The cellular distribution of Cd was analysed in protoplasts from abcc3 mutants and AtABCC3 overexpressors grown in the presence of Cd, by means of the Cd-specific fluorochromes 5-nitrobenzothiazole coumarin (BTC-5N) and Leadmium™ Green AM dye. This analysis revealed that Cd is mostly localized in the cytosol of abcc3 mutant protoplasts whereas there is an increase in vacuolar Cd in protoplasts from AtABCC3ox plants. Overexpression of AtABCC3 in cad1-3 mutant seedlings defective in PC production and in plants treated with l-buthionine sulphoximine (BSO), an inhibitor of PC biosynthesis, had no effect on Cd tolerance, suggesting that AtABCC3 acts via PCs. In addition, overexpression of AtABCC3 in atabcc1 atabcc2 mutant seedlings defective in the Cd transporters AtABCC1 and AtABCC2 complements the Cd sensitivity of double mutants, but not in the presence of BSO. Accordingly, the level of AtABCC3 transcript in wild type seedlings was lower than that of AtABCC1 and AtABCC2 in the absence of Cd but higher after Cd exposure, and even higher in atabcc1 atabcc2 mutants. The results point to AtABCC3 as a transporter of PC-Cd complexes, and suggest that its activity is regulated by Cd and is co-ordinated with the activity of AtABCC1/AtABCC2.

  18. Changes in the levels of phytochelatins and related metal-binding peptides in chickpea seedlings exposed to arsenic and different heavy metal ions.

    Science.gov (United States)

    Gupta, Dharmendra K; Tohoyama, Hiroshi; Joho, Masanori; Inouhe, Masahiro

    2004-06-01

    Phytochelatin-related peptides were analyzed in chickpea plants exposed to six different heavy-metal ions. Cadmium and arsenic stimulated phytochelatin and homophytochelatin synthesis in roots but other metals did not. These metals, however, caused an overall increase in the precursors, glutathione, homoglutathione and cysteine. These changes may be different biochemical indexes for heavy-metal contamination.

  19. Identification and quantification of glutathione and phytochelatins from Chlorella vulgaris by RP-HPLC ESI-MS/MS and oxygen-free extraction.

    Science.gov (United States)

    Simmons, Denina B D; Hayward, Allison R; Hutchinson, Thomas C; Emery, R J Neil

    2009-10-01

    Phytochelatins are short, cysteine-containing, detoxification peptides produced by plants, algae, and fungi in response to heavy metal exposure. These peptides auto-oxidize easily. Current extraction protocols do not adequately address losses of phytochelatins because of their oxidation and the use of indirect methods for quantification. Method enhancements include the use of an argon environment during extraction to reduce auto-oxidation, the use of glycine-(13)C2-labeled glutathione as an internal standard, and an electrospray ionization source with a triple quadrupole mass spectrometer as a detector. The method-detection limits were 0.081 microM for glutathione, 0.440 microM for phytochelatin 2, and 0.120 microM for phytochelatin 3. These detection limits were comparable to similar studies and were not compromised incorporating these adjustments. The use of a labeled internal standard and an inert gaseous environment during sample preparation greatly improved calibration linearity and sensitivity. Furthermore, phytochelatin degradation was significantly reduced and more accurately tracked. Previous studies involving phytochelatin analyses have likely been subject to higher variability caused by this propensity for phytochelatins to degrade rapidly in air. The method adjustments were simple and cost-effective and allowed phytochelatin analyses to be performed for hours at a time with minimal auto-oxidation.

  20. Environmental cadmium levels increase phytochelatin and glutathione in lettuce grown in a chelator-buffered nutrient solution.

    Science.gov (United States)

    Maier, Elizabeth A; Matthews, Rosalyn D; McDowell, Jennifer A; Walden, Rebecca R; Ahner, Beth A

    2003-01-01

    Phytochelatins are enzymatically synthesized peptides involved in metal detoxification and have been measured in plants grown at very high Cd concentrations, but few studies have examined the response of plants at lower environmentally relevant Cd concentrations. Using an ethylenediaminetetraacetic acid (EDTA)-buffered nutrient medium, we have varied Cd exposure and measured phytochelatin and glutathione concentrations in romaine lettuce (Lactuca sativa L. var. longifolia Lam. var. Parris Island) grown in a flow-through hydroponic (FTH) system. Very low free ionic Cd (10(-9.6) M) increased average phytochelatin concentrations above those of controls, and increasing Cd resulted in increased phytochelatin production, though increases were tissue dependent. Glutathione concentrations also increased with increasing Cd. In other standard hydroponic experiments, the media were manipulated to vary total Cd concentration while the ionic Cd was fixed. We found that the total amount of Cd (primarily EDTA bound) in the medium altered thiol production in roots, whereas thiols in leaves remained constant. The Cd uptake into roots and translocation to old leaves was also influenced by the total concentration in the medium. Cadmium in all tissues was lower and in some tissues thiol concentrations were higher than in FTH-grown plants grown in identical medium, suggesting that nutrient delivery technique is also an important variable. Though phytochelatin and glutathione production can be sensitive to changes in bioavailable Cd, thiol concentrations will not necessarily reflect the Cd content of the plant tissues.

  1. Response of phytochelatins and their relationship with cadmium toxicity in a floating macrophyte Pistia stratiotes L. at environmentally relevant concentrations.

    Science.gov (United States)

    Wang, C; Wang, L Y; Sun, Q

    2010-02-01

    An indoor experiment was undertaken to investigate the response of phytochelatins and their relationship to cadmium toxicity in Pistia stratiotes L., a free-floating macrophyte, exposed to low concentrations of cadmium typically found in realistic environments. Cadmium concentrations of 0.01 to 0.08 microM had no toxic effects on the growth of this plant, as indicated by no significant changes in the fresh weights of leaves and roots and the slight induction of phytochelatins in plant tissues, whereas cadmium concentrations of 0.16 to 1 microM were toxic, and cadmium toxicity increased with the increase of cadmium concentrations in solutions, accompanied by the dramatic production of phytochelatins in plant tissues, especially in roots. There was a positive correction between root phytochelatin levels and cadmium toxicity, as measured by the growth inhibition rate of the root fresh weight. The results suggested that phytochelatins in aquatic macrophytes can serve as sensitive biomarkers for heavy metal toxicity in a moderately polluted water environment.

  2. Physiological characterization of cadmium-exposed Chlamydomonas reinhardtii.

    Science.gov (United States)

    Bräutigam, Anja; Schaumlöffel, Dirk; Preud'homme, Hugues; Thondorf, Iris; Wesenberg, Dirk

    2011-12-01

    Chlamydomonas reinhardtii is a common model organism for investigation of metal stress. This green alga produces phytochelatins in the presence of metal ions. The influence of cadmium is of main interest, because it is a strong activator of phytochelatin synthase. Cell wall bound and intracellular cadmium content was determined after exposition to 70 µm CdCl(2), showing the main portion of the metal outside the cell. Nevertheless, imported cadmium was sufficient to cause significant changes in thiolpeptide metabolism and its transcriptional regulation. Modern analytical approaches enable new insights into phytochelatin (PC) distribution. A new rapid and precise UPLC-MS method allowed high-throughput PC quantification in algal samples after 1, 4, 24 and 48 h cadmium stress. Initially, canonic PCs were synthesized in C. reinhardtii during cadmium exposition, but afterwards CysPCs became the major thiolpeptides. Thus, after 48 h the concentration of the PC-isoforms CysPC(2-3) and CysGSH attained between 105 and 199 nmol g(-1) fresh weight (FW), whereas the PC(2-3) concentrations were only 15 nmol g(-1) FW. The relative quantification of γ-glutamyl transpeptidase (γ-GT) mRNA suggests the generation of CysPCs by glutamate cleavage from canonic PCs by γ-GT. Furthermore, a homology model of C. reinhardtii phytochelatin synthase was constructed to verify the use of crystal structures from Anabaena sp. phytochelatin synthase (PCS) for docking studies with canonical PCs and CysPCs. From the difference in energy scores, we hypothesize that CysPC may prevent the synthesis of canonical PCs by blocking the binding pocket. Finally, possible physiological reasons for the high abundance of CysPC compared with their canonic precursors are discussed.

  3. Characterization of PCS1055, a novel muscarinic M4 receptor antagonist.

    Science.gov (United States)

    Croy, Carrie H; Chan, Wai Y; Castetter, Andrea M; Watt, Marla L; Quets, Anne T; Felder, Christian C

    2016-07-05

    Identification of synthetic ligands selective for muscarinic receptor subtypes has been challenging due to the high sequence identity and structural homology among the five muscarinic acetylcholine receptors. Here, we report the pharmacological characterization of PCS1055, a novel muscarinic M4 receptor antagonist. PCS1055 inhibited radioligand [(3)H]-NMS binding to the M4 receptor with a Ki=6.5nM. Though the potency of PCS1055 is lower than that of pan-muscarinic antagonist atropine, it has better subtype selectivity over previously reported M4-selective reagents such as the muscarinic-peptide toxins (Karlsson et al., 1994; Santiago and Potter, 2001a) at the M1 subtype, and benzoxazine ligand PD102807 at the M3-subtype (Bohme et al., 2002). A detailed head-to-head comparison study using [(3)H]-NMS competitive binding assays characterizes the selectivity profiles of PCS1055 to that of other potent muscarinic-antagonist compounds PD102807, tropicamide, AF-DX-384, pirenzapine, and atropine. In addition to binding studies, the subtype specificity of PCS1055 is also demonstrated by functional receptor activation as readout by GTP-γ-[(35)S] binding. These GTP-γ-[(35)S] binding studies showed that PCS1055 exhibited 255-, 69.1-, 342- and >1000-fold greater inhibition of Oxo-M activity at the M4 versus the M1-, M2(-), M3-or M5 receptor subtypes, respectively. Schild analyses indicates that PCS1055 acts as a competitive antagonist to muscarinic M4 receptor, and confirms the affinity of the ligand to be low nanomolar, Kb=5.72nM. Therefore, PCS1055 represents a new M4-preferring antagonist that may be useful in elucidating the roles of M4 receptor signaling.

  4. Learner-centred mathematics and statistics education using netbook tablet PCs

    Science.gov (United States)

    Loch, Birgit; Galligan, Linda; Hobohm, Carola; McDonald, Christine

    2011-10-01

    Tablet technology has been shown to support learner-centred mathematics education when this technology is available to both the lecturer and the students. However, cost is often the barrier to students' use of tablet PCs for their university studies. This article argues that more affordable netbook PCs with tablet capabilities can be viable alternatives to full-sized tablet PCs to enhance active and collaborative learning in mathematics and statistics. For a whole teaching semester, netbook tablet PCs were given to volunteer students from two different cohorts. Students were enrolled in nursing mathematics or introductory statistics in non-mathematics majors at an Australian university. The aims were to gauge the suitability of this technology and to identify what active and collaborative learning emerged in these first-year classes. While the netbook tablet PCs were actively promoted in their tutorials, of additional interest was students' use of the technology for any aspect of their studies both inside and outside the classroom. The outcome of this study was to inform a university decision to provide inexpensive tablet technology to larger cohorts of students. The results highlight different approaches required in the mathematics and statistics classes to achieve collaborative and active learning facilitated through the technology. Environmental variables such as the tutor, student, learning space, availability of other technologies and subject content had an impact on the nature of learning. While learner-centred education can be facilitated by inexpensive netbook tablet PCs, we caution that the savings may come at the expense of computing power.

  5. Synthetic effects of heavy metal ions on the phytochelatin induction in rice callus; Ine no phytochelatin yudo ni oyobosu jukinzoku ion no kyozon koka

    Energy Technology Data Exchange (ETDEWEB)

    Takatera, K.; Ono, Y.; Watanabe, T. [The University of Tokyo, Tokyo (Japan). Institute of Industrial Science

    1998-03-01

    This paper describes the result of investigating synergistic effects of heavy metal ions on phytochelatin (PC) in rice callus. The following matters were made clear: with regard to rice callus cultured in culture media added with Cd, Cu, Zn and Hg independently, measurements were carried out on heavy metals contained in SH group of PC and in PC by using a method that combines high pressure liquid chromatography (HPLC) with induction bonded plasma mass analysis; PC induction can be recognized in those cultured under presence of Cd and Cu, but PC incorporating heavy metals is not induced in those cultured under presence of Hg and Zn; and when PC synthesis amount was measured by gel filtration - HPLC with regard to rice callus cultured in a culture medium in which Cd coexists with any of Zn, Cu, Pb, Hg and Ni, the effect of impeding PC synthesis increases in the order of Zn, Pb, Cu, Ni and Hg. 7 figs., 1 tab.

  6. Crystallization and preliminary crystallographic analysis of a novel plant type III polyketide synthase that produces pentaketide chromone

    Energy Technology Data Exchange (ETDEWEB)

    Morita, Hiroyuki [Mitsubishi Kagaku Institute of Life Sciences (MITILS), 11 Minamiooya, Machida, Tokyo 194-8511 (Japan); Kondo, Shin [ZOEGENE Corporation, 1000 Kamoshida, Aoba, Yokohama, Kanagawa 227-8502 (Japan); Abe, Tsuyoshi; Noguchi, Hiroshi [School of Pharmaceutical Sciences and the COE21 Program, University of Shizuoka, Shizuoka 422-8526 (Japan); Sugio, Shigetoshi, E-mail: ssugio@rc.m-kagaku.co.jp [ZOEGENE Corporation, 1000 Kamoshida, Aoba, Yokohama, Kanagawa 227-8502 (Japan); Abe, Ikuro, E-mail: ssugio@rc.m-kagaku.co.jp [School of Pharmaceutical Sciences and the COE21 Program, University of Shizuoka, Shizuoka 422-8526 (Japan); PRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012 (Japan); Kohno, Toshiyuki, E-mail: ssugio@rc.m-kagaku.co.jp [Mitsubishi Kagaku Institute of Life Sciences (MITILS), 11 Minamiooya, Machida, Tokyo 194-8511 (Japan)

    2006-09-01

    Pentaketide chromone synthase from A. arborescens has been overexpressed in E. coli, purified and crystallized. Diffraction data have been collected to 1.6 Å. Pentaketide chromone synthase (PCS) from Aloe arborescens is a novel plant-specific type III polyketide synthase that catalyzes the formation of 5,7-dihydroxy-2-methylchromone from five molecules of malonyl-CoA. Recombinant PCS expressed in Escherichia coli was crystallized by the hanging-drop vapour-diffusion method. The crystals belonged to space group P2{sub 1}, with unit-cell parameters a = 73.2, b = 88.4, c = 70.0 Å, α = γ = 90.0, β = 95.6°. Diffraction data were collected to 1.6 Å resolution using synchrotron radiation at BL24XU of SPring-8.

  7. Biochemistry: Acetohydroxyacid Synthase

    Directory of Open Access Journals (Sweden)

    Pham Ngoc Chien

    2010-02-01

    Full Text Available Acetohydroxyacid synthase (AHAS, EC 2.2.1.6; formerly known as acetolactate synthase, ALS is a thiamin-and FAD-dependent enzyme which catalyses the first common step in the biosynthesis of the branched-chain amino acids (BCAA isoleucine, leucine and valine. The enzyme is inhibited by several commercial herbicides and has been studied over the last 20 to 30 years. A short introductory note about acetohydroxyacid synthase has been provided.

  8. Preparing for ICD-10-CM/PCS implementation: impact on productivity and quality.

    Science.gov (United States)

    Stanfill, Mary H; Hsieh, Kang Lin; Beal, Kathleen; Fenton, Susan H

    2014-01-01

    Coding productivity is expected to drop significantly during the lead-up to and in the initial stages of ICD-10-CM/PCS implementation, now expected to be delayed until October 1, 2015. This study examined the differences in coding productivity between ICD-9-CM and ICD-10-CM/PCS for hospital inpatient cases matched for complexity and severity. Additionally, interrater reliability was calculated to determine the quality of the coding. On average, coding of an inpatient record took 17.71 minutes (69 percent) longer with ICD-10-CM/PCS than with ICD-9-CM. A two-tailed T-test for statistical validity for independent samples was significant (p = .001). No coder characteristics such as years of experience or educational level were found to be a significant factor in coder productivity. Coders who had received more extensive training were faster than coders who had received only basic training. Though this difference was not statistically significant, it provides a strong indication of significant return on investment for staff training time. Coder interrater reliability was substantial for ICD-9-CM but only moderate for ICD-10-CM/PCS, though some ICD-10-CM/PCS cases had complete interrater (coder) agreement. Time spent coding a case was negatively correlated with interrater reliability (-0.425 for ICD-10-CM and -0.349 for ICD-10-PCS). This finding signals that increased time per case does not necessarily translate to higher quality. Adequate training for coders, as well as guidance regarding time invested per record, is important. Additionally, these findings indicate that previous estimates of initial coder productivity loss with ICD-10-CM/PCS may have been understated.

  9. [The role of Cd-binding proteins and phytochelatins in the formation of cadmium resistance in Nicotiana plumbaginifolia cell lines].

    Science.gov (United States)

    Fenik, S I; Solodushko, V G; Kaliniak, T B; Blium, Ia B

    2007-01-01

    Nicotiana plumbaginifolia callus lines with the equal resistance to cadmium have been produced under different selective conditions--either without inhibition of the phytochelatin synthesis (line Cd-R) or in the presence of the inhibitor butionine sulfoximine (line Cd-Ri). The level of phytochelatin synthesis in the line Cd-R five-fold exceeded the control value and in the line Cd-Ri it was twice as much as in the control. It was shown that in the control line mainly three cadmium-binding proteins are expressed of the molecular weihgts 41, 34 and 19 kD. The common feature of the both resistant lines is the expression of the cadmium-binding proteins of 40, 37 and 19 kD. The resistant lines differ with respect to the synthesis of relatively low-molecular cadmium-binding proteins. The proteins of the molecular weights 12.5, 11.5 and 9 kD are expressed in the line Cd-R, while the proteins of 13 and 10 kD are expressed in the line Cd-Ri. It was supposed that both the phytochelatins and the Cd-binding proteins contribute to the resisitance of N. plumbaginifolia callus lines to cadmium and the lack of the phytochelatins can be equilibrated by the changes in the low-molecular Cd-binding protein synthesis.

  10. Genotoxic effects and induction of phytochelatins in the presence of cadmium in Vicia faba roots.

    Science.gov (United States)

    Béraud, Eric; Cotelle, Sylvie; Leroy, Pierre; Férard, Jean-François

    2007-10-01

    This study investigates different effects in roots of Vicia faba (broad bean) after exposure to cadmium. Genotoxic effects were assessed by use of the well-known Vicia root tip micronucleus assay. Cytotoxic effects were evaluated by determining the mitotic index in root tip cells. Finally, molecular induction mechanisms were evaluated by measuring phytochelatins with HPLC. After hydroponical exposure of V. faba roots to a range of cadmium concentrations and during different exposure times, the results of this approach showed large variations, according to the endpoint measured: after 48 h of exposure, genotoxic effects were found between 7.5 x 10(-8) and 5 x 10(-7)M CdCl(2), and cytotoxic effects were observed between 2.5 x 10(-7) and 5 x 10(-7)M CdCl(2). Statistically significant phytochelatin (PC) concentrations were measured at >or=10(-6)M CdCl(2) for PC(2), and at >or=10(-5)M CdCl(2) for PC3 and PC4.

  11. Tablet PCs in engineering mathematics courses at the J.B. Speed School of Engineering

    Science.gov (United States)

    Hieb, Jeffrey L.; Ralston, Patricia A. S.

    2010-06-01

    In fall 2007, J.B. Speed School of Engineering at the University of Louisville joined the ranks of universities requiring the purchase of Tablet PCs for all new entering students. This article presents a description of how the Department of Engineering Fundamentals incorporated Tablet PCs into their instruction, a review of the literature pertaining to the use of Tablet PCs for instruction and preliminary survey results from the students in engineering mathematics courses at the end of the first year, after students had been exposed to Tablet PCs for 1 year. Results show that a large majority of students in the Department of Engineering Fundamentals agree that presentation of engineering mathematics material using the Tablet PCs and DyKnow software is a vast improvement over overhead projector, blackboard, or PowerPoint lectures and course packs. However, students are split as to whether the Tablet PC is something they actually want to use for their own note-taking. Finally, a plan for assessment of tablet impact on student learning is presented.

  12. Synthesis of ceramic precursor polycarbosi-lane (PCS) under supercritical fluids (SCFs) state

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Using xylene as supercritical fluids (SCFs) media and polydimethylsilane (PDMS) as raw material, the ceramic precursor polycarbosilane (PCS) with moderate mo-lecular weight and narrow molecular weights distribution was synthesized under high pressure and temperature in autoclave. Softening-point test, Fourier transform infrared spectroscopy (FT-IR), gel permeation chromatography (GPC), element analysis and the yield test of PCS were adopted to characterize the SCFs-method and compared with two other PCS synthesis methods named normal-pressure high-temperature (NP) method and high-pressure hith-temperature (HP) method. The results indicate that the PCS maintains a high Si-H content and its chemical formula is SiC1.91H7.27O0.05. The yield is 63.5 wt% which has been improved by about 20 wt%, the softening-point is 180―220 ℃, the number average molecular weight is 1477 and the molecular weights distribution index is 1.61. The synthesis method via supercritical fluids with good diathermancy and reaction uniformity is a new feasi-ble method of synthesizing PCS with good qualities.

  13. Poplar maintains zinc homeostasis with heavy metal genes HMA4 and PCS1

    Science.gov (United States)

    Adams, Joshua P.; Adeli, Ardeshir; Hsu, Chuan-Yu; Harkess, Richard L.; Page, Grier P.; dePamphilis, Claude W.; Schultz, Emily B.; Yuceer, Cetin

    2011-01-01

    Perennial woody species, such as poplar (Populus spp.) must acquire necessary heavy metals like zinc (Zn) while avoiding potential toxicity. Poplar contains genes with sequence homology to genes HMA4 and PCS1 from other species which are involved in heavy metal regulation. While basic genomic conservation exists, poplar does not have a hyperaccumulating phenotype. Poplar has a common indicator phenotype in which heavy metal accumulation is proportional to environmental concentrations but excesses are prevented. Phenotype is partly affected by regulation of HMA4 and PCS1 transcriptional abundance. Wild-type poplar down-regulates several transcripts in its Zn-interacting pathway at high Zn levels. Also, overexpressed PtHMA4 and PtPCS1 genes result in varying Zn phenotypes in poplar; specifically, there is a doubling of Zn accumulation in leaf tissues in an overexpressed PtPCS1 line. The genomic complement and regulation of poplar highlighted in this study supports a role of HMA4 and PCS1 in Zn regulation dictating its phenotype. These genes can be altered in poplar to change its interaction with Zn. However, other poplar genes in the surrounding pathway may maintain the phenotype by inhibiting drastic changes in heavy metal accumulation with a single gene transformation. PMID:21504875

  14. Synthesis of ceramic precursor polycarbosilane (PCS) under supercritical fluids (SCFs) state

    Institute of Scientific and Technical Information of China (English)

    YANG DaXiang; SONG YongCai; LI ZongYuan

    2008-01-01

    Using xylene as supercritical fluids (SCFs) media and polydimethylsilane (PDMS) as raw material, the ceramic precursor polycarbosilane (PCS) with moderate mo- lecular weight and narrow molecular weights distribution was synthesized under high pressure and temperature in autoclave. Softening-point test, Fourier transform infrared spectroscopy (FT-IR), gel permeation chromatography (GPC), element analysis and the yield test of PCS were adopted to characterize the SCFs-method and compared with two other PCS synthesis methods named normal-pressure high-temperature (NP) method and high-pressure hith-temperature (HP) method. The results indicate that the PCS maintains a high Si-H content and its chemical formula is SiC1.91H7.27O0.05. The yield is 63.5 wt% which has been improved by about 20 wt%, the softening-point is 180-220℃, the number average molecular weight is 1477 and the molecular weights distribution index is 1.61. The synthesis method via supercritical fluids with good diathermancy and reaction uniformity is a new feasi- ble method of synthesizing PCS with good qualities.

  15. Proposal for the award of blanket contracts for the supply of Intel-based desktop PCs, display monitors and portable PCs

    CERN Document Server

    2000-01-01

    This document concerns the award of blanket contracts for the supply of the three following categories of equipment for the period 2001-2004: a) desktop PCs (complete PC systems but without display monitors), b) display monitors (conventional CRTs or flat screen LCDs) and c) portable PCs (also called notebooks or laptops). Following a market survey carried out among 41 firms in fourteen Member States, an invitation to tender (IT-2692/IT) was sent on 19 May 2000 to 12 firms and three consortia, each consisting of two firms, in five Member States. By the closing date, CERN had received seven tenders, all from the Swiss subsidiaries of the firms and consortia. The Finance Committee is invited to agree to the negotiation of - blanket contracts with VOBIS (CH), ELONEX (CH) and FUJITSU-SIEMENS (CH), the three lowest bidders complying with the specification, for the supply of Desktop PCs; - blanket contracts with VOBIS (CH), SYNOPTIC (CH) and ELONEX (CH), the three lowest bidders offering display monitors manufactur...

  16. Study on operation of a research reactor during one PCS pump failure accident

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Kyoung Woo; Yoon, Hyu Ngi; Kim, Seong Hoon; Chi, Dae Young; Yoon, Juh Yeon [KAERI, Daejeon (Korea, Republic of)

    2012-10-15

    The Primary cooling system (PCS) of a research reactor is designed to provide adequate cooling to the reactor core with a reasonable margin during all operation modes. The PCS consists of pumps, heat exchangers, and all necessary interconnecting pipes, valves, and instruments. The number of pumps is determined from a safety and economic point of view. As the number of pump trains increase, the cost increases according to the increase in safety class equipment. However, it is impossible to install one pump for a PCS because a zero flow can instantaneously occur during a pump failure such as a pump seizure. Thus, a PCS frequently consists of two parallel 50% capacity pumps and heat exchangers. In addition, check valves are generally installed to prevent a reversal flow when multiple pumps are designed to operate. However, if a swing type check valve is used, it should be estimated whether the slam due to instantaneous closing of the valve affects the system vibration. To reduce the vibration by a slam phenomenon, additional equipment such as a damper will be installed in the valve. The purpose of the check valve in PCS is to prevent the flow path when a reverse flow occurs. The installation of additional equipment will make it difficult to perform this function. In this study, it is estimated whether the PCS can operate without check valves. First, a flow analysis using Flowmaster was compared and verified by the calculation employing a empirical correlation. Second, the simulation for a one pump failure accident was performed and analyzed.

  17. Synbiotic functional drink from Jerusalem artichoke juice fermented by probiotic Lactobacillus plantarum PCS26

    OpenAIRE

    Dimitrovski, Darko; Velickova, Elena; Dimitrovska, Maja; Langerholc, Tomaz; Winkelhausen, Eleonora

    2015-01-01

    A probiotic strain Lactobacillus plantarum PCS26 was used to ferment Jerusalem artichoke juice. Growth kinetics of the bacterial strain was followed during juice fermentation both in flask and in laboratory fermentor. Jerusalem artichoke showed to be an excellent source of nutrients for L. plantarum PCS26 growth. The culture grew very well reaching more than 1010 cfu/ml in just 12 h. The pH changed from the initial 6.5 to 4.6 at the end of fermentation. The culture hydrolyzed fructooligosacch...

  18. Cost-benefit analysis of permanent change of duty station (PCS) modes of travel for moves to Alaska

    OpenAIRE

    Massaquoi, Mohamed B.

    2013-01-01

    Approved for public release; distribution is unlimited. This research examines whether permanent change of station (PCS) travel by privately owned vehicle (POV) to Alaska is to the governments advantage. The objectives of this research are to determine estimated total costs of PCS travel to Alaska by various modes of travel; to identify which of the PCS cost drivers has the greatest impact on government expenditures; and to calculate and project potential cost savings to the government bas...

  19. Characterization of lead-phytochelatin complexes by nano-electrospray ionization mass spectrometry

    Directory of Open Access Journals (Sweden)

    Christian eScheidegger

    2012-02-01

    Full Text Available The role of phytochelatins (PCn, metal-binding oligopeptides with the general structure (γGlu - Cysn - Gly (n = 2- 11 in metal detoxification is assumed to be based on immobilization of metals, which prevents binding of metals to important biomolecules. Although induction of phytochelatin synthesis has often been observed in algae upon exposure to metals, direct evidence for binding of the inducing metal to phytochelatins is scarce. In this study, a nano-electrospray ionization mass spectrometry (nano-ESI-MS method is developed for identification and characterization of Pb(II-PCn and Zn(II-PCn complexes. Complexes of Pb(II with standard PCn (n = 2 - 4 were examined by nano-ESI-MS with respect to their stoichiometry. Pb-PCn mass spectra indicated the presence of the [M+H]+ peak of PCn and complexes with various stoichiometries. Analysis of Pb-PC2 allowed the identification of four different complexes observed at m/z 746.10, 952.06, 1285.24 and 1491.20, corresponding to [Pb-PC2]+, [Pb2-PC2]+, [Pb-(PC22]+ and [Pb2-(PC22]+. Their m/z indicated coordination of Pb(II by PC2 through the thiol groups of PC cysteine and possibly carboxylic groups. For each of the standard PC3 and PC4, two different complexes were observed, corresponding to Pb-PC3, Pb2-PC3, Pb-PC4 and Pb2-PC4. The measured isotopic patterns were for all complexes identical to the theoretical isotopic patterns. Addition of Zn(II to previously formed Pb-PC2 complexes showed the appearance of the [Zn-PC2]+ complexes at m/z 602.6 and the decrease of the [Pb-PC2]+ peak. These findings corroborate the postulated Pb-PC complexes from a previous study using size exclusion chromatography of PC extracted from algae, as well as the concurrent formation of Pb-, Zn- and Cu-PC complexes in algae.

  20. Phytochelatin formation kinetics and toxic effects in the freshwater alga Chlamydomonas reinhardtii upon short- and long-term exposure to lead(II).

    Science.gov (United States)

    Scheidegger, Christian; Behra, Renata; Sigg, Laura

    2011-01-25

    Phytochelatins (PC) are metal-binding ligands synthesized by algae in response to elevated concentrations of various metals, such as Pb. Kinetics of PC synthesis and Pb accumulation in Chlamydomonas reinhardtii were investigated as a function of [Pb(2+)]=10(-11)-10(-7)M (pPb11-pPb7.1) in the exposure medium for up to 6h. The role of PC in Pb detoxification was explored by relating PC synthesis to the effects of Pb on growth and photosynthetic yield upon exposure to pPb9 and pPb8.3 for up to 72h. Pb accumulation increased with increasing [Pb(2+)], reaching a maximum concentration of 596±77amol/cell (intracellular concentration 2.98mM) at pPb7.1. Low concentrations of PC(2)-PC(4) were present in C. reinhardtii grown in control media without Pb addition. Upon short-term exposure, PC(2) and PC(3) synthesis was induced within minutes at [Pb(2+)]≥pPb8 and PC(4) synthesis after a lag phase at pPb7.1. Cellular PC(2)-PC(4) concentrations increased with time over 6h and with increasing [Pb(2+)]. PC concentrations after 6h exposure to pPb7.1 were 28.5±0.2amol/cell (142μM) PC(2), 2.8±0.05amol/cell (14μM) PC(3) and 0.30±0.01amol/cell (1.5μM) PC(4). Upon long-term exposure, induction of PC synthesis was detected at pPb9 and synthesis of PCs with a higher degree of polymerization was observed (PC(5)). PC concentrations were lower than intracellular Pb and were thus not present at sufficiently high concentrations to immobilize accumulated Pb. Inhibition of photosynthesis and growth up to 100% was observed upon long-term exposure, whereas in short-term experiments no inhibitory effects were detected.

  1. Tablet PCs and Reconceptualizing Learning with Technology: A Case Study in Higher Education

    Science.gov (United States)

    van Oostveen, R.; Muirhead, William; Goodman, William M.

    2011-01-01

    Purpose: This study aims to examine the experience of 31 university students who were issued tablet PCs for their use during an academic year. The primary research problem which drove this project revolved around the student perceptions of the benefits of technology to provide opportunities to restructure their learning experiences.…

  2. Tablet PCs as Instructional Tools in English as a Foreign Language Education

    Science.gov (United States)

    Savas, Perihan

    2014-01-01

    The main goal of the study presented in this paper was to investigate the perceptions of 40 pre-service English as a Foreign Language (EFL) teachers about the effectiveness of Tablet PCs as instructional tools in EFL classes. The study was conducted at a state university in Turkey and the data collection was mainly done via two questionnaires.…

  3. Managing and Ever Increasing Number of Linux—PCs at DESY

    Institute of Scientific and Technical Information of China (English)

    A.Gellrich; M.Ernst; 等

    2001-01-01

    An ever increasing number of computer systems-mainly PCs-require elaborated management strategies and tools.In the contribution to CHEP'01 we will present and discuss new concepts and developments concerning directory services and asset management,We will in particular report on first experiences with systems currently being implemented.

  4. Information Technologies Pre-Service Teachers' Acceptance of Tablet PCs as an Innovative Learning Tool

    Science.gov (United States)

    Cuhadar, Cem

    2014-01-01

    The current study is aimed to determine if Turkish IT pre-service teachers' acceptance of tablet PCs is within the framework of the Technology Acceptance Model. The research was patterned as a phenomenological study which is among the qualitative research methods. Participants were eight pre-service teachers studying in Trakya University, Faculty…

  5. Tablet PCs in a Paperless Classroom: Student and Teacher Perceptions on Screen Size

    Science.gov (United States)

    Runnels, Judith; Rutson-Griffiths, Arthur

    2013-01-01

    A paperless classroom, when all materials required to complete a class are available in an electronic form, has been shown to have positive impacts on student and teacher motivation, engagement, productivity, and efficiency. Recent trends suggest that of all of the technological tools available, tablet PCs can support many aspects of a paperless…

  6. PCs for Families: A Study of Early Intervention Using Networked Computing in Education.

    Science.gov (United States)

    Reaux, Ray A.; Ehrich, Roger W.; McCreary, Faith; Rowland, Keith; Hood, Susan

    1998-01-01

    Discusses the PCs for Families experiment, a longitudinal quantitative and ethnographic study of networked computing in the fifth-grade classroom examining how networked computing affects students' educational achievements, attitude and professional development of teachers and support instructors, and how families support students and react to the…

  7. Exploring the use of tablet PCs in veterinary medical education: opportunity or obstacle?

    Science.gov (United States)

    Wang, Hong; Rush, Bonnie R; Wilkerson, Melinda; van der Merwe, Deon

    2014-01-01

    A tablet PC is a laptop computer with a touch screen and a digital pen or stylus that can be used for handwritten notes and drawings. The use of tablet PCs has been investigated in many disciplines such as engineering, mathematics, science, and education. The purpose of this article is to explore student and faculty attitudes toward and experiences with tablet PCs 6 years after the implementation of a tablet PC program in the College of Veterinary Medicine (CVM) at Kansas State University (K-State). This study reports that the use of tablet PCs has enhanced students' learning experiences through learner-interface interaction, learner-content interaction, learner-instructor interaction, and learner-learner interaction. This study also identifies digital distraction as the major negative experience with tablet PCs during class time. The tablet PC program provides CVM faculty the potential to pursue technology integration strategies that support expected learning outcomes and provides students the potential to develop self-monitoring and self-discipline skills that support learning with digital technologies.

  8. 47 CFR 1.20007 - Additional assistance capability requirements for wireline, cellular, and PCS telecommunications...

    Science.gov (United States)

    2010-10-01

    ... 47 Telecommunication 1 2010-10-01 2010-10-01 false Additional assistance capability requirements for wireline, cellular, and PCS telecommunications carriers. 1.20007 Section 1.20007 Telecommunication... telecommunications carriers. (a) Definition—(1) Call-identifying information. Call identifying information...

  9. Quantification of phytochelatins in plants by reversed-phase HPLC-ESI-MS-MS.

    Science.gov (United States)

    El-Zohri, M H A; Cabala, R; Frank, H

    2005-08-01

    An on-line HPLC-ESI-MS-MS method has been developed for determination of glutathione and phytochelatins (PC) in plant tissues. For sample pretreatment, dithiothreitol (DTT) must be added at the very beginning, as an anti-oxidant. Optimization of instrumental conditions i.e. composition of HPLC mobile phase, ionization efficiency of the electrospray interface, and MS-MS detection in the multiple ion-monitoring mode, are the central aspects of this work. A polystyrene-packed column was found to be superior to a standard silica-packed reversed-phase column. A concave quadratic gradient of ammonium formate buffer and acetonitrile was found to be optimum. The limits of quantitation were 0.2 micromol kg(-1) plant tissue for glutathione and PC. The method has been applied to analysis of tissue samples from Vicia faba grown in Cd-containing nutrient solutions.

  10. Absence of pneumococcal PcsB is associated with overexpression of LysM domain-containing proteins.

    Science.gov (United States)

    Giefing-Kröll, Carmen; Jelencsics, Kira E; Reipert, Siegfried; Nagy, Eszter

    2011-07-01

    The streptococcal protein required for cell separation B (PcsB) is predicted to play an important role in peptidoglycan metabolism, based on sequence motifs and altered phenotypes of gene deletion mutant cells exhibiting defects in cell separation. However, no enzymic activity has been demonstrated for PcsB so far. By generating gene deletion mutant strains in four different genetic backgrounds we could demonstrate that pcsB is not essential for cell survival in Streptococcus pneumoniae, but is essential for proper cell division. Deletion mutant cells displayed cluster formation due to aberrant cell division, reduced growth and antibiotic sensitivity that were fully reverted by transformation with a plasmid carrying pcsB. Immunofluorescence staining revealed that PcsB was localized to the cell poles, similarly to PBP3 and LytB, enzymes with demonstrated peptidoglycan-degrading activity required for daughter cell separation. Similarly to other studies with PcsB homologues, we could not detect peptidoglycan-lytic activity with recombinant or native pneumococcal PcsB in vitro. In addition to defects in septum placement and separation, the absence of PcsB induced an increased release of several proteins, such as enolase, MalX and the SP0107 LysM domain protein. Interestingly, genes encoding both LysM domain-containing proteins that are present in the pneumococcal genome (SP0107 and SP2063) and predicted to be involved in cell wall metabolism were found to be highly overexpressed (14-33-fold increase) in ΔpcsB cells in two different genetic backgrounds. Otherwise, we detected very few changes in the global gene expression profile of cells lacking PcsB. Thus our data suggest that LysM domain proteins partially compensate for the lack of PcsB function and allow the survival and slow growth of the pneumococcus.

  11. PCS: a pallet costing system for wood pallet manufacturers (version 1.0 for Windows®)

    Science.gov (United States)

    A. Jefferson, Jr. Palmer; Cynthia D. West; Bruce G. Hansen; Marshall S. White; Hal L. Mitchell

    2002-01-01

    The Pallet Costing System (PCS) is a computer-based, Microsoft Windows® application that computes the total and per-unit cost of manufacturing an order of wood pallets. Information about the manufacturing facility, along with the pallet-order requirements provided by the customer, is used in determining production cost. The major cost factors addressed by PCS...

  12. Use of Tablet PCs to Enhance Instruction and Promote Group Collaboration in a Course to Prepare Future Mathematics Specialists

    Science.gov (United States)

    Ellington, Aimee J.; Wilson, Jill H.; Nugent, Jeffrey S.

    2011-01-01

    This article details the use of tablet PCs in a mathematics content course for future Mathematics Specialists. Instructors used tablet PCs instead of a traditional whiteboard to capture demonstration and discussion. Students were grouped for collaborative problem solving and exploration exercises. Each group was provided with a tablet PC for…

  13. Crystallization and preliminary crystallographic analysis of a novel plant type III polyketide synthase that produces pentaketide chromone

    Science.gov (United States)

    Morita, Hiroyuki; Kondo, Shin; Abe, Tsuyoshi; Noguchi, Hiroshi; Sugio, Shigetoshi; Abe, Ikuro; Kohno, Toshiyuki

    2006-01-01

    Pentaketide chromone synthase (PCS) from Aloe arborescens is a novel plant-specific type III polyketide synthase that catalyzes the formation of 5,7-dihydroxy-2-methylchromone from five molecules of malonyl-CoA. Recombinant PCS expressed in Escherichia coli was crystallized by the hanging-drop vapour-diffusion method. The crystals belonged to space group P21, with unit-cell parameters a = 73.2, b = 88.4, c = 70.0 Å, α = γ = 90.0, β = 95.6°. Diffraction data were collected to 1.6 Å resolution using synchrotron radiation at BL24XU of SPring-8. PMID:16946474

  14. Assessing the planning and implementation strategies for the ICD-10-CM/PCS coding transition in Alabama hospitals.

    Science.gov (United States)

    Houser, Shannon H; Morgan, Darius; Clements, Kay; Hart-Hester, Susan

    2013-01-01

    Health information management (HIM) professionals play a significant role in transitioning from ICD-9-CM to ICD-10-CM/PCS. ICD-10-CM/PCS coding will impact many operational aspects of healthcare facilities, such as physicians' documentation in health records, coders' process for review of clinical information, the billing process, and the payers' reimbursement to the healthcare facilities. This article examines the level of readiness and planning for ICD-10-CM/PCS implementation among hospitals in Alabama, identifies training methods/approaches to be used by the hospitals, and discusses the challenges to the ICD-10-CM/PCS coding transition. A 16-question survey was distributed to 116 Alabama hospital HIM directors in December 2011 with follow-up through February 2012. Fifty-three percent of respondent hospitals began the planning process in 2011, and most facilities were halfway or less than halfway to completion of specific implementation tasks. Hospital coders will be or are being trained using in-house training, through seminars/webinars, or by consultants. The impact of ICD-10-CM/PCS implementation can be minimized by training coders in advance, hiring new coders, and adjusting coders' productivity measures. Three major challenges to the transition were identified: the need to interact with physicians and other providers more often to obtain information needed to code in ICD-10-CM/PCS systems, education and training of coders and other ICD-10-CM/PCS users, and dependence on vendors for major technology upgrades for ICD-10-CM/PCS systems. Survey results provide beneficial information for HIM professionals and other users of coded data to assist in establishing sound practice standards for ICD-10-CM/PCS coding implementation. Adequate planning and preparation will be essential to the successful implementation of ICD-10-CM/PCS.

  15. Effect of Hg, As and Pb on biomass production, photosynthetic rate, nutrients uptake and phytochelatin induction in Pfaffia glomerata.

    Science.gov (United States)

    Gupta, D K; Huang, H G; Nicoloso, F T; Schetinger, M R; Farias, J G; Li, T Q; Razafindrabe, B H N; Aryal, N; Inouhe, M

    2013-11-01

    Plantlets of Pfaffia glomerata (Spreng.) were exposed for 28 days to three different metal/metalloid (Hg, Pb and As) with different levels (Hg 1; As 25, 50, 100 and Pb 100 and 400 μM) to analyze the possible phytochelatin initiation and affects on growth and photosynthetic pigments vis-à-vis metal accumulation potential of plants. The plantlets showed significant Hg, As and Pb accumulation in roots (150, 1267.67 and 2129 μg g(-1) DW respectively); however, a low root to shoot metal translocation was observed. It was interesting to note that all tested macronutrient (Mg, K, Ca) was higher in shoots and just opposite in case of micronutrients (Cu, Fe, Zn), was recorded highest in roots. The growth of plantlets (analyzed in terms of length and dry weight) was negatively affected by various metal treatments. In addition, the level of photosynthetic pigments alters significantly in response to all metal/metalloid treatment. In response to all tested metal/metalloids in plants only As induced phytochelatins (PC2, PC3 and PC4) in roots, and in shoots, GSH was observed in all tested metal/metalloids. In conclusion, P. glomerata plantlets could not cooperatively induce phytochelatins under any of Hg and Pb levels.

  16. Determination and characterization of cysteine, glutathione and phytochelatins (PC₂₋₆) in Lolium perenne L. exposed to Cd stress under ambient and elevated carbon dioxide using HPLC with fluorescence detection.

    Science.gov (United States)

    Ju, Xue Hai; Tang, Shirong; Jia, Yan; Guo, Junkang; Ding, Yongzhen; Song, Zhengguo; Zhao, Yujie

    2011-06-15

    Metal-binding thiols, involved in detoxification mechanisms in plant and other organism under heavy metal stress, are receiving more and more attentions, and various methods have been developed to determine related thiols such as cysteine (Cys), glutathione (GSH) and phytochelatins (PCs). In present study, an HPLC method was established for simultaneous determination of Cys GSH and PC(2-6) after treatment with disulfide reductant of tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and thiolyte reagent of monobromobimane (mBBr). The separation of thiol derivatives was performed on an Agilent Zorbax Eclipse XDB-C18 column (4.6 mm × 30 mm, 1.8 μm) with a linear gradient elution of 0.1% (v/v) trifluoroacetic acid (TFA)-acetonitrile (ACN) at 0.8 mL min(-1). The temperature of the column was maintained at 25°C. The excitation and emission wavelengths were set at 380 and 470 nm, respectively. The thiol derivatives were well separated in 19 min, and the total analysis time was 30 min. The established method was proved selective, specific and reproducible, and could be applicable to determine Cys, GSH and PC(2-6) and to evaluate their roles in detoxification mechanisms in Cd-treated Lolium perenne L. under ambient and elevated carbon dioxide (CO(2)). It was found that the total SH contents and proportions of thiols in roots and shoots were dependent on Cd concentration, whereas the total SH contents decreased and the proportions of thiols altered without significance at elevated CO(2) level.

  17. The Capacity of ICD-10-CM/PCS to Characterize Surgical Care.

    Science.gov (United States)

    Utter, Garth H; Schuster, Kevin M; Miller, Preston R; Mowery, Nathan T; Agarwal, Suresh K; Winchell, Robert J; Crandall, Marie L

    2017-05-22

    The International Classification of Diseases, 10th edition, Clinical Modification and Procedure Coding System (ICD-10-CM/PCS) have been in effect since October 1, 2015 and offer much greater capacity than previous classifications to characterize the nature and treatment of surgical conditions. However, the diagnosis codes of ICD-10-CM are unevenly specific across the spectrum of surgical conditions and currently do not adequately allow description of important distinctions in disease severity for many common surgical conditions. Through our work on the Patient Assessment Committee of the American Association for the Surgery of Trauma, we sought changes to ICD-10-CM to allow for better characterization of the severity of several common emergency general surgical conditions, such as acute appendicitis and acute pancreatitis. The ICD Coordination and Maintenance Committee periodically entertains requests to improve ICD-10-CM/PCS through a formal process with public input; the Centers for Medicare and Medicaid services, in coordination with other agencies and organizations, issue annual updates to these classifications. We describe some potential improvements to ICD-10-CM/PCS as well as how individuals and organizations can propose and effect such changes. With the next parent classification for mortality reporting (ICD-11) currently in development by the World Health Organization, now is also an opportune time to request changes that eventually would be incorporated into the future U.S. version of ICD-11 for morbidity reporting.

  18. Effects of low-dose midazolam with propofol in patient-controlled sedation (PCS) for apicectomy.

    Science.gov (United States)

    Küçükyavuz, Zuhal; Cambazoğlu, Mine

    2004-06-01

    We studied the effects of low-dose midazolam with propofol for patient control sedation (PCS) in 30 healthy (ASA grade I) patients who were randomly allocated into two equal groups (n = 15 in each). They were given a propofol infusion of 2mg/kg/h after a bolus dose of 0.7 mg/kg. The second group was given the 2mg/kg/h propofol infusion after a dose of midazolam 0.03 mg/kg and a bolus dose of propofol 0.7 mg/kg. The standard dose for PCS was propofol 0.2mg/kg in both groups. Clinical data were taken and haemodynamic variables, and oxygen saturation were recorded before and on the 5th, 10th, 20th, and 30th minutes during the operations. The level of sedation, amnesia and conditions of each patient were evaluated during the study. Patients' satisfaction was recorded using a modified visual analogue scale (VAS). All results were evaluated statistically. We conclude that low-dose midazolam with propofol during PCS neither reduced oxygen saturation nor prolonged the time of discharge. Low-dose midazolam with propofol also improved the acceptability and comfort for patients and made the operation easier, which makes it preferable to propofol alone.

  19. Robust method for the analysis of phytochelatins in rice by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry based on polymeric column materials.

    Science.gov (United States)

    Yu, Shasha; Bian, Yingfang; Zhou, Rong; Mou, Renxiang; Chen, Mingxue; Cao, Zhaoyun

    2015-12-01

    A sensitive and robust high-performance liquid chromatography coupled with electrospray tandem mass spectrometry method for the identification and quantification of glutathione and phytochelatins from rice was developed. Homogenized samples were extracted with water containing 100 mM dithiothreitol, and solid-phase extraction using polymer anion exchange resin was employed for sample purification. Chromatography was performed on a polymeric column with acetonitrile and water containing 0.1% formic acid as the mobile phase at the flow rate of 300 μL/min. The limit of quantitation was 6-100 nM. This assay showed excellent linearity for both glutathione and phytochelatins over physiological normal ranges, with correlation coefficients (r) > 0.9976. Recoveries for four biothiols were within the range of 76-118%, within relative standard deviations less than 15%. The intraday precision (n = 7) was 2.1-13.3%, and the interday precision over 15 days was 4.3-15.2%. The optimized method was applied to analyze tissue samples from rice grown using nutrient solutions with three different cadmium concentrations (0, 50, and 100 μM). With increasing cadmium concentrations, the content of phytochelatin 2 and phytochelatin 3 in rice roots increased, in contrast to most phytochelatins, and the content of glutathione in rice stems and roots decreased significantly.

  20. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, R.B.; Wildung, M.R.; Burke, C.C.; Gershenzon, J.

    1999-03-02

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). 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 geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate. 5 figs.

  1. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wildung, Mark Raymond (Colfax, WA); Burke, Charles Cullen (Moscow, ID); Gershenzon, Jonathan (Jena, DE)

    1999-01-01

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). 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 geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate.

  2. An Arabidopsis callose synthase

    DEFF Research Database (Denmark)

    Ostergaard, Lars; Petersen, Morten; Mattsson, Ole

    2002-01-01

    unclear whether callose synthases can also produce cellulose and whether plant cellulose synthases may also produce beta-1,3-glucans. We describe here an Arabidopsis gene, AtGsl5, encoding a plasma membrane-localized protein homologous to yeast beta-1,3-glucan synthase whose expression partially......Beta-1,3-glucan polymers are major structural components of fungal cell walls, while cellulosic beta-1,4-glucan is the predominant polysaccharide in plant cell walls. Plant beta-1,3-glucan, called callose, is produced in pollen and in response to pathogen attack and wounding, but it has been...... in the Arabidopsis mpk4 mutant which exhibits systemic acquired resistance (SAR), elevated beta-1,3-glucan synthase activity, and increased callose levels. In addition, AtGsl5 is a likely target of salicylic acid (SA)-dependent SAR, since AtGsl5 mRNA accumulation is induced by SA in wild-type plants, while...

  3. De novo Transcriptome Analysis of Sinapis alba in Revealing the Glucosinolate and Phytochelatin Pathways

    Science.gov (United States)

    Zhang, Xiaohui; Liu, Tongjin; Duan, Mengmeng; Song, Jiangping; Li, Xixiang

    2016-01-01

    Sinapis alba is an important condiment crop and can also be used as a phytoremediation plant. Though it has important economic and agronomic values, sequence data, and the genetic tools are still rare in this plant. In the present study, a de novo transcriptome based on the transcriptions of leaves, stems, and roots was assembled for S. alba for the first time. The transcriptome contains 47,972 unigenes with a mean length of 1185 nt and an N50 of 1672 nt. Among these unigenes, 46,535 (97%) unigenes were annotated by at least one of the following databases: NCBI non-redundant (Nr), Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, Gene Ontology (GO), and Clusters of Orthologous Groups of proteins (COGs). The tissue expression pattern profiles revealed that 3489, 1361, and 8482 unigenes were predominantly expressed in the leaves, stems, and roots of S. alba, respectively. Genes predominantly expressed in the leaf were enriched in photosynthesis- and carbon fixation-related pathways. Genes predominantly expressed in the stem were enriched in not only pathways related to sugar, ether lipid, and amino acid metabolisms but also plant hormone signal transduction and circadian rhythm pathways, while the root-dominant genes were enriched in pathways related to lignin and cellulose syntheses, involved in plant-pathogen interactions, and potentially responsible for heavy metal chelating, and detoxification. Based on this transcriptome, 14,727 simple sequence repeats (SSRs) were identified, and 12,830 pairs of primers were developed for 2522 SSR-containing unigenes. Additionally, the glucosinolate (GSL) and phytochelatin metabolic pathways, which give the characteristic flavor and the heavy metal tolerance of this plant, were intensively analyzed. The genes of aliphatic GSLs pathway were predominantly expressed in roots. The absence of aliphatic GSLs in leaf tissues was due to the shutdown of BCAT4, MAM1, and CYP79F1 expressions. Glutathione was extensively

  4. De novo transcriptome analysis of Sinapis alba in revealing the glucosinolate and phytochelatin pathways

    Directory of Open Access Journals (Sweden)

    Xiaohui eZhang

    2016-03-01

    Full Text Available Sinapis alba is an important condiment crop and can also be used as a phytoremediation plant. Though it has important economic and agronomic values, sequence data and the genetic tools are still rare in this plant. In the present study, a de novo transcriptome based on the transcriptions of leaves, stems and roots was assembled for S. alba for the first time. The transcriptome contains 47,972 unigenes with a mean length of 1,185 nt and an N50 of 1,672 nt. Among these unigenes, 46,535 (97% unigenes were annotated by at least one of the following databases: NCBI non-redundant (Nr, Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG pathway, Gene Ontology (GO, and Clusters of Orthologous Groups of proteins (COGs. The tissue expression pattern profiles revealed that 3,489, 1,361 and 8,482 unigenes were predominantly expressed in the leaves, stems and roots of S. alba, respectively. Genes predominantly expressed in the leaf were enriched in photosynthesis- and carbon fixation-related pathways. Genes predominantly expressed in the stem were enriched in not only pathways related to sugar, ether lipid and amino acid metabolisms but also plant hormone signal transduction and circadian rhythm pathways, while the root-dominant genes were enriched in pathways related to lignin and cellulose syntheses, involved in plant-pathogen interactions, and potentially responsible for heavy metal chelating and detoxification. Based on this transcriptome, 14,727 simple sequence repeats (SSRs were identified, and 12,830 pairs of primers were developed for 2,522 SSR-containing unigenes. Additionally, the glucosinolate (GSL and phytochelatin metabolic pathways, which give the characteristic flavor and the heavy metal tolerance of this plant, were intensively analyzed. The genes of aliphatic GSLs pathway were predominantly expressed in roots. The absence of aliphatic GSLs in leaf tissues was due to the shutdown of BCAT4, MAM1 and CYP79F1 expressions. Glutathione was

  5. Electrochemical survey of the chain length influence in phytochelatins competitive binding by cadmium.

    Science.gov (United States)

    Gusmão, Rui; Ariño, Cristina; Díaz-Cruz, José Manuel; Esteban, Miquel

    2010-11-01

    Multivariate curve resolution with alternating least squares (MCR-ALS) was applied to voltammetric data obtained in the analysis of the competitive binding of glutathione (GSH) and phytochelatins [(gammaGlu-Cys)(n)-Gly, PC(n), n=2-5] by Cd(2+). The displacements between ligands and chain length influence on the competitive binding of PC(n) toward Cd(2+) were investigated. The analysis of the resulting pure voltammograms and concentration profiles of the resolved components suggests that ligands containing more thiol groups are able to displace the shortest chain ligands from their metal complexes, whereas the opposite does not happen. However, when the length of the chain surpasses that of PC(3), the binding capacity of the molecule still increases (i.e., it can bind more metal ions), but the position and shape of the voltammetric signals practically rest unchanged. This suggests that at this level, the stability of metal binding could depend more on the nature of the binding sites separately than on the quantity of the sites (i.e., the chain length).

  6. From cysteine to longer chain thiols: thermodynamic analysis of cadmium binding by phytochelatins and their fragments.

    Science.gov (United States)

    Chekmeneva, Elena; Gusmão, Rui; Díaz-Cruz, José Manuel; Ariño, Cristina; Esteban, Miquel

    2011-08-01

    Isothermal Titration Calorimetry (ITC) was used to study the binding of Cd(2+) by phytochelatins ((γGlu-Cys)(n)-Gly, PC(n); n = 1-5) and their selected fragments (Cys, Cys-Gly and γGlu-Cys) in order to understand the influence of the chain length on the complex stabilities and the origin of the enhanced affinities in Tris buffer at pH 7.5 and 8.5 and at 25 °C. Different complexes are formed with glutathione (GSH) and its fragments, Cys, Cys-Gly and γGlu-Cys, and their stabilities depend on the corresponding pK(a) value of the thiol group in the ligands. The stability of Cd-PC(n) complexes increases moving towards higher PC(2-5), as well as the complexing capacity expressed as the number of metal ions that can be bound by one ligand molecule. The affinity of Cd(2+) for the PC(n) can be described by the following GSH < PC(2) < PC(3)≤ PC(4)≤ PC(5) sequence. On the basis of these thermodynamic data it is possible to explain the abundance of certain Cd-PC(n) complexes found in nature. The comprehension of the thermodynamic rules that govern the interactions of Cd(2+) with PC(n) and their constituents is of great service in the research with real plant samples subjected to metal stress and in the development of new strategies of bio/phytoremediation.

  7. Cross-cultural adaptation and validation of the South African Pain Catastrophizing Scale (SA-PCS among patients with fibromyalgia

    Directory of Open Access Journals (Sweden)

    Morris Linzette D

    2012-11-01

    Full Text Available Abstract Background Pain catastrophization has recently been recognized as a barrier to the healthy development of physical functioning among chronic pain patients. Levels of pain catastrophization in chronic pain patients are commonly measured using the Pain Catastrophizing Scale (PCS. Objective To cross-culturally adapt and validate the South African PCS (SA-PCS among English-, Afrikaans- and Xhosa-speaking patients with fibromyalgia living in the Cape Metropole area, Western Cape, South Africa. Methods The original PCS was cross-culturally adapted in accordance with international standards to develop an English, Afrikaans and Xhosa version of the SA-PCS using a repeated measures study design. Psychometric testing included face/content validity, internal consistency (Cronbach’s alpha-α, test-retest reliability (intraclass coefficient correlations-ICC, sensitivity-to-change and cross-sectional convergent validity (by comparing the adapted SA-PCS to related constructs. Results The cross-culturally adapted English, Afrikaans and Xhosa SA-PCS showed good face and content validity, excellent internal consistency (with Chronbach’s α = 0.98, 0.98 and 0.97 for the English, Afrikaans and Xhosa SA-PCS, as a whole, respectively, excellent test-retest reliability (with ICC’s of 0.90, 0.91 and 0.89 for the English, Afrikaans and Xhosa SA-PCS, respectively; as well as satisfactory sensitivity-to-change (with a minimum detectable change of 8.8, 9.0 and 9.3 for the English, Afrikaans and Xhosa SA-PCS, respectively and cross-sectional convergent validity (when compared to pain severity as well as South African versions of the Tampa scale for Kinesiophobia and the revised Fibromyalgia Impact Questionnaire. Conclusion The SA-PCS can therefore be recommended as simple, efficient, valid and reliable tool which shows satisfactory sensitivity-to-change and cross-sectional convergent validity, for use among English, Afrikaans and Xhosa-speaking patients with

  8. An approach to effective UHF (S/L band) data communications for satellite Personal Communication Service (PCS)

    Science.gov (United States)

    Hayase, Joshua Y.

    1995-01-01

    Reliable signaling information transfer is fundamental in supporting the needs of data communication PCS via LMS (Land Mobile Service) SSs (satellite systems). The needs of the system designer can be satisfied only through the collection of media information that can be brought to bear on the pertinent design issues. We at ISI hope to continue our dialogue with fading media experts to address the unique data communications needs of PCS via LMS SSs.

  9. Hybrid polyketide synthases

    Energy Technology Data Exchange (ETDEWEB)

    Fortman, Jeffrey L.; Hagen, Andrew; Katz, Leonard; Keasling, Jay D.; Poust, Sean; Zhang, Jingwei; Zotchev, Sergey

    2016-05-10

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing an even-chain or odd-chain diacid or lactam or diamine. The present invention also provides for a host cell comprising the PKS and when cultured produces the even-chain diacid, odd-chain diacid, or KAPA. The present invention also provides for a host cell comprising the PKS capable of synthesizing a pimelic acid or KAPA, and when cultured produces biotin.

  10. Arsenic availability, toxicity and direct role of GSH and phytochelatins in As detoxification in the green alga Stichococcus bacillaris.

    Science.gov (United States)

    Pawlik-Skowrońska, B; Pirszel, J; Kalinowska, R; Skowroński, T

    2004-12-10

    Accumulation and toxicity of inorganic arsenic forms As(III) and As(V) to the green microalga Stichococcus bacillaris depended on environmental variables. pH of exposure and to a lesser extent elevated concentrations of humic acid, chloride and orthophosphate ions affected arsenic accumulation and its toxicity. As(V) was more toxic than As(III), especially at the near neutral pH 6.8. Intracellular As(V) uptake by algal cells was greater at pH 6.8 than at 8.2. In response to As(III) and As(V) the alga produced phytochelatins (PC(2-3)), but at As(V) exposure, their levels in cells were higher than with As(III), suggesting higher As(V) than As(III) availability and uptake. Arsenic in algal cells occurred in various complexes with non-protein SH groups. Some of these complexes dissociated under acidic conditions, but others were able to dissociate only at an alkaline pH. The former consisted of SH groups of phytochelatins. Those dissociating at an alkaline pH involved SH groups from both glutathione (GSH) and phytochelatins (PC) or their derivatives. In the predominant acid-stable mixed As-SH complex, the ratio of SH (PC(2)) to SH (GSH) was 2:1, which suggests that one molecule of PC(2) (containing two SH groups) together with one molecule of GSH were involved in intracellular complexation of each As atom. This is the first demonstration of GSH involvement in arsenic complexation, in vivo. The intracellular concentration of As was greater than that of non-protein SH groups which suggests that not all the arsenic in algal cells was complexed and detoxified by thiol groups.

  11. Arsenic availability, toxicity and direct role of GSH and phytochelatins in As detoxification in the green alga Stichococcus bacillaris

    Energy Technology Data Exchange (ETDEWEB)

    Pawlik-Skowronska, B. [Centre for Ecological Research, Polish Academy of Sciences at Dziekanow Lesny, Experimental Station, Niecala 18/3, 20-080 Lublin (Poland)]. E-mail: pawlik@golem.umcs.lublin.pl; Pirszel, J. [Centre for Ecological Research, Polish Academy of Sciences at Dziekanow Lesny, Experimental Station, Niecala 18/3, 20-080 Lublin (Poland); Kalinowska, R. [Centre for Ecological Research, Polish Academy of Sciences at Dziekanow Lesny, Experimental Station, Niecala 18/3, 20-080 Lublin (Poland); Skowronski, T. [Centre for Ecological Research, Polish Academy of Sciences at Dziekanow Lesny, Experimental Station, Niecala 18/3, 20-080 Lublin (Poland)

    2004-12-10

    Accumulation and toxicity of inorganic arsenic forms As(III) and As(V) to the green microalga Stichococcus bacillaris depended on environmental variables. pH of exposure and to a lesser extent elevated concentrations of humic acid, chloride and orthophosphate ions affected arsenic accumulation and its toxicity. As(V) was more toxic than As(III), especially at the near neutral pH 6.8. Intracellular As(V) uptake by algal cells was greater at pH 6.8 than at 8.2. In response to As(III) and As(V) the alga produced phytochelatins (PC{sub 2-3}), but at As(V) exposure, their levels in cells were higher than with As(III), suggesting higher As(V) than As(III) availability and uptake. Arsenic in algal cells occurred in various complexes with non-protein -SH groups. Some of these complexes dissociated under acidic conditions, but others were able to dissociate only at an alkaline pH. The former consisted of -SH groups of phytochelatins. Those dissociating at an alkaline pH involved -SH groups from both glutathione (GSH) and phytochelatins (PC) or their derivatives. In the predominant acid-stable mixed As-SH complex, the ratio of -SH (PC{sub 2}) to -SH (GSH) was 2:1, which suggests that one molecule of PC{sub 2} (containing two -SH groups) together with one molecule of GSH were involved in intracellular complexation of each As atom. This is the first demonstration of GSH involvement in arsenic complexation, in vivo. The intracellular concentration of As was greater than that of non-protein -SH groups which suggests that not all the arsenic in algal cells was complexed and detoxified by thiol groups.

  12. The phytochelatin transporters AtABCC1 and AtABCC2 mediate tolerance to cadmium and mercury.

    Science.gov (United States)

    Park, Jiyoung; Song, Won-Yong; Ko, Donghwi; Eom, Yujin; Hansen, Thomas H; Schiller, Michaela; Lee, Tai Gyu; Martinoia, Enrico; Lee, Youngsook

    2012-01-01

    Heavy metals such as cadmium (Cd) and mercury (Hg) are toxic pollutants that are detrimental to living organisms. Plants employ a two-step mechanism to detoxify toxic ions. First, phytochelatins bind to the toxic ion, and then the metal-phytochelatin complex is sequestered in the vacuole. Two ABCC-type transporters, AtABCC1 and AtABCC2, that play a key role in arsenic detoxification, have recently been identified in Arabidopsis thaliana. However, it is unclear whether these transporters are also implicated in phytochelatin-dependent detoxification of other heavy metals such as Cd(II) and Hg(II). Here, we show that atabcc1 single or atabcc1 atabcc2 double knockout mutants exhibit a hypersensitive phenotype in the presence of Cd(II) and Hg(II). Microscopic analysis using a Cd-sensitive probe revealed that Cd is mostly located in the cytosol of protoplasts of the double mutant, whereas it occurs mainly in the vacuole of wild-type cells. This suggests that the two ABCC transporters are important for vacuolar sequestration of Cd. Heterologous expression of the transporters in Saccharomyces cerevisiae confirmed their role in heavy metal tolerance. Over-expression of AtABCC1 in Arabidopsis resulted in enhanced Cd(II) tolerance and accumulation. Together, these results demonstrate that AtABCC1 and AtABCC2 are important vacuolar transporters that confer tolerance to cadmium and mercury, in addition to their role in arsenic detoxification. These transporters provide useful tools for genetic engineering of plants with enhanced metal tolerance and accumulation, which are desirable characteristics for phytoremediation.

  13. Identification of phytochelatins in the cadmium-stressed conjugating green alga Micrasterias denticulata.

    Science.gov (United States)

    Volland, Stefanie; Schaumlöffel, Dirk; Dobritzsch, Dirk; Krauss, Gerd-Joachim; Lütz-Meindl, Ursula

    2013-04-01

    Aquatic environments like peat bogs are affected by anthropogenic metal input into the environment. These ecosystems are inhabited by unicellular green algae of the class Zygnematophyceae. In this study the desmid Micrasterias denticulata was stressed with 600 nM Cd, 10 μM Cr and 300 nM Cu for 3 weeks. GSH levels were measured with HPLC and did not differ between the different treatments or the control. According to the metallo-thiolomics concept, mass spectrometry was used as a method for unambiguous thiol peptide identification. PC2, PC3 and PC4 were clearly identified in the Cd stressed sample with UPLC-MS by their MS spectrum and molecular masses. PC2 and PC3 were determined to be the main thiol compounds, while PC4 was only abundant in traces in Micrasterias. In addition, the identity of PC2 and PC3 was confirmed by MS/MS. No PCs were detected in the Cu stressed algae sample. However, in the Cr stressed sample traces of PC2 were indicated by a peak in UPLC-MS at the retention time of the PC2 standard, but the intensity was too low to acquire reliable MS and MS/MS spectra. In this study PCs have been detected for the first time in a green alga of the division Streptophyta, a close relative to higher plants.

  14. iRadioactivity — Possibilities and Limitations for Using Smartphones and Tablet PCs as Radioactive Counters

    Science.gov (United States)

    Kuhn, Jochen; Molz, Alexander; Gröber, Sebastian; Frübis, Jan

    2014-09-01

    A study conducted in 2013 showed that about 70-80% of teens and young adults in the United States own a smartphone.1 Furthermore the number of tablet PC users in the United States will increase up to more than 80% by 2015.2 As a result, these devices have increasingly become everyday tools, particularly for the younger generation. In recent years, various articles have been published about the use of smartphones and tablet PCs as experimental tools especially in the physics classroom. This is possible because today's smartphones and tablet PCs are equipped with many sensors, which can be used to perform quantitative measurements of sound, acceleration, magnetic flux density, air pressure, light intensity, humidity, angular velocity, temperature, or position on Earth (GPS). While previous articles mainly present experiments on mechanics or acoustics, in which the acceleration sensor or the microphone is used (for a synopsis of different examples, see Ref. 3; for recent papers, see Refs.), in this article we focus on experiments for studying radioactivity using the camera sensor.

  15. Student’s Opinions for Using Tablet PCs in Education: A Case Study

    Directory of Open Access Journals (Sweden)

    İlhan VARANK

    2014-04-01

    Full Text Available Tablet PCs may produce solutions to some problems experienced in the traditional classes. The purpose of this qualitative case study is to evaluate students' suggestions about a tablet-enhanced learning environment which aims to increase the efficiency of the instructional process in the classroom. The participants of the study were 20 (62.5% male and 12 (37.5% female pre-service teachers. Two open-ended questions were directed to the participants: (I What are your suggestions about the tablet-enhanced learning environment? and (II How could tablet PCs be used more effectively during the lessons? Also, information regarding the pre-service teachers’ genders and their experiences with touch screen devices was collected. It was found that students’ opinions about the tablet-enhanced instruction were negative with respect to the five themes found (instructional approach, student experience, time management, technical problems and ownership and they faced some problems during the instructions in the classroom.

  16. Solid-State Disk with Double Data Rate DRAM Interface for High-Performance PCs

    Science.gov (United States)

    Kim, Dong; Bang, Kwanhu; Ha, Seung-Hwan; Park, Chanik; Chung, Sung Woo; Chung, Eui-Young

    We propose a Solid-State Disk (SSD) with a Double Data Rate (DDR) DRAM interface for high-performance PCs. Traditional SSDs simply inherit the interface protocol of Hard Disk Drives (HDD) such as Parallel Advanced Technology Attachment (PATA) or Serial-ATA (SATA) for maintaining the compatibility. However, SSD itself provides much higher performance than HDD, hence the interface also needs to be enhanced. Unlike the traditional SSDs, the proposed SSD with DDR DRAM interface is placed in the North Bridge which provides two or more DDR DRAM interface ports in high-performance PCs. The novelty of our work is on DQS signaling scheme which allows arbitrary Column Address Strobe (CAS) latency unlike typical DDR DRAM interface scheme. The experimental results show that the proposed SSD maximally outperforms the traditional SSD by 8.7 times in read mode, by 1.5 times in write mode. Also, for synthetic workloads, the proposed scheme shows performance improvement over the conventional architecture by a factor of 1.6 times.

  17. Strain and plastic composite support (PCS) selection for vitamin K (Menaquinone-7) production in biofilm reactors.

    Science.gov (United States)

    Mahdinia, Ehsan; Demirci, Ali; Berenjian, Aydin

    2017-06-30

    Menaquinone-7 (MK-7), a subtype of vitamin K, has received a significant attention due to its effect on improving bone and cardiovascular health. Current fermentation strategies, which involve static fermentation without aeration or agitation, are associated with low productivity and scale-up issues and hardly justify the commercial production needs of this vitamin. Previous studies indicate that static fermentation is associated with pellicle and biofilm formations, which are critical for MK-7 secretion while posing significant operational issues. Therefore, the present study is undertaken to evaluate the possibility of using a biofilm reactor as a new strategy for MK-7 fermentation. Bacillus species, namely, Bacillus subtilis natto, Bacillus licheniformis, and Bacillus amyloliquifaciens as well as plastic composite, supports (PCS) were investigated in terms of MK-7 production and biofilm formation. Results show the possibility of using a biofilm reactor for MK-7 biosynthesis. Bacillus subtilis natto and soybean flour yeast extract PCS in glucose medium were found as the most potent combination for production of MK-7 as high as 35.5 mg/L, which includes both intracellular and extracellular MK-7.

  18. Induction of lead-binding phytochelatins in vetiver grass [Vetiveria zizanioides (L.)].

    Science.gov (United States)

    Andra, Syam S; Datta, Rupali; Sarkar, Dibyendu; Makris, Konstantinos C; Mullens, Conor P; Sahi, Shivendra V; Bach, Stephan B H

    2009-01-01

    Elevated lead (Pb) concentrations in residential houseyards around house walls painted with Pb-based pigments pose serious human health risks, especially to children. Vetiver grass (Vetiveria zizanioides L.) has shown promise for use in in situ Pb phytoremediation efforts. However, little is known about the biochemical mechanisms responsible for the observed high Pb tolerance by vetiver. We hypothesized that vetiver exposure to Pb induced the synthesis of phytochelatins (PC(n)) and the formation of Pb-PC(n) complexes, alleviating the phytotoxic effects of free Pb ions. Our main objective was to identify PC(n) and Pb-PC(n) complexes in root and shoot compartments of vetiver grass using high-performance liquid chromatography coupled to electrospray mass spectrometry (HPLC-ES-MS). After 7 d of exposure to Pb, vetiver accumulated up to 3000 mg Pb kg(-1) in shoot tissues, but much higher Pb concentrations were measured in root ( approximately 20,000 mg kg(-1)), without phytotoxic symptoms. Scanning electron micrographs showed Pb deposition in the vascular tissues of root and shoot, suggesting Pb translocation to shoot. Collision-induced dissociation analyses in MS/ MS mode during HPLC-ES-MS analysis allowed for the confirmation of four unique PC(n) (n = 1-4) based on their respective amino acid sequence. The high tolerance of vetiver grass to Pb was attributed to the formation of PC(n) and Pb-PC(n) complexes within the plant tissues, using ES-MS and Pb mass isotopic patterns. These data illustrate the mechanism of high Pb tolerance by vetiver grass, suggesting its potential usefulness for the remediation of Pb-contaminated residential sites.

  19. Lead tolerance mechanism in Conyza canadensis: subcellular distribution, ultrastructure, antioxidative defense system, and phytochelatins.

    Science.gov (United States)

    Li, Ying; Zhou, Chuifan; Huang, Meiying; Luo, Jiewen; Hou, Xiaolong; Wu, Pengfei; Ma, Xiangqing

    2016-03-01

    We used hydroponic experiments to examine the effects of different concentrations of lead (Pb) on the performance of the Pb-tolerable plant Conyza canadensis. In these experiments, most of the Pb was accumulated in the roots; there was very little Pb accumulated in stems and leaves. C. canadensis is able to take up significant amounts of Pb whilst greatly restricting its transportation to specific parts of the aboveground biomass. High Pb concentrations inhibited plant growth, increased membrane permeability, elevated antioxidant enzyme activity in roots, and caused a significant increase in root H2O2 and malondialdehyde content. Analysis of Pb content at the subcellular level showed that most Pb was associated with the cell wall fraction, followed by the nucleus-rich fraction, and with a minority present in the mitochondrial and soluble fractions. Furthermore, transmission electron microscopy and energy dispersive X-ray analysis of root cells revealed that the cell wall and intercellular space in C. canadensis roots are the main locations of Pb accumulation. Additionally, high Pb concentrations adversely affected the cellular structure of C. canadensis roots. The increased enzyme activity suggests that the antioxidant system may play an important role in eliminating or alleviating Pb toxicity in C. canadensis roots. However, the levels of non-protein sulfhydryl compounds, glutathione, and phytochelatin did not significantly change in either the roots or leaves under Pb-contaminated treatments. Our results provide strong evidence that cell walls restrict Pb uptake into the root and act as an important barrier protecting root cells, while demonstrating that antioxidant enzyme levels are correlated with Pb exposure. These findings demonstrate the roles played by these detoxification mechanisms in supporting Pb tolerance in C. canadensis.

  20. Can arsenic-phytochelatin complex formation be used as an indicator for toxicity in Helianthus annuus?

    Science.gov (United States)

    Raab, Andrea; Ferreira, Katia; Meharg, Andrew A; Feldmann, Jörg

    2007-01-01

    The formation of arsenic-phytochelatin (As-PC) complexes is thought to be part of the plant detoxification strategy for arsenic. This work examines (i) the arsenic (As) concentration-dependent formation of As-PC complex formation and (ii) redistribution and metabolism of As after arrested As uptake in Helianthus annuus. HPLC with parallel ICP-MS/ES-MS detection was used to identify and quantify the species present in plant extracts exposed to arsenate (As(V)) (between 0 and 66.7 micromol As l-1 for 24 h). At As concentrations below the EC50 value for root growth (22 micromol As l-1) As uptake is exponential, but it is reduced at concentrations above. Translocation between root and shoot seemed to be limited to the uptake phase of arsenic. No redistribution of As between root and shoot was observed after arresting As exposure. The formation of As-PC complexes was concentration-dependent. The amount and number of As-PC complexes increased exponentially with concentration up to 13.7 micromol As l-1. As(III)-PC3 and GS-As(III)-PC2 complexes were the dominant species in all samples. The ratio of PC-bound As to unbound As increased up to 1.3 micromol As l-1 and decreased at higher concentrations. Methylation of inorganic As was only a minor pathway in H. annuus with about 1% As methylated over a 32 d period. The concentration dependence of As-PC complex formation, amount of unbound reduced and oxidized PC2, and the relative uptake rate showed that As starts to influence the cellular metabolism of H. annuus negatively at As concentrations well below the EC50 value determined by more traditional means. Generally, As-PC complexes and PC-synthesis rate seem to be the more sensitive parameters to be studied when As toxicity values are to be estimated.

  1. Monoterpene synthases from common sage (Salvia officinalis)

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wise, Mitchell Lynn (Pullman, WA); Katahira, Eva Joy (Pullman, WA); Savage, Thomas Jonathan (Christchurch 5, NZ)

    1999-01-01

    cDNAs encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase from common sage (Salvia officinalis) have been isolated and sequenced, and the corresponding amino acid sequences has been determined. Accordingly, isolated DNA sequences (SEQ ID No:1; SEQ ID No:3 and SEQ ID No:5) are provided which code for the expression of (+)-bornyl diphosphate synthase (SEQ ID No:2), 1,8-cineole synthase (SEQ ID No:4) and (+)-sabinene synthase SEQ ID No:6), respectively, from sage (Salvia officinalis). In other aspects, replicable recombinant cloning vehicles are provided which code for (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase, or for a base sequence sufficiently complementary to at least a portion of (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene 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 (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant monoterpene synthases that may be used to facilitate their production, isolation and purification in significant amounts. Recombinant (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase may be used to obtain expression or enhanced expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase in plants in order to enhance the production of monoterpenoids, or may be otherwise employed for the regulation or expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase, or the production of their products.

  2. Carbon nanotubes and graphene modified screen-printed carbon electrodes as sensitive sensors for the determination of phytochelatins in plants using liquid chromatography with amperometric detection.

    Science.gov (United States)

    Dago, Àngela; Navarro, Javier; Ariño, Cristina; Díaz-Cruz, José Manuel; Esteban, Miquel

    2015-08-28

    Nanomaterials are of great interest for the development of electrochemical sensors. Multi-walled carbon nanotubes and graphene were used to modify the working electrode surface of different screen-printed carbon electrodes (SPCE) with the aim of improving the sensitivity of the SPCE and comparing it with the conventional glassy carbon electrode. To assay the usability of these sensors, a HPLC methodology with amperometric detection was developed to analyze several phytochelatins in plants of Hordeum vulgare and Glycine max treated with Hg(II) or Cd(II) giving detection limits in the low μmolL(-1) range. Phytochelatins are low molecular weight peptides with the general structure γ-(Glu-Cys)n-Gly (n=2-5) which are synthesized in plants in the presence of heavy metal ions. These compounds can chelate heavy metal ions by the formation of complexes which, are transported to the vacuoles, where the toxicity is not threatening. For this reason phytochelatins are essential in the detoxification of heavy metal ions in plants. The developed HPLC method uses a mobile phase of 1% of formic acid in water with KNO3 or NaCl (pH=2.00) and 1% of formic acid in acetonitrile. Electrochemical detection at different carbon-based electrodes was used. Among the sensors tested, the conventional glassy carbon electrode offers the best sensitivity although modification improves the sensitivity of the SPCE. Glutathione and several isoforms of phytochelatin two were found in plant extracts of both studied species.

  3. Melatonin mitigates cadmium phytotoxicity through modulation of phytochelatins biosynthesis, vacuolar sequestration and antioxidant potential in Solanum lycopersicum L.

    Directory of Open Access Journals (Sweden)

    Jie eZhou

    2015-08-01

    Full Text Available Melatonin is a ubiquitous signal molecule, playing crucial roles in plant growth and stress tolerance. Recently, toxic metal cadmium (Cd has been reported to regulate melatonin content in rice; however, the function of melatonin under Cd stress, particularly in higher plants, still remains elusive. Here, we show that optimal dose of melatonin could effectively ameliorate Cd-induced phytotoxicity in tomato. The contents of Cd and melatonin were gradually increased over time under Cd stress. However, such increase in endogenous melatonin was incapable to reverse detrimental effects of Cd. Meanwhile, supplementation with melatonin conferred Cd tolerance as evident by plant biomass and photosynthesis. In addition to notable increase in antioxidant enzymes activity, melatonin-induced Cd stress mitigation was closely associated with enhanced H+-ATPase activity and the contents of glutathione and phytochelatins. Although exogenous melatonin had no effect on root Cd content, it significantly reduced leaf Cd content, indicating its role in Cd transport. Analysis of Cd in different subcellular compartments revealed that melatonin increased cell wall and vacuolar fractions of Cd. Our results suggest that melatonin-induced enhancements in antioxidant potential, phytochelatins biosynthesis and subsequent Cd sequestration might play a critical role in plant tolerance to Cd. Such a mechanism may have potential implication in safe food production.

  4. Physiological analyses indicate superoxide dismutase, catalase, and phytochelatins play important roles in Pb tolerance in Eremochloa ophiuroides.

    Science.gov (United States)

    Li, Xi; Cen, Huameng; Chen, Youxiang; Xu, Siying; Peng, Lingli; Zhu, Hanmingyue; Li, Yiqiao

    2016-01-01

    Phytoremediation is considered to be a promising approach to restore or stabilize soil contaminated by lead (Pb). Turfgrasses, due to their high biomass yields, are considered to be suitable for use in phytoextraction of soil contaminated with heavy metal. It has been demonstrated that centipedegrass (Eremochloa ophiuroides (Munro) Hack., Poaceae) is a good turfgrass for restore of soil contaminated by Pb. However, the enhanced tolerant mechanisms in metallicolous (M) centipedegrass accessions remain unknown. In this study, we made a comparative study of growth performance, Pb accumulation, antioxidant levels, and phytochelatin concentrations in roots and shoots from M and nonmetallicolous (NM) centipedegrass accessions. Results showed that turf quality and growth rate were less repressed in M accessions than in NM accession. Pb stress caused generation of reactive oxygen species in centipedegrass with relatively lower levels in M accessions. Antioxidant activity analysis indicated that superoxide dismutase and catalase played important roles in Pb tolerance in M accessions. M accessions accumulated more Pb in roots and shoots. Greatly increased phytochelatins and less repressed sulfur contents in roots and shoots of M accessions indicated that they correlated with Pb accumulation and tolerance in centipedegrass.

  5. Melatonin mitigates cadmium phytotoxicity through modulation of phytochelatins biosynthesis, vacuolar sequestration, and antioxidant potential in Solanum lycopersicum L.

    Science.gov (United States)

    Hasan, Md Kamrul; Ahammed, Golam Jalal; Yin, Lingling; Shi, Kai; Xia, Xiaojian; Zhou, Yanhong; Yu, Jingquan; Zhou, Jie

    2015-01-01

    Melatonin is a ubiquitous signal molecule, playing crucial roles in plant growth and stress tolerance. Recently, toxic metal cadmium (Cd) has been reported to regulate melatonin content in rice; however, the function of melatonin under Cd stress, particularly in higher plants, still remains elusive. Here, we show that optimal dose of melatonin could effectively ameliorate Cd-induced phytotoxicity in tomato. The contents of Cd and melatonin were gradually increased over time under Cd stress. However, such increase in endogenous melatonin was incapable to reverse detrimental effects of Cd. Meanwhile, supplementation with melatonin conferred Cd tolerance as evident by plant biomass and photosynthesis. In addition to notable increase in antioxidant enzymes activity, melatonin-induced Cd stress mitigation was closely associated with enhanced H(+)-ATPase activity and the contents of glutathione and phytochelatins. Although exogenous melatonin had no effect on root Cd content, it significantly reduced leaf Cd content, indicating its role in Cd transport. Analysis of Cd in different subcellular compartments revealed that melatonin increased cell wall and vacuolar fractions of Cd. Our results suggest that melatonin-induced enhancements in antioxidant potential, phytochelatins biosynthesis and subsequent Cd sequestration might play a critical role in plant tolerance to Cd. Such a mechanism may have potential implication in safe food production.

  6. Capillary zone electrophoresis for analysis of phytochelatins and other thiol peptides in complex biological samples derivatized with monobromobimane.

    Science.gov (United States)

    Perez-Rama, Mónica; Torres Vaamonde, Enrique; Abalde Alonso, Julio

    2005-02-01

    A new method to improve the analysis of phytochelatins and their precursors (cysteine, gamma-Glu-Cys, and glutathione) derivatized with monobromobimane (mBrB) in complex biological samples by capillary zone electrophoresis is described. The effects of the background electrolyte pH, concentration, and different organic additives (acetonitrile, methanol, and trifluoroethanol) on the separation were studied to achieve optimum resolution and number of theoretical plates of the analyzed compounds in the electropherograms. Optimum separation of the thiol peptides was obtained with 150 mM phosphate buffer at pH 1.60. Separation efficiency was improved when 2.5% v/v methanol was added to the background electrolyte. The electrophoretic conditions were 13 kV and capillary dimensions with 30 cm length from the inlet to the detector (38 cm total length) and 50 microm inner diameter. The injection was by pressure at 50 mbar for 17 s. Under these conditions, the separation between desglycyl-peptides and phytochelatins was also achieved. We also describe the optimum conditions for the derivatization of biological samples with mBrB to increase electrophoretic sensitivity and number of theoretical plates. The improved method was shown to be simple, reproducible, selective, and accurate in measuring thiol peptides in complex biological samples, the detection limit being 2.5 microM glutathione at a wavelength of 390 nm.

  7. SIMATIC PCS7在CSTR中的设计方案及其应用%Design Scheme for SIMATIC PCS7 in CSTR and Its Application

    Institute of Scientific and Technical Information of China (English)

    张兴越; 杨国田; 陈峭; 秦宇飞

    2008-01-01

    过程工业常见的连续搅拌釜式反应系统(CSTR)其内部化学反应不稳定,直接影响生产的安全和效率,控制比较复杂.为实现对CSTR的稳定控制,介绍了应用传统控制算法而设计的针对CSTR的控制策略并通过仿真实验验证其性能.传统控制算法具有实现简单、控制性能稳定的优点,为了适应CSTR 化学反应的复杂性,本文灵活应用传统控制算法,严格控制反应物的进料时序,进而控制釜内反应的速度,实现CSTR 的安全稳定性和生产效率可控制性.同时,利用西门子新一代工业可编程控制器--SIMATIC PCS7 BOX作为控制器,在多功能过程控制仿真实验系统(MPCE-1000)上构建的CSTR仿真对象上实现稳定控制.仿真结果表明该控制策略在一定程度上能够适应CSTR 的复杂反应.

  8. Intra-operative Patient-Controlled Sedation (PCS:Propofol versus Midazolam Supplementation During Epidural Analgesia (Clinical and Hormonal Study

    Directory of Open Access Journals (Sweden)

    Hassan S Al-khayat

    2008-01-01

    Full Text Available This study was done on sixty adult males scheduled to have an epidural analgesia for elective inguinal hernia repair. The study was designed to compare propofol and midazolam with regard to their suitability for the patient-controlled sedation (PCS technique during epidural analgesia. Patients were divided into three equal groups and premedicated with 0.2mg.kg -1 oral midazolam. Group I (G1 served as control. Using PCS technique, the pump was programmed to deliver on demand a bolus dose of 0.5 mg.kg- 1 of propofol in Group II (G2 or 0.1mg.kg -1 midazolam in Group III(G3. Patient′s sedation status was assessed by sedation score, comfort scale and by psychometric testing. The total delivered dose of each tested drug was calculated. Serum concentrations of propfol and midazolam, plasma cortisol and free fatty acids were measured. Propofol and midazolam PCS technique produced excellent and easily controllable sedation. The dose needed to produce steady state sedation was 2.8±1.42 and 0.11±0.6 mg.kg -1 .h- 1 for propofol and midazolam respectively. Propofol was more suitable than midazolam for PCS because of its rapid onset, favorable recovery profile and low side effects. PCS proved to be a stress-free and acceptable technique.

  9. Prenyldiphosphate synthases and gibberellin biosynthesis

    NARCIS (Netherlands)

    van Schie, C.C.N.; Haring, M.A.; Schuurink, R.C.; Bach, T.J.; Rohmer, M.

    2013-01-01

    Gibberellins are derived from the diterpene precursor geranylgeranyl diphophosphate (GGPP). GGPP is converted to ent-kaurene, which contains the basic structure of gibberellins, in the plastids by the combined actions of copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). Generally, ge

  10. QSAR studies of bioconcentration factors of polychlorinated biphenyls (PCBs) using DFT, PCS and CoMFA.

    Science.gov (United States)

    Liu, Hui; Liu, Hongxia; Sun, Ping; Wang, Zunyao

    2014-11-01

    The bioconcentration factors (BCFs) of 58 polychlorinated biphenyls (PCBs) were modeled by quantitative structure-activity relationship (QSAR) using density functional theory (DFT), the position of Cl substitution (PCS) and comparative molecular field analysis (CoMFA) methods. All the models were robust and predictive, and especially, the best CoMFA model was significant with a correlation coefficient (R(2)) of 0.926, a cross-validation correlation coefficient (Q(2)) of 0.821 and a root mean square error estimated (RMSE) of 0.235. The results indicate that the electrostatic descriptors play a more significant role in BCFs of PCBs. Additionally, a test set was used to compare the predictive ability of our models to others, and results show that our CoMFA model present the lowest RMSE. Thus, the models obtain in this work can be used to predict the BCFs of remaining 152 PCBs without available experimental values.

  11. Bandwidth optimization of compact microstrip antenna for PCS/DCS/bluetooth application

    Science.gov (United States)

    Singh, Vinod; Ali, Zakir; Ayub, Shahanaz; Singh, Ashutosh

    2014-09-01

    A novel compact broadband microstrip patch antenna is presented for various wireless applications. The proposed antenna has been fabricated and the impedance bandwidth and radiation pattern are measured. The simulated and measured antenna characteristics along with radiation pattern and gain are presented. It is stated that the proposed designed antenna can completely cover the required band widths of Digital communication system (DCS 1.71-1.88 GHz), Personal communication system (PCS 1.85-1.88 GHz) and IEEE 802.11b/g (2.4-2.485 GHz) with satisfactory radiation characteristics. The Experimental result shows that the proposed antenna presents a bandwidth 60.25% covering the range of 1.431-2.665 GHz with the maximum radiation efficiency 90%.

  12. Synbiotic functional drink from Jerusalem artichoke juice fermented by probiotic Lactobacillus plantarum PCS26.

    Science.gov (United States)

    Dimitrovski, Darko; Velickova, Elena; Dimitrovska, Maja; Langerholc, Tomaz; Winkelhausen, Eleonora

    2016-01-01

    A probiotic strain Lactobacillus plantarum PCS26 was used to ferment Jerusalem artichoke juice. Growth kinetics of the bacterial strain was followed during juice fermentation both in flask and in laboratory fermentor. Jerusalem artichoke showed to be an excellent source of nutrients for L. plantarum PCS26 growth. The culture grew very well reaching more than 10(10) cfu/ml in just 12 h. The pH changed from the initial 6.5 to 4.6 at the end of fermentation. The culture hydrolyzed fructooligosaccharides present in the Jerusalem artichoke juice, yielding fructose which was presumably consumed along with the malic acid as energy and carbon source. Lactic acid was the main metabolite produced in concentration of 4.6 g/L. Acetic and succinic acid were also identified. Sensory evaluation of the fermented Jerusalem artichoke juice and its mixtures with blueberry juice showed that the 50/50 % v/v mixture would be very well accepted by the consumers. Above 80 % of the panelists would buy this drink, and over 60 % were willing to pay more for it. Culture survivability in the fermented juices during storage at 4-7 °C was assayed by the Weibullian model. The product shelf-life was extended from 19.70 ± 0.50 days of pure Jerusalem artichoke juice to 35.7 ± 6.4 days of the mixture containing 30 % blueberry juice.

  13. Design and Verification for PCS Layer of PCI Express Using VHDL

    Directory of Open Access Journals (Sweden)

    Hemant Kumar Soni Mr. Vikas Gupta Mrs. Deepti Agrawal

    2011-12-01

    Full Text Available The aim of this paper is to design and verify the physical layer implementation by using ISE 8.1 from Xilinx and Spartan 3 FPGA to reduce the cost and hardware as well without affecting the performance and reliability of PCI Express. PCI is the third generation high performance I/O bus used to interconnect peripheral devices in applications such as computing and communicating platform. PCI Express is an all in compassing I/O device interconnect bus that has application in the Mobile, Desktop, Workstation, Server, Embedded computing and communication platform. PCS is the sub layer of the physical layer of PCI Express 1.0. The major constituents of this layer are transmitter and receiver. Transmitter comprises of 8b/10b encoder. The Primary purpose of this scheme is to embed a clock into the serial bit stream of transmitter lanes with advancement in the design by reducing the utilized hardware resources within FPGA. No clock is transmitted along with the serial data bit stream. This eliminates EMI noise and provides DC balance. Receiver comprises of special symbol detector, elastic buffer and 8b/10b decoder. . 8b/10b decoder gives 8bit character and data/control signals. Disparity error and Decode error can be known though this module. If any error is present in the received data then loopback signal is generated. This work uses VHDL to model different blocks of the PCS of physical layer of PCI Express. The RTL code is simulated, synthesized and implemented using the ISE 8.1 from Xilinx and the Spartan 3 FPGA was targeted for implementation. In this paper we have reduced the hardware as well as cost of total system without affecting the speed of the PCI Express.

  14. Complexation of heavy metals by phytochelatins: voltammetric study of the binding of Cd2+ and Zn2+ ions by the phytochelatin (gamma-Glu-Cys)3Gly assisted by multivariate curve resolution.

    Science.gov (United States)

    Cruz, Boris H; Díaz-Cruz, José Manuel; Ariño, Cristina; Esteban, Miquel

    2005-02-01

    The complexation of Cd2+, Zn2+, and both together with the phytochelatin (gamma-Glu-Cys)3Gly is studied by differential pulse polarography, and data are analyzed by multivariate curve resolution by alternating least squares (MCR-ALS). MCR-ALS yields the respective unitary voltammograms and concentration profiles of the resolved components, which contain information on the relative stabilities and stoichiometries of the formed complexes. The analysis of these results shows, for the Cd2+/(gamma-Glu-Cys)3Gly system, the presence of different kinds of bound Cd2+. For the Zn2+/ (gamma-Glu-Cys)3Gly system, the poor definition of the reduction signals of the complexes prevents a clear discrimination among differently bound Zn2+ ions. Atentative complexation/ electrochemical model is proposed for when both metal ions, Cd2+ and Zn2+, compete toward complexation, and some of the corresponding equilibrium constants are estimated.

  15. Resource Allocation in a Frequency Hopping PCS1900/GSM/DCS1800 Type of Network

    DEFF Research Database (Denmark)

    Nielsen, Thomas Toftegaard; Wigard, Jeroen; Michaelsen, Per-Henrik

    1999-01-01

    Resource allocation in a frequency hopping network is even more problematic than in a traditional network. The combined effect from all serving frequencies has to be considered directly in the allocation process. An algorithm doing this for a PCS1900/GSM/DCS1800 type of network is presented...

  16. Cadmium binding in mixtures of phytochelatins and their fragments: a voltammetric study assisted by multivariate curve resolution and mass spectrometry.

    Science.gov (United States)

    Gusmão, Rui; Ariño, Cristina; Díaz-Cruz, José Manuel; Esteban, Miquel

    2010-01-01

    Phytochelatins (PC(n), (gamma-Glu-Cys)(n)Gly) are cysteine-rich peptides synthesized by plants which are involved in metal bioregulation and phytoremediation. Multivariate Curve Resolution by Alternating Least Squares (MCR-ALS) is applied to voltammetric data obtained from the analysis of the competitive binding of Cys or Cys-Gly with PC(2) or PC(3) by Cd(2+). The displacements between ligands, the chain length dependence on the competitive binding to PC(n) and the possible existence of mixed ligand metal-complexes are investigated. The shape analysis of the resulting pure voltammograms and concentration profiles of the components resolved by MCR-ALS suggests that ligands containing more thiol groups are able to displace the shorter chain ligands from their metal complexes, whereas the opposite does not happen. Electrochemical results are compared with ESI-MS measurements.

  17. Analysis of phytochelatin complexes in the lead tolerant vetiver grass [Vetiveria zizanioides (L.)] using liquid chromatography and mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Andra, Syam S., E-mail: syam.andra@gmail.co [Environmental Geochemistry Laboratory, University of Texas at San Antonio, One UTSA Circle, San Antonio, TX (United States); Datta, Rupali [Biological Sciences, Michigan Technological University, Houghton, MI (United States); Sarkar, Dibyendu [Department of Earth and Environmental Studies, Montclair State University, Montclair, NJ (United States); Saminathan, Sumathi K.M. [Environmental Geochemistry Laboratory, University of Texas at San Antonio, One UTSA Circle, San Antonio, TX (United States); Mullens, Conor P.; Bach, Stephan B.H. [Department of Chemistry, University of Texas at San Antonio, San Antonio, TX (United States)

    2009-07-15

    Ethylenediamene tetraacetic acid (EDTA) has been used to mobilize soil lead (Pb) and enhance plant uptake for phytoremediation. Chelant bound Pb is considered less toxic compared to free Pb ions and hence might induce less stress on plants. Characterization of possible Pb complexes with phytochelatins (PC{sub n}, metal-binding peptides) and EDTA in plant tissues will enhance our understanding of Pb tolerance mechanisms. In a previous study, we showed that vetiver grass (Vetiveria zizanioides L.) can accumulate up to 19,800 and 3350 mg Pb kg{sup -1} dry weight in root and shoot tissues, respectively; in a hydroponics set-up. Following the basic incubation study, a greenhouse experiment was conducted to elucidate the efficiency of vetiver grass (with or without EDTA) in remediating Pb-contaminated soils from actual residential sites where Pb-based paints were used. The levels of total thiols, PC{sub n}, and catalase (an antioxidant enzyme) were measured in vetiver root and shoot following chelant-assisted phytostabilization. In the presence of 15 mM kg {sup -1} EDTA, vetiver accumulated 4460 and 480 mg Pb kg{sup -1} dry root and shoot tissue, respectively; that are 15- and 24-fold higher compared to those in untreated controls. Despite higher Pb concentrations in the plant tissues, the amount of total thiols and catalase activity in EDTA treated vetiver tissues was comparable to chelant unamended controls, indicating lowered Pb toxicity by chelation with EDTA. The identification of glutathione (referred as PC{sub 1}) (m/z 308.2), along with chelated complexes like Pb-EDTA (m/z 498.8) and PC{sub 1}-Pb-EDTA (m/z 805.3) in vetiver root tissue using electrospray tandem mass spectrometry (ES-MS) highlights the possible role of such species towards Pb tolerance in vetiver grass. - Chelated lead in conjunction with phytochelatins synthesis and complexation reduces stress in the lead tolerant vetiver grass.

  18. Catalytic performance of symmetrical and unsymmetrical sulfur-containing pincer complexes: synthesis and tandem catalytic activity of the first PCS-pincer palladium complex

    NARCIS (Netherlands)

    Gagliardo, M.; Selander, N.; Mehendale, N.C.; van Koten, G.; Klein Gebbink, R.J.M.; Szabó, K.J.

    2008-01-01

    The synthesis and catalytic applications of a new aryl-based unsymmetrical PCS-pincer complex are reported. Preparation of the robust air- and moisture-stable PCS-pincer palladium complex 5[X] started from the symmetrical ,-dibromo-meta-xylene and involved the selective substitution of one bromide b

  19. Using wireless (Pocket)PCs in Large Introductory Courses to Expand Discourse and Interactivity

    Science.gov (United States)

    van der Pluijm, B. A.; Knoop, P. A.; Samson, P. J.; Teasley, S. D.

    2005-12-01

    Teaching methods in introductory, undergraduate courses traditionally rely on static textbooks and/or course packs, with presentation delivered as a monologue in front of a mostly passive, large audience. The concepts presented in class are often best illustrated using visualizations and/or demonstrations, but even the most stunning of images or spectacular exhibits, while motivating, offer students only passive participation in the learning process. Add to this the advent of course websites with lecture notes and PowerPoint presentations and the students are left with little incentive to attend, much less participate. Clearly this model does not provide much opportunity or motivation for today's students to learn and think more critically about the arguments being developed. What is needed is a coupling of the rich imagery of many fields with advances in technology and in learning, toward revitalizing pedagogical approaches in survey-level courses and student-instructor interaction. Our IT-enhanced classroom project couples the use of peer instruction techniques in large classes (as originally described by Mazur, 1997) with the use of interactive spatial concept challenges, utilizing wireless PocketPCs (handhelds) or student-owned wireless-enabled laptops. The technologies employed (web, PocketPC/laptop, WiFi) are off-the-shelf technologies and the Peer Instruction technique is increasingly documented in undergraduate science classes. However, the combination is not employed due to its initial cost, wrongly perceived level of effort to implement, availability of engaging activities and modest volume of data on student learning. We'll show our development, implementation and preliminary cognitive assessment efforts of this IT-enhanced classroom experience, involving interactive image quizzes and data manipulation in large introductory classes at the University of Michigan.

  20. Properties of phosphorylated thymidylate synthase

    DEFF Research Database (Denmark)

    Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr;

    2015-01-01

    Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichin......Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat...

  1. Biphenyl synthase, a novel type III polyketide synthase.

    Science.gov (United States)

    Liu, B; Raeth, T; Beuerle, T; Beerhues, L

    2007-05-01

    Biphenyls and dibenzofurans are the phytoalexins of the Maloideae, a subfamily of the economically important Rosaceae. The carbon skeleton of the two classes of antimicrobial secondary metabolites is formed by biphenyl synthase (BIS). A cDNA encoding this key enzyme was cloned from yeast-extract-treated cell cultures of Sorbus aucuparia. BIS is a novel type III polyketide synthase (PKS) that shares about 60% amino acid sequence identity with other members of the enzyme superfamily. Its preferred starter substrate is benzoyl-CoA that undergoes iterative condensation with three molecules of malonyl-CoA to give 3,5-dihydroxybiphenyl via intramolecular aldol condensation. BIS did not accept CoA-linked cinnamic acids such as 4-coumaroyl-CoA. This substrate, however, was the preferential starter molecule for chalcone synthase (CHS) that was also cloned from S. aucuparia cell cultures. While BIS expression was rapidly, strongly and transiently induced by yeast extract treatment, CHS expression was not. In a phylogenetic tree, BIS grouped together closely with benzophenone synthase (BPS) that also uses benzoyl-CoA as starter molecule but cyclizes the common intermediate via intramolecular Claisen condensation. The molecular characterization of BIS thus contributes to the understanding of the functional diversity and evolution of type III PKSs.

  2. Genetics Home Reference: GM3 synthase deficiency

    Science.gov (United States)

    ... Facebook Share on Twitter Your Guide to Understanding Genetic Conditions Search MENU Toggle navigation Home Page Search ... Conditions Genes Chromosomes & mtDNA Resources Help Me Understand Genetics Home Health Conditions GM3 synthase deficiency GM3 synthase ...

  3. Mycocerosic acid synthase exemplifies the architecture of reducing polyketide synthases.

    Science.gov (United States)

    Herbst, Dominik A; Jakob, Roman P; Zähringer, Franziska; Maier, Timm

    2016-03-24

    Polyketide synthases (PKSs) are biosynthetic factories that produce natural products with important biological and pharmacological activities. Their exceptional product diversity is encoded in a modular architecture. Modular PKSs (modPKSs) catalyse reactions colinear to the order of modules in an assembly line, whereas iterative PKSs (iPKSs) use a single module iteratively as exemplified by fungal iPKSs (fiPKSs). However, in some cases non-colinear iterative action is also observed for modPKSs modules and is controlled by the assembly line environment. PKSs feature a structural and functional separation into a condensing and a modifying region as observed for fatty acid synthases. Despite the outstanding relevance of PKSs, the detailed organization of PKSs with complete fully reducing modifying regions remains elusive. Here we report a hybrid crystal structure of Mycobacterium smegmatis mycocerosic acid synthase based on structures of its condensing and modifying regions. Mycocerosic acid synthase is a fully reducing iPKS, closely related to modPKSs, and the prototype of mycobacterial mycocerosic acid synthase-like PKSs. It is involved in the biosynthesis of C20-C28 branched-chain fatty acids, which are important virulence factors of mycobacteria. Our structural data reveal a dimeric linker-based organization of the modifying region and visualize dynamics and conformational coupling in PKSs. On the basis of comparative small-angle X-ray scattering, the observed modifying region architecture may be common also in modPKSs. The linker-based organization provides a rationale for the characteristic variability of PKS modules as a main contributor to product diversity. The comprehensive architectural model enables functional dissection and re-engineering of PKSs.

  4. The construction of an engineered bacterium to remove cadmium from wastewater.

    Science.gov (United States)

    Chang, S; Shu, H

    2014-01-01

    The removal of cadmium (Cd) from wastewater before it is released from factories is important for protecting human health. Although some researchers have developed engineered bacteria, the resistance of these engineered bacteria to Cd have not been improved. In this study, two key genes involved in glutathione synthesis (gshA and gshB), a serine acetyltransferase gene (cysE), a Thlaspi caerulescens phytochelatin synthase gene (TcPCS1), and a heavy metal ATPase gene (TcHMA3) were transformed into Escherichia coli BL21. The resistance of the engineered bacterium to Cd was significantly greater than that of the initial bacterium and the Cd accumulation in the engineered bacterium was much higher than in the initial bacterium. In addition, the Cd resistance of the bacteria harboring gshB, gshA, cysE, and TcPCS1 was higher than that of the bacteria harboring gshA, cysE, and TcPCS1. This finding demonstrated that gshB played an important role in glutathione synthesis and that the reaction catalyzed by glutathione synthase was the limiting step for producing phytochelatins. Furthermore, TcPCS1 had a greater specificity and a higher capacity for removing Cd than SpPCS1, and TcHMA3 not only played a role in T. caerulescens but also functioned in E. coli.

  5. Biosynthesis of phytochelatins and arsenic accumulation in the marine microalga Phaeodactylum tricornutum in response to arsenate exposure.

    Science.gov (United States)

    Morelli, Elisabetta; Mascherpa, Marco Carlo; Scarano, Gioacchino

    2005-12-01

    The arsenate-induced synthesis of phytochelatins (PC), intracellular cysteine-rich metal-binding peptides, and its relationship with toxicity and with As accumulation in the cell have been studied in laboratory cultures of the marine microalga Phaeodactylum tricornutum. The time course of cellular PC and As in short-term exposures showed that the involvement of PC in the As detoxification as well as the pathway of cellular As depend on the extent of As accumulation and on the rate of PC synthesis. At arsenate concentrations causing As accumulation at a rate exceeding that of PC synthesis, cells seem to activate a mechanism of release of As mainly in a chemical form not complexed with PC. At arsenate concentrations at which the synthesis of PC occurs at a rate sufficient to allow a significant portion of As accumulated in the cell to be bound, the fate of cellular As seems to be mainly controlled by PC. The occurrence of these different pathways of As detoxification was discussed to explain the pattern of cellular As and PC in cells grown for three days at growth-inhibitory and at no growth-inhibitory concentration of arsenate.

  6. Elucidation of the defence mechanism in microalgae Chlorella sorokiniana under mercury exposure. Identification of Hg-phytochelatins.

    Science.gov (United States)

    Gómez-Jacinto, Verónica; García-Barrera, Tamara; Gómez-Ariza, José Luis; Garbayo-Nores, Inés; Vílchez-Lobato, Carlos

    2015-08-01

    Algae and aquatic macrophytes are capable of accumulating heavy metals up to concentrations several orders of magnitude higher than those existing in their surrounding environment. Investigation of mercury toxicology in microalgae is of great interest from ecological point of view, since they could be used as bioindicator to evaluate aquatic ecosystems affected by Hg pollution. In this study, we have performed an exposure experiment focused on the biological response of microalgae Chlorella sorokiniana, a unicellular model organism, to Hg-induced toxicity. The culture was exposed to different concentrations of this element for nine days, namely 0.5, 1, 5 and 10mg L(-1) of HgCl2 (as Hg). To achieve a better understanding of the biological mechanisms triggered by Hg-induced toxicity in this alga a metallomic approach based on SEC-ICP-ORS-MS was applied to survey biomarkers of biological response to mercury contamination in surface water. In addition, the combination of RP-HPLC-ICP-ORS-MS and RP-HPLC-ESI-QqQ-TOF-MS was applied to identify, for the first time, two Hg-binding phytochelatins in this aquatic organism, using cell extracts from microalgae exposed to inorganic mercury.

  7. Accumulation of arsenic, lead, copper, and zinc, and synthesis of phytochelatins by indigenous plants of a mining impacted area.

    Science.gov (United States)

    Machado-Estrada, Blenda; Calderón, Jaqueline; Moreno-Sánchez, Rafael; Rodríguez-Zavala, José S

    2013-06-01

    Several native plants, able to grow in an unconfined mining impacted area that is now in close vicinity with urban areas, were evaluated for their ability to accumulate heavy metals. The main soil contaminants were As, Pb, Cu, and Zn. Sampling of the rhizospheric metal polluted soil showed that Euphorbia prostrata Aiton, Parthenium incanum Kunth, and Zinnia acerosa (DC.) A. Gray were able to grow in the presence of high amounts of mixtures of these elements. The plants accumulated the metals in the above ground parts and increased the synthesis of thiol molecules. E. prostrata showed the highest capacity for accumulation of the mixture of elements (588 μg g DW(-1)). Analysis of the thiol-molecules profile showed that these plants synthesized high amounts of long-chain phytochelatins, accompanied by low amounts of monothiol molecules, which may be related to their higher resistance to As and heavy metals. The three plants showed translocation factors from roots to leaves >1 for As, Pb, Cu, and Zn. Thus, by periodically removing aerial parts, these plants could be useful for the phytoremediation of semi-arid and arid mining impacted areas, in which metal hyper-accumulator plants are not able to grow.

  8. Sphingomyelin synthase SMS2 displays dual activity as ceramide phosphoethanolamine synthase[S

    Science.gov (United States)

    Ternes, Philipp; Brouwers, Jos F. H. M.; van den Dikkenberg, Joep; Holthuis, Joost C. M.

    2009-01-01

    Sphingolipids are vital components of eukaryotic membranes involved in the regulation of cell growth, death, intracellular trafficking, and the barrier function of the plasma membrane (PM). While sphingomyelin (SM) is the major sphingolipid in mammals, previous studies indicate that mammalian cells also produce the SM analog ceramide phosphoethanolamine (CPE). Little is known about the biological role of CPE or the enzyme(s) responsible for CPE biosynthesis. SM production is mediated by the SM synthases SMS1 in the Golgi and SMS2 at the PM, while a closely related enzyme, SMSr, has an unknown biochemical function. We now demonstrate that SMS family members display striking differences in substrate specificity, with SMS1 and SMSr being monofunctional enzymes with SM and CPE synthase activity, respectively, and SMS2 acting as a bifunctional enzyme with both SM and CPE synthase activity. In agreement with the PM residency of SMS2, we show that both SM and CPE synthase activities are enhanced at the surface of SMS2-overexpressing HeLa cells. Our findings reveal an unexpected diversity in substrate specificity among SMS family members that should enable the design of specific inhibitors to target the biological role of each enzyme individually. PMID:19454763

  9. Effects assessment of 10 functioning years on the main components of the molten salt PCS experimental facility of ENEA

    Science.gov (United States)

    Gaggioli, Walter; Di Ascenzi, Primo; Rinaldi, Luca; Tarquini, Pietro; Fabrizi, Fabrizio

    2016-05-01

    In the frame of the Solar Thermodynamic Laboratory, ENEA has improved CSP Parabolic Trough technologies by adopting new advanced solutions for linear tube receivers and by implementing a binary mixture of molten salt (60% NaNO3 and 40% KNO3) [1] as both heat transfer fluid and heat storage medium in solar field and in storage tanks, thus allowing the solar plants to operate at high temperatures up to 550°C. Further improvements have regarded parabolic mirror collectors, piping and process instrumentation. All the innovative components developed by ENEA, together with other standard parts of the plant, have been tested and qualified under actual solar operating conditions on the PCS experimental facility at the ENEA Casaccia Research Center in Rome (Italy). The PCS (Prova Collettori Solari, i.e. Test of Solar Collectors) facility is the main testing loop built by ENEA and it is unique in the world for what concerns the high operating temperature and the fluid used (mixture of molten salt). It consists in one line of parabolic trough collectors (test section of 100 m long life-size solar collectors) using, as heat transfer fluid, the aforesaid binary mixture of molten salt up to 10 bar, at high temperature in the range 270° and 550°C and a flow rate up to 6.5 kg/s. It has been working since early 2004 [2] till now; it consists in a unique closed loop, and it is totally instrumented. In this paper the effects of over ten years qualification tests on the pressurized tank will be presented, together with the characterization of the thermal losses of the piping of the molten salt circuit, and some observations performed on the PCS facility during its first ten years of operation.

  10. Effect of heparin on hemorheology and inflammatory cytokines in patients with spinal trauma after PCS pedicle screw fixation

    Institute of Scientific and Technical Information of China (English)

    Nan-Xiang Huang; Hong Lin; Wei Li

    2016-01-01

    Objective:To explore the effect of low molecular weight heparin (LMWH) on the hemorheology, inflammatory cytokines, and coagulation funtion in patients with spinal trauma after PCS pedicle screw fixation.Methods: A total of 80 patients with spinal trauma who were admitted in our hospital were included in the study and randomized into the treatment group and the control group with 40 cases in each group. The patients in the two groups were performed with PCS pedicle screw fixation. The patients in the treatment group were given subcutaneous injection of LMWH after operation. The hemorheology indicators, inflammatory cytokines levels, and coagulation function change before and after operation in the two groups were compared.Results: The difference of RBV (low shear, middle shear, and high shear) (mPa•s), PAR, and PAR (1 min) (%) before operation between the two groups was not statistically significant (P>0.05), the above indicators 24 h after operation in the two groups were significantly elevated when compared with before operation (P<0.05), and the above indicators 24 h after operation in the treatment group were significantly lower than those in the control group (P<0.05). The above indicators 48 h and 1 week after operation in the control group were significantly elevated, while those in the treatment group were significantly reduced when compared with before treatment (P<0.05). CRP, IL-6, and IL-10 levels after operation in the treatment group were significantly lower than those in the control group (P<0.05). PT, APTT, and FIB after operation in the treatment group were significantly lower than those in the control group (P<0.05).Conclusions:LMWH can effectively reduce the blood viscosity, inhibit the inflammatory reaction, and contribute to postoperative rehabilitation in patients with spinal trauma after PCS pedicle screw fixation.

  11. Application of Siemens PCS 7 in The 6500T Ammonium Molybdate Process%西门子PCS7系统在6500T钼酸铵工艺中的应用

    Institute of Scientific and Technical Information of China (English)

    聂巨峰

    2013-01-01

    This paper introduces the process of the 6500T new ammonium molybdate, describes applications of Siemens S7-400H PLC, WINCC V6.0, ET200M,Y-LINK,MicroMaster 430 series converter , ABB ACS800 series converter and SIMOCODE pro etc in the control part of the 6500T ammonium molybdate process, it is first applied in domestic; and highlightes applications of Siemens PCS7 advanced function library.%本文简要介绍了4酸铵的工艺流程,描述了Siemens S7-400H PLC、WINCC V6.0、ET200M、Y-LINK、MicroMaster430系列变频器、ABB ACS800变频器和SIMOCODE pro等产品在国内首家6500T钼酸铵工艺中控制部分的应用,重点介绍了Siemens PCS7高级功能库的应用.

  12. Determination of cadmium and lead species and phytochelatins in pea (Pisum sativum) by HPLC-ICP-MS and HPLC-ESI-MSn.

    Science.gov (United States)

    Barałkiewicz, Danuta; Kózka, Małgorzata; Piechalak, Aneta; Tomaszewska, Barbara; Sobczak, Paweł

    2009-07-15

    An analytical approach based on hyphenated techniques was used for studying the speciation of cadmium and lead in Pisum sativum. Proper preservation conditions were employed to avoid the oxidation of -SH groups and corresponding decomposition of metal-binding complexes. SEC column was washed with 5 mM beta-mercaptoethanol and then samples were analysed using ICP-MS as a detector. Results showed that cadmium is the inhibitor of lead uptake. HPLC-ESI-MS(n) assays revealed fragmentation pathways of phytochelatins.

  13. Cd-induced phytochelatin synthesis in Dittrichia viscosa (L.) Greuter is determined by the dilution of the culture medium.

    Science.gov (United States)

    Fernández, R; Fernández-Fuego, D; Rodríguez-González, P; Alonso, J I García; Bertrand, A; González, A

    2014-01-01

    In this paper, we examined Cd accumulation and PC synthesis in two clones of Dittrichia viscosa, one with a metallicolous (DV-A) and the other with a non-metallicolous origin (DV-W). The clones were cultured in vitro with 0 and 10 mg Cd L(-1) in both short-term treatments (up to 72 h) and over 10 days. We also examined the influence of the culture medium dilution and the PC-synthesis inhibitor, L-buthionine-sulfoximine (BSO), on these parameters. Similar Cd accumulation values were found in the two clones. No synthesis of new thiolic compounds was observed in Cd-treated plants cultured in vitro in Murashige and Skoog medium up to 72 h when compared to controls. Dilution of the culture medium affected PC production, increasing it in 1/2 MS and especially in 1/4 MS. Cd uptake did not increase in the same way, but still hyperaccumulation levels were exceeded in all Cd treatments. BSO addition increased the sensitivity of D. viscosa to Cd and diminished Cd accumulation. Nevertheless, a poor correlation between PCs and Cd accumulation capacity was observed since the highest Cd content did not correspond to the highest PC levels. All these results obtained suggest that PCs are important in Cd accumulation and detoxification in D. viscosa and also that other mechanisms might be involved in these traits.

  14. Toward Automated FAÇADE Texture Generation for 3d Photorealistic City Modelling with Smartphones or Tablet Pcs

    Science.gov (United States)

    Wang, S.

    2012-07-01

    An automated model-image fitting algorithm is proposed in this paper for generating façade texture image from pictures taken by smartphones or tablet PCs. The façade texture generation requires tremendous labour work and thus, has been the bottleneck of 3D photo-realistic city modelling. With advanced developments of the micro electro mechanical system (MEMS), camera, global positioning system (GPS), and gyroscope (G-sensors) can all be integrated into a smartphone or a table PC. These sensors bring the possibility of direct-georeferencing for the pictures taken by smartphones or tablet PCs. Since the accuracy of these sensors cannot compared to the surveying instruments, the image position and orientation derived from these sensors are not capable of photogrammetric measurements. This paper adopted the least-squares model-image fitting (LSMIF) algorithm to iteratively improve the image's exterior orientation. The image position from GPS and the image orientation from gyroscope are treated as the initial values. By fitting the projection of the wireframe model to the extracted edge pixels on image, the image exterior orientation elements are solved when the optimal fitting achieved. With the exact exterior orientation elements, the wireframe model of the building can be correctly projected on the image and, therefore, the façade texture image can be extracted from the picture.

  15. Analysis of phytochelatin complexes in the lead tolerant vetiver grass [Vetiveria zizanioides (L.)] using liquid chromatography and mass spectrometry.

    Science.gov (United States)

    Andra, Syam S; Datta, Rupali; Sarkar, Dibyendu; Saminathan, Sumathi K M; Mullens, Conor P; Bach, Stephan B H

    2009-07-01

    Ethylenediamene tetraacetic acid (EDTA) has been used to mobilize soil lead (Pb) and enhance plant uptake for phytoremediation. Chelant bound Pb is considered less toxic compared to free Pb ions and hence might induce less stress on plants. Characterization of possible Pb complexes with phytochelatins (PCn, metal-binding peptides) and EDTA in plant tissues will enhance our understanding of Pb tolerance mechanisms. In a previous study, we showed that vetiver grass (Vetiveria zizanioides L.) can accumulate up to 19,800 and 3350 mg Pb kg(-1) dry weight in root and shoot tissues, respectively; in a hydroponics set-up. Following the basic incubation study, a greenhouse experiment was conducted to elucidate the efficiency of vetiver grass (with or without EDTA) in remediating Pb-contaminated soils from actual residential sites where Pb-based paints were used. The levels of total thiols, PCn, and catalase (an antioxidant enzyme) were measured in vetiver root and shoot following chelant-assisted phytostabilization. In the presence of 15 mM kg (-1) EDTA, vetiver accumulated 4460 and 480 mg Pb kg(-1) dry root and shoot tissue, respectively; that are 15- and 24-fold higher compared to those in untreated controls. Despite higher Pb concentrations in the plant tissues, the amount of total thiols and catalase activity in EDTA treated vetiver tissues was comparable to chelant unamended controls, indicating lowered Pb toxicity by chelation with EDTA. The identification of glutathione (referred as PC1) (m/z 308.2), along with chelated complexes like Pb-EDTA (m/z 498.8) and PC(1)-Pb-EDTA (m/z 805.3) in vetiver root tissue using electrospray tandem mass spectrometry (ES-MS) highlights the possible role of such species towards Pb tolerance in vetiver grass.

  16. Enhanced arsenate reduction by a CDC25-like tyrosine phosphatase explains increased phytochelatin accumulation in arsenate-tolerant Holcus lanatus.

    Science.gov (United States)

    Bleeker, Petra M; Hakvoort, Henk W J; Bliek, Mattijs; Souer, Erik; Schat, Henk

    2006-03-01

    Decreased arsenate [As(V)] uptake is the major mechanism of naturally selected As(V) hypertolerance in plants. However, As(V)-hypertolerant ecotypes also show enhanced rates of phytochelatin (PC) accumulation, suggesting that improved sequestration might additionally contribute to the hypertolerance phenotype. Here, we show that enhanced PC-based sequestration in As(V)-hypertolerant Holcus lanatus is not due to an enhanced capacity for PC synthesis as such, but to increased As(V) reductase activity. Vacuolar transport of arsenite-thiol complexes was equal in both ecotypes. Based on homology with the yeast As(V) reductase, Acr2p, we identified a Cdc25-like plant candidate, HlAsr, and confirmed the As(V) reductase activity of both HlAsr and the homologous protein from Arabidopsis thaliana. The gene appeared to be As(V)-inducible and its expression was enhanced in the As(V)-hypertolerant H. lanatus ecotype, compared with the non-tolerant ecotype. Homologous ectopic overexpression of the AtASR cDNA in A. thaliana produced a dual phenotype. It improved tolerance to mildly toxic levels of As(V) exposure, but caused hypersensitivity to more toxic levels. Arabidopsis asr T-DNA mutants showed increased As(V) sensitivity at low exposure levels and enhanced arsenic retention in the root. It is argued that, next to decreased uptake, enhanced expression of HlASR might act as an additional determinant of As(V) hypertolerance and As transport in H. lanatus.

  17. Electrochemical behavior of phytochelatins and related peptides at the hanging mercury drop electrode in the presence of cobalt(II) ions.

    Science.gov (United States)

    Dorcák, Vlastimil; Sestáková, Ivana

    2006-01-01

    Direct current voltammetry and differential pulse voltammetry have been used to investigate the electrochemical behaviour of two phytochelatins: heptapeptide (gamma-Glu-Cys)3-Gly and pentapeptide (gamma-Glu-Cys)2-Gly, tripeptide glutathione gamma-Glu-Cys-Gly and its fragments: dipeptides Cys-Gly and gamma-Glu-Cys at the hanging mercury drop electrode in the presence of cobalt(II) ions. Most interesting results were obtained with direct current voltammetry in the potential region of -0.80 V up to -1.80 V. Differential pulse voltammetry of the same solutions of Co(II) with peptides gives more complicated voltammograms with overlapping peaks, probably in connection with the influence of adsorption at slow scan rates necessarily used in this method. However, in using Brdicka catalytic currents for analytical purposes, differential pulse voltammograms seem to be more helpful. Presented investigations have shown that particularly the prewave of cobalt(II) allows distinguishing among phytochelatins, glutathione, and its fragments.

  18. Producing biofuels using polyketide synthases

    Science.gov (United States)

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2013-04-16

    The present invention provides for a non-naturally occurring polyketide synthase (PKS) capable of synthesizing a carboxylic acid or a lactone, and a composition such that a carboxylic acid or lactone is included. The carboxylic acid or lactone, or derivative thereof, is useful as a biofuel. The present invention also provides for a recombinant nucleic acid or vector that encodes such a PKS, and host cells which also have such a recombinant nucleic acid or vector. The present invention also provides for a method of producing such carboxylic acids or lactones using such a PKS.

  19. Design and Realize CSTR Control System Based on Siemens PCS7%基于西门子PCS7的搅拌反应釜连续反应控制系统

    Institute of Scientific and Technical Information of China (English)

    董立新; 万小华; 顾幸生

    2011-01-01

    针对搅拌反应釜连续反应过程,采用多功能过程和实验系统(MPCE)中的连续搅拌反应过程(CSTR)进行控制系统设计。进料流量采用比值控制,液位采用串级控制,升温过程采用预测模糊控制,恒温阶段采用模糊免疫PID控制,并设计了压力安全联锁控制系统。基于西门子PCS7产品进行工程实现,运行结果表明:控制系统中各种控制策略操作简单,控制效果、安全性和可行性较好。%For the reaction process in the continuous stirred tank reactor,the continuous stirred tank reactor(CSTR) in multifunction process and experiment system is utilized to design the control system.The ratio control is adopted for flow,and the cascade control is utilized for level.Besides,the process of rising temperature is controlled via fuzzy predictive method,and the stage of constant temperature is controlled by using fuzzy immunity PID mehtod.Moroever,safety interlock control for pressure is also designed.The above project is implemented by means of SIMATIC PCS7,which attain better control performance,security,and feasibility.

  20. High-Quality Learning Environments for Engineering Design: Using Tablet PCs and Guidelines from Research on How People Learn

    Directory of Open Access Journals (Sweden)

    Enrique Palou

    2012-04-01

    Full Text Available A team of several faculty members and graduate students at Universidad de las Amricas Puebla is improving engineering design teaching and learning by creating richer learning environments that promote an interactive classroom while integrating formative assessment into classroom practices by means of Tablet PCs and associated technologies. Learning environments that are knowledge-, learner-, community-, and assessment-centered as highlighted by the How People Learn framework, have been developed. To date, the redesign of the undergraduate course entitled Introduction to Engineering Design has signicantly (p<0.05 increased student participation; formative assessment and feedback are more common and rapid; and instructors are utilizing the information gained through real-time formative assessments to tailor instruction to meet student needs. Particularly important have been opportunities to make student thinking visible and to give them chances to revise, as well as opportunities for "what if" thinking.

  1. Heterooligomeric phosphoribosyl diphosphate synthase of Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    2004-01-01

    The yeast Saccharomyces cerevisiae contains five phosphoribosyl diphosphate (PRPP) synthase-homologous genes (PRS1-5), which specify PRPP synthase subunits 1-5. Expression of the five S. cerevisiae PRS genes individually in an Escherichia coli PRPP-less strain (Deltaprs) showed that a single PRS...

  2. Molecular evolution and sequence divergence of plant chalcone synthase and chalcone synthase-Like genes.

    Science.gov (United States)

    Han, Yingying; Zhao, Wenwen; Wang, Zhicui; Zhu, Jingying; Liu, Qisong

    2014-06-01

    Plant chalcone synthase (CHS) and CHS-Like (CHSL) proteins are polyketide synthases. In this study, we evaluated the molecular evolution of this gene family using representative types of CHSL genes, including stilbene synthase (STS), 2-pyrone synthase (2-PS), bibenzyl synthase (BBS), acridone synthase (ACS), biphenyl synthase (BIS), benzalacetone synthase, coumaroyl triacetic acid synthase (CTAS), and benzophenone synthase (BPS), along with their CHS homologs from the same species of both angiosperms and gymnosperms. A cDNA-based phylogeny indicated that CHSLs had diverse evolutionary patterns. STS, ACS, and 2-PS clustered with CHSs from the same species (late diverged pattern), while CTAS, BBS, BPS, and BIS were distant from their CHS homologs (early diverged pattern). The amino-acid phylogeny suggested that CHS and CHSL proteins formed clades according to enzyme function. The CHSs and CHSLs from Polygonaceae and Arachis had unique evolutionary histories. Synonymous mutation rates were lower in late diverged CHSLs than in early diverged ones, indicating that gene duplications occurred more recently in late diverged CHSLs than in early diverged ones. Relative rate tests proved that late diverged CHSLs had unequal rates to CHSs from the same species when using fatty acid synthase, which evolved from the common ancestor with the CHS superfamily, as the outgroup, while the early diverged lineages had equal rates. This indicated that late diverged CHSLs experienced more frequent mutation than early diverged CHSLs after gene duplication, allowing obtaining new functions in relatively short period of time.

  3. Design of DCS System for Copper and Cobalt Recovery Process Based on PCS7%基于PCS7的铜钴回收冗余控制系统设计

    Institute of Scientific and Technical Information of China (English)

    巩运迎; 吴明亮; 宗秀玲; 郜鹏鹏

    2012-01-01

    According to a new technology of copper and cobalt recovery was still in the manual operation, low degree of automation, design of copper and cobalt recovery redundancy control system based on Siemens PCS7 process control system to the entire production process automatic monitoring,for copper cobalt recovery thickener process of complicated mechanism, poor operating environment, has great inertia, nonlinear and material properties of wave analysis of frequent faults, design thickener structure, parametes self -turning fuzzy PID control. In Qinghai, mining group500000 tons of copper and cobalt recovery projects in the practical application proves that the control system can be very good to meet the control requirement, the comprehensive recovery efficiency improved, greatly reduce the labor intensity of the workers,the realization of safe and stable and reliable automated production.%针对新型铜钴回收工艺仍然处于人工手动操作、自动化程度低的问题,该文在西门子PCS7过程控制系统下设计铜钴回收冗余控制系统对整个生产流程进行自动化监控,针对铜钴回收浓密机工艺机理复杂,操作环境恶劣,具有大惯性、非线性以及来料性质波动频繁等缺点,分析浓密机的结构,设计浓密机的参数自整定模糊PID控制.通过在青海某矿业集团50万吨铜钴回收项目的实际应用证明,该系统能很好地满足控制要求.浓密机运行安全可靠,提高了综合回收效率,减轻了劳动强度,实现了安全稳定的自动化生产.

  4. 47 CFR Appendix I to Subpart E of... - A Procedure for Calculating PCS Signal Levels at Microwave Receivers (Appendix E of the...

    Science.gov (United States)

    2010-10-01

    ... at Microwave Receivers (Appendix E of the Memorandum Opinion and Order) I Appendix I to Subpart E of... PERSONAL COMMUNICATIONS SERVICES Broadband PCS Pt. 24, Subpt. E, App. I Appendix I to Subpart E of Part 24... station and the characteristics of each receiver, i.e., adjacent channel susceptibility, antenna...

  5. Which Teaching Strategy Is Better for Enhancing Anti-Phishing Learning Motivation and Achievement? The Concept Maps on Tablet PCs or Worksheets?

    Science.gov (United States)

    Sun, Jerry Chih-Yuan; Lee, Kuan-Hsien

    2016-01-01

    The purpose of this study is to evaluate the feasibility of the integration of concept maps and tablet PCs in anti-phishing education for enhancing students' learning motivation and achievement. The subjects were 155 students from grades 8 and 9. They were divided into an experimental group (77 students) and a control group (78 students). To begin…

  6. Soil bacterium Pseudomonas sp.593 synthesizes phosphatidylcholine via phosphatidylcholine synthase pathway%土壤假单胞菌593使用Pcs途径合成磷酯酰胆碱

    Institute of Scientific and Technical Information of China (English)

    熊敏; 吴彬; 何火光; 李洋; 王行国

    2011-01-01

    [Objective]Prokaryotes synthesize phosphotidylcholine by using phospholipid N-methylation or phosphatidylcholine synthase pathway or both.To confirm which pathway the soil bacterium Pseudomonas sp.593 utilizes, we tested its phosphotidylcholine synthesis, cloned the pcs gene encoding phosphatidylcholine synthase, examined Pcs activity, and constructed a pcs- mutant.[Methods]To clone the pcs gene from Pseudomonas sp.593 genomic DNA, we firstly aligned amino acid sequences of phosphatidylcholine synthases in different pseudomonas strains reported in databases.Then we designed degenerate primers based on two amino acid segments conserved in sequences of phosphatidylcholine synthases.A partial fragment of the pcs gene was finally amplified from Pseudomonas sp.593 genomic DNA.The amplified partial fragment was labeled with digoxigenin-dUTP ( DIG) as a probe, sub-cloning library of Pseudomonas sp.593 genomic DNA was prepared and then screened using DIG-labelled probe via in situ colony hybridization.DNA homologous recombination in vivo was preformed to delete pcs gene of Pseudomonas sp.593.Thinlayer chromatography ( TLC) assay was used to analyze total phospholipids, detect phosphotidylcholine content and determine pcs gene activity.[Results]TLC analysis revealed that Pseudomonas sp.593 growing in the M9 or LB medium with choline was able to synthesize phosphotidylcholine, but wasn't without addition of choline.A 894 bp DNA fragment coded a protein with phosphatidylcholine synthase activity was cloned from Pseudomonas sp.593.The pcs- mutant obtained from in vivo mutagenesis was unable to form phosphotidylcholine, no matter choline was presented in the medium or not.[Conclusion]Phosphatidylcholine synthase pathway is a sole way for phosphotidylcholine synthesis in soil bacterium Pseudomonas sp.593 or other Pseudomonas strains.%[目的]原核生物有两条代谢途径N-甲基化途径(Pmt途径)和磷脂酰胆碱合酶途径(Pcs途径)合成磷脂酰胆

  7. Selected Morphological Characteristics, Lead Uptake and Phytochelatin Synthesis by Coffeeweed (Sesbania exaltata Raf. Grown in Elevated Levels of Lead-Contaminated Soil

    Directory of Open Access Journals (Sweden)

    Maria F. T. Begonia

    2011-06-01

    Full Text Available Remediation of lead-contaminated soil is significant due to the inherent toxicity of lead (Pb, and the quantity of Pb discharged into the soil. One of the most cost-effective and environmentally sound technologies for the cleanup of metal-contaminated soils is through the use of plants. While much is known about the ecological evolution of metal tolerance in plants, the physiological, biochemical, and genetic mechanisms of tolerance is not well understood in the majority of resistant ecotypes such as the legume, Sesbania exaltata Raf. This study was therefore conducted to determine the morphological and physiological characteristics of Sesbania that had been grown in Pb-contaminated soil, and to assess phytochelatin synthesis as a way of elucidating its relative Pb tolerance. Sesbania plants were grown in the greenhouse and exposed to various levels of Pb: 0, 1000, and 2000 mg Pb/kg soil. Plants were harvested after 6, 8, and 10 weeks of growth and morphological characteristics (e.g., root and shoot biomass, root length, number of root nodules, shoot height, number of leaves, number of flowers, number and length of pods were recorded. Generally, there were no statistical differences in morphological characteristics among the treatments. Further, no discernible phytotoxic symptoms, such as chlorosis, wilting, or necrotic lesions, in neither roots nor shoots were observed. We concluded that while Sesbania did not fit the model of a hyperaccumulator, the plant was, nonetheless, tolerant to elevated Pb levels. Our assessment for phytochelatin synthesis as a tolerance mechanism was inconclusive and further investigations of tolerance mechanisms are warranted.

  8. Selected morphological characteristics, lead uptake and phytochelatin synthesis by coffeeweed (Sesbania exaltata Raf.) grown in elevated levels of lead-contaminated soil.

    Science.gov (United States)

    Miller, Gloria; Begonia, Gregorio; Begonia, Maria F T

    2011-06-01

    Remediation of lead-contaminated soil is significant due to the inherent toxicity of lead (Pb), and the quantity of Pb discharged into the soil. One of the most cost-effective and environmentally sound technologies for the cleanup of metal-contaminated soils is through the use of plants. While much is known about the ecological evolution of metal tolerance in plants, the physiological, biochemical, and genetic mechanisms of tolerance is not well understood in the majority of resistant ecotypes such as the legume, Sesbania exaltata Raf. This study was therefore conducted to determine the morphological and physiological characteristics of Sesbania that had been grown in Pb-contaminated soil, and to assess phytochelatin synthesis as a way of elucidating its relative Pb tolerance. Sesbania plants were grown in the greenhouse and exposed to various levels of Pb: 0, 1000, and 2000 mg Pb/kg soil. Plants were harvested after 6, 8, and 10 weeks of growth and morphological characteristics (e.g., root and shoot biomass, root length, number of root nodules, shoot height, number of leaves, number of flowers, number and length of pods) were recorded. Generally, there were no statistical differences in morphological characteristics among the treatments. Further, no discernible phytotoxic symptoms, such as chlorosis, wilting, or necrotic lesions, in neither roots nor shoots were observed. We concluded that while Sesbania did not fit the model of a hyperaccumulator, the plant was, nonetheless, tolerant to elevated Pb levels. Our assessment for phytochelatin synthesis as a tolerance mechanism was inconclusive and further investigations of tolerance mechanisms are warranted.

  9. A Single Amino Acid Substitution Converts Benzophenone Synthase into Phenylpyrone Synthase*

    OpenAIRE

    Klundt, Tim; Bocola, Marco; Lütge, Maren; Beuerle, Till; Liu, Benye; Beerhues, Ludger

    2009-01-01

    Benzophenone metabolism provides a number of plant natural products with fascinating chemical structures and intriguing pharmacological activities. Formation of the carbon skeleton of benzophenone derivatives from benzoyl-CoA and three molecules of malonyl-CoA is catalyzed by benzophenone synthase (BPS), a member of the superfamily of type III polyketide synthases. A point mutation in the active site cavity (T135L) transformed BPS into a functional phenylpyrone synthase (PPS). The dramatic ch...

  10. Water quality benchmarking (WQB) and priority control screening (PCS) of persistent toxic substances (PTSs) in China: necessity, method and a case study.

    Science.gov (United States)

    He, Wei; Qin, Ning; Kong, Xiang-Zhen; Liu, Wen-Xiu; He, Qi-Shuang; Wang, Qing-Mei; Yang, Chen; Jiang, Yu-Jiao; Yang, Bin; Wu, Wen-Jing; Xu, Fu-Liu

    2014-02-15

    The priority control screening (PCS) and water quality benchmarking (WQB) of toxic chemicals in water are key steps to ensure the safety of drinking water and aquatic ecosystem that is the crucial goal of water environment management. Owing to the different levels of social-economic development in different countries and regions, the PCS and WQB of toxic chemicals must be determined in accordance with their specific water environment situations. However, in China, the PCS and WQB of toxic chemicals in water were mainly introduced from the other countries. A method for the PCS and WQB of toxic chemicals in water based on the ecological risks was proposed, and a platform named Bayesian Matbugs Calculator (BMC) was developed. As a case study, the WQB and PCS of sixty-nine PTSs based their ecological risks were performed on the basis of one-year monthly monitoring in Lake Chaohu. The results showed that the current national water quality criteria (WQC) would underestimate the toxicological risk to organisms in this aquatic ecosystem. It appears necessary to develop new WQC for the protection of aquatic organisms in Lake Chaohu. Four grades of priority control chemicals (PCCs) in Lake Chaohu were proposed. The highest priority was assigned to organonitrogen-phosphorus pesticides, including parathion, dichlorvos, malathion, omethoate, and di-n-butyl phthalate. However, the national "blacklist" of toxic compounds only covered 7 of 20 PCCs, indicating that the other 13 PCCs would not be controlled efficiently. Because the pollution pattern of PTSs in various water bodies might be quite different, we appealed to the governments to screen the regional PPC lists or develop a more comprehensive national list for aquatic ecosystem protection in China.

  11. The lumazine synthase/riboflavin synthase complex: shapes and functions of a highly variable enzyme system.

    Science.gov (United States)

    Ladenstein, Rudolf; Fischer, Markus; Bacher, Adelbert

    2013-06-01

    The xylene ring of riboflavin (vitamin B2 ) is assembled from two molecules of 3,4-dihydroxy-2-butanone 4-phosphate by a mechanistically complex process that is jointly catalyzed by lumazine synthase and riboflavin synthase. In Bacillaceae, these enzymes form a structurally unique complex comprising an icosahedral shell of 60 lumazine synthase subunits and a core of three riboflavin synthase subunits, whereas many other bacteria have empty lumazine synthase capsids, fungi, Archaea and some eubacteria have pentameric lumazine synthases, and the riboflavin synthases of Archaea are paralogs of lumazine synthase. The structures of the molecular ensembles have been studied in considerable detail by X-ray crystallography, X-ray small-angle scattering and electron microscopy. However, certain mechanistic aspects remain unknown. Surprisingly, the quaternary structure of the icosahedral β subunit capsids undergoes drastic changes, resulting in formation of large, quasi-spherical capsids; this process is modulated by sequence mutations. The occurrence of large shells consisting of 180 or more lumazine synthase subunits has recently generated interest for protein engineering topics, particularly the construction of encapsulation systems.

  12. Pteris vittata - revisited: uptake of As and its speciation, impact of P, role of phytochelatins and S.

    Science.gov (United States)

    Vetterlein, Doris; Wesenberg, Dirk; Nathan, Petra; Bräutigam, Anja; Schierhorn, Angelika; Mattusch, Jürgen; Jahn, Reinhold

    2009-11-01

    Pteris vittata is known to hyperaccumulate As but the mechanism is poorly understood. We found an increase of As concentration with increasing soil solution As concentrations, but P application had no impact, although plant P concentrations responded to different rates of P supply. As in fronds was dominantly (82-89%) present in the form of AsIII. In roots we detected 45% as AsIII which is higher than reported in previous studies and supports substantial As-reduction to take place in roots. We detected PC2/3GS-AsIII, PC2-GS-AsIII and (PC2)2-AsIII in increasing amounts with application of As. The total amount of PC was in the range reported previously and far too small to assign a significant role in As detoxification to PCs. The close correlation between S and As in fronds and the lack of data on sulphur uptake and metabolism indicates the need for a detailed investigation on sulphur nutritional status and As metabolism in P. vittata.

  13. Pteris vittata - Revisited: Uptake of As and its speciation, impact of P, role of phytochelatins and S

    Energy Technology Data Exchange (ETDEWEB)

    Vetterlein, Doris, E-mail: doris.vetterlein@ufz.d [UFZ Helmholtz Centre for Environmental Research, Department of Soil Physics, Theodor-Lieser-Str. 4, 06120 Halle (Germany); Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle/Saale (Germany); Wesenberg, Dirk; Nathan, Petra; Braeutigam, Anja; Schierhorn, Angelika [Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle/Saale (Germany); Mattusch, Juergen [UFZ Helmholtz Centre for Environmental Research, Department of Analytical Chemistry, Permoserstr. 15, 04318 Leipzig (Germany); Jahn, Reinhold [Soil Science and Soil Protection Group, Institute of Agriculture and Nutritional Sciences, Martin Luther University Halle-Wittenberg, Weidenplan 14, 06108 Halle/Saale (Germany)

    2009-11-15

    Pteris vittata is known to hyperaccumulate As but the mechanism is poorly understood. We found an increase of As concentration with increasing soil solution As concentrations, but P application had no impact, although plant P concentrations responded to different rates of P supply. As in fronds was dominantly (82-89%) present in the form of AsIII. In roots we detected 45% as AsIII which is higher than reported in previous studies and supports substantial As-reduction to take place in roots. We detected PC2/3GS-AsIII, PC2-GS-AsIII and (PC2)2-AsIII in increasing amounts with application of As. The total amount of PC was in the range reported previously and far too small to assign a significant role in As detoxification to PCs. The close correlation between S and As in fronds and the lack of data on sulphur uptake and metabolism indicates the need for a detailed investigation on sulphur nutritional status and As metabolism in P. vittata. - As-PC complexes were detected in increasing amounts with increasing As availability, but total amounts were small and do not explain the close correlation between S and As in fronds.

  14. WinLALS for a linked-atom least-square refinement program for helical polymers on WINDOWS PCs.

    Science.gov (United States)

    Okada, Kenji; Noguchi, Keiichi; Okuyama, Kenji; Arnott, Struther

    2003-07-01

    Fiber diffraction dada from polymers are sufficiently different in kind and quantity from single crystal data as to warrant analyses with a different emphasis: refinement of competing molecular models where torsion angles and bond angles are the explicit variables rather than atomic coordinates. The first linked-atom least-squares (LALS) refinement program had been devolved at Arnott's laboratories at King's College London [Arnott, S., Wonacott, A.J., 1966a. Polymer 7, 157] on mainframe and several revised versions were maintained at Purdue University [Smith, P.J.C., Arnott, S., 1978. Acta Crystallogr. Sect. A 34, 3; Chandrasekan, R. 2000. LALS Users Manual, Whistler Centre for Carbohydrate Researchm Purdue University, West Lafayette, IN] on workstation. Today the LALS users have to choose correctly any one program that they want to use, trigonometric or Bessel functions, from some versions. To develop a new WinLALS program based on the dimensioned version of the latest LALS2000 program [LALS Users Manual (2000)], we reviewed all the mathematical expressions and corrected the optimization of the non-bonded atomic contact terms. The WinLALS is coded with FORTRAN 90 and runs on MICROSOFT-WINDOWS PCs and the many amendments including changing input/output assignments, expanding array sizes, arranging that the update files have all output parameters of each cycle, and correcting several bugs are performed. This paper describes the mathematical expressions in detail employed in WinLALS and compares results of its applications obtained with those obtained earlier.

  15. The social benefits of WEEE re-use schemes. A cost benefit analysis for PCs in Spain.

    Science.gov (United States)

    González, Xose Manuel; Rodríguez, Miguel; Pena-Boquete, Yolanda

    2017-03-16

    One goal of the new European legislation set out in WEEE Directive 2012/19/UE is the promotion of WEEE re-use schemes. However, some authors are rather sceptical about the contribution of WEEE re-use schemes to improve resource efficiency. In order to evaluate and to design adequate policy instruments, some authors recommend the cost-benefit analysis (CBA) as a compulsory first step. In this context, the main contribution of this paper is to enlarge the empirical literature by providing a CBA of re-use schemes versus recycling processes of PCs. The analysis is made for Spain by quantifying in monetary terms the social damages of environmental impacts such as climate change, human toxicity, particulate matter formation, metal depletion, etc. Our results suggest that promoting re-use against recycling (and consequently the need for manufacturing a new PC from raw materials) may reduce environmental costs by 45.20€ per PC. Those social benefits are mainly generated in the re-use preparation process and distribution activities, whereas the re-use scenario displays a worse performance in energy consumption. The difference in the distribution stage during the second life cycle originates from the fact that the ready to re-use product is produced locally, while the brand new product is manufactured and distributed from abroad, mainly Asia. These results provide valuable information to policymakers and think tanks willing to design support schemes for re-use over recycling operations.

  16. Mammalian N-acetylglutamate synthase.

    Science.gov (United States)

    Morizono, Hiroki; Caldovic, Ljubica; Shi, Dashuang; Tuchman, Mendel

    2004-04-01

    N-Acetylglutamate synthase (NAGS, E.C. 2.3.1.1) is a mitochondrial enzyme that catalyzes the formation of N-acetylglutamate (NAG), an essential allosteric activator of carbamylphosphate synthetase I (CPSI). The mouse and human NAGS genes have been identified based on similarity to regions of NAGS from Neurospora crassa and cloned from liver cDNA libraries. These genes were shown to complement an argA- (NAGS) deficient Escherichia coli strain, and enzymatic activity of the proteins was confirmed by a new stable isotope dilution assay. The deduced amino acid sequence of mammalian NAGS contains a putative mitochondrial-targeting signal at the N-terminus. The mouse NAGS preprotein was overexpressed in insect cells to determine post-translational modifications and two processed proteins with different N-terminal truncations have been identified. Sequence analysis using a hidden Markov model suggests that the vertebrate NAGS protein contains domains with a carbamate kinase fold and an acyl-CoA N-acyltransferase fold, and protein crystallization experiments are currently underway. Inherited NAGS deficiency results in hyperammonemia, presumably due to the loss of CPSI activity. We, and others, have recently identified mutations in families with neonatal and late-onset NAGS deficiency and the identification of the gene has now made carrier testing and prenatal diagnosis feasible. A structural analog of NAG, carbamylglutamate, has been shown to bind and activate CPSI, and several patients have been reported to respond favorably to this drug (Carbaglu).

  17. Terpene synthases from Cannabis sativa.

    Science.gov (United States)

    Booth, Judith K; Page, Jonathan E; Bohlmann, Jörg

    2017-01-01

    Cannabis (Cannabis sativa) plants produce and accumulate a terpene-rich resin in glandular trichomes, which are abundant on the surface of the female inflorescence. Bouquets of different monoterpenes and sesquiterpenes are important components of cannabis resin as they define some of the unique organoleptic properties and may also influence medicinal qualities of different cannabis strains and varieties. Transcriptome analysis of trichomes of the cannabis hemp variety 'Finola' revealed sequences of all stages of terpene biosynthesis. Nine cannabis terpene synthases (CsTPS) were identified in subfamilies TPS-a and TPS-b. Functional characterization identified mono- and sesqui-TPS, whose products collectively comprise most of the terpenes of 'Finola' resin, including major compounds such as β-myrcene, (E)-β-ocimene, (-)-limonene, (+)-α-pinene, β-caryophyllene, and α-humulene. Transcripts associated with terpene biosynthesis are highly expressed in trichomes compared to non-resin producing tissues. Knowledge of the CsTPS gene family may offer opportunities for selection and improvement of terpene profiles of interest in different cannabis strains and varieties.

  18. Induced lead binding phytochelatins in Brassica juncea and Sesuvium portulacastrum investigated by orthogonal chromatography inductively coupled plasma-mass spectrometry and matrix assisted laser desorption ionization-time of flight-mass spectrometry.

    Science.gov (United States)

    Zaier, H; Mudarra, A; Kutscher, D; Fernández de la Campa, M R; Abdelly, C; Sanz-Medel, A

    2010-06-25

    The accumulation and transport of lead in Brassica juncea and Sesuvium portulacastrum plants and the possible formation of complexes of this element with bioligands such as phytochelatins was studied in roots and shoots of plants exposed to different amounts of Pb(NO(3))(2). Speciation studies on the plant extracts were conducted using size exclusion liquid chromatography and ion pair liquid chromatography coupled to UV and ICP-MS to monitor lead and sulphur. The identification of the species separated by chromatography was performed by MALDI-TOF-MS. In both types of exposed plants it was possible to identify the presence of the phytochelatin isoform PC(3). The results obtained suggest that both types of plants can be useful in studies of phytoremediation but the ability of S. portulacastrum to accumulate and redistribute Pb from root to shoot is more effective than B. juncea.

  19. Critical aspartic acid residues in pseudouridine synthases.

    Science.gov (United States)

    Ramamurthy, V; Swann, S L; Paulson, J L; Spedaliere, C J; Mueller, E G

    1999-08-01

    The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine at particular positions in certain RNA molecules. Genomic data base searches and sequence alignments using the first four identified pseudouridine synthases led Koonin (Koonin, E. V. (1996) Nucleic Acids Res. 24, 2411-2415) and, independently, Santi and co-workers (Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996) Nucleic Acids Res. 24, 3756-3762) to group this class of enzyme into four families, which display no statistically significant global sequence similarity to each other. Upon further scrutiny (Huang, H. L., Pookanjanatavip, M., Gu, X. G., and Santi, D. V. (1998) Biochemistry 37, 344-351), the Santi group discovered that a single aspartic acid residue is the only amino acid present in all of the aligned sequences; they then demonstrated that this aspartic acid residue is catalytically essential in one pseudouridine synthase. To test the functional significance of the sequence alignments in light of the global dissimilarity between the pseudouridine synthase families, we changed the aspartic acid residue in representatives of two additional families to both alanine and cysteine: the mutant enzymes are catalytically inactive but retain the ability to bind tRNA substrate. We have also verified that the mutant enzymes do not release uracil from the substrate at a rate significant relative to turnover by the wild-type pseudouridine synthases. Our results clearly show that the aligned aspartic acid residue is critical for the catalytic activity of pseudouridine synthases from two additional families of these enzymes, supporting the predictive power of the sequence alignments and suggesting that the sequence motif containing the aligned aspartic acid residue might be a prerequisite for pseudouridine synthase function.

  20. An investigation into eukaryotic pseudouridine synthases.

    Science.gov (United States)

    King, Ross D; Lu, Chuan

    2014-08-01

    A common post-transcriptional modification of RNA is the conversion of uridine to its isomer pseudouridine. We investigated the biological significance of eukaryotic pseudouridine synthases using the yeast Saccharomyces cerevisiae. We conducted a comprehensive statistical analysis on growth data from automated perturbation (gene deletion) experiments, and used bi-logistic curve analysis to characterise the yeast phenotypes. The deletant strains displayed different alteration in growth properties, including in some cases enhanced growth and/or biphasic growth curves not seen in wild-type strains under matched conditions. These results demonstrate that disrupting pseudouridine synthases can have a significant qualitative effect on growth. We further investigated the significance of post-transcriptional pseudouridine modification through investigation of the scientific literature. We found that (1) In Toxoplasma gondii, a pseudouridine synthase gene is critical in cellular differentiation between the two asexual forms: Tachyzoites and bradyzoites; (2) Mutation of pseudouridine synthase genes has also been implicated in human diseases (mitochondrial myopathy and sideroblastic anemia (MLASA); dyskeratosis congenita). Taken together, these results are consistent with pseudouridine synthases having a Gene Ontology function of "biological regulation".

  1. The Portable Chemical Sterilizer (PCS), D-FENS, and D-FEND ALL: novel chlorine dioxide decontamination technologies for the military.

    Science.gov (United States)

    Doona, Christopher J; Feeherry, Florence E; Setlow, Peter; Malkin, Alexander J; Leighton, Terrence J

    2014-06-29

    There is a stated Army need for a field-portable, non-steam sterilizer technology that can be used by Forward Surgical Teams, Dental Companies, Veterinary Service Support Detachments, Combat Support Hospitals, and Area Medical Laboratories to sterilize surgical instruments and to sterilize pathological specimens prior to disposal in operating rooms, emergency treatment areas, and intensive care units. The following ensemble of novel, 'clean and green' chlorine dioxide technologies are versatile and flexible to adapt to meet a number of critical military needs for decontamination(6,15). Specifically, the Portable Chemical Sterilizer (PCS) was invented to meet urgent battlefield needs and close critical capability gaps for energy-independence, lightweight portability, rapid mobility, and rugged durability in high intensity forward deployments(3). As a revolutionary technological breakthrough in surgical sterilization technology, the PCS is a Modern Field Autoclave that relies on on-site, point-of-use, at-will generation of chlorine dioxide instead of steam. Two (2) PCS units sterilize 4 surgical trays in 1 hr, which is the equivalent throughput of one large steam autoclave (nicknamed "Bertha" in deployments because of its cumbersome size, bulky dimensions, and weight). However, the PCS operates using 100% less electricity (0 vs. 9 kW) and 98% less water (10 vs. 640 oz.), significantly reduces weight by 95% (20 vs. 450 lbs, a 4-man lift) and cube by 96% (2.1 vs. 60.2 ft(3)), and virtually eliminates the difficult challenges in forward deployments of repairs and maintaining reliable operation, lifting and transporting, and electrical power required for steam autoclaves.

  2. 基于百度PCS实施SaaS架构的应用分析%Applied Analysis of Deploying SaaS Structure Based on Baidu PCS

    Institute of Scientific and Technical Information of China (English)

    张伟

    2015-01-01

    To develop application systems via cloud computing platform is efficient and low-cost. Besides, the best model of constructing the SaaS (Software-as-a-Service) structure enterprise application system is to de-ploy application systems based on the cloud computing platform. Aiming to research the development issues of enterprise SaaS application system based on Baidu PCS (Personal Cloud Storage) platform, the functional ad-vantages of deploying the SaaS structure of application systems based on Baidu PCS was analyzed. On this basis, combined with the Android developing environment, the integrated development method of SaaS struc-ture based on PCS was investigated. With help of SDK (Software Development Kit) offered by PCS, this paper raises a cloud storage solution of realizing structuring data processing and can provide those who develop the application systems with some references.%发挥云计算平台开发应用系统高效率、低成本的优势,基于云计算平台部署应用系统是实施SaaS(Software-as-a-Service软件即服务)架构企业应用系统的最佳模式。为探索基于百度个人云存储PCS(Personal Cloud Storage)平台的企业SaaS应用系统开发问题,文章分析了基于百度PCS实施应用系统SaaS架构的功能优势,在此基础上,结合Android开发环境探讨基于PCS的SaaS架构的集成开发方法,利用PCS所提供的SDK(Software Development Kit),给出了实现结构化数据处理的云存储解决方案。

  3. Evolution of Oswestry 2.0 questionnaire and the physical component (PCS) of SF-36 during the first postoperative year of lumbar spine fusion in degenerative diseases

    OpenAIRE

    Alexandre Henrique Silveira Bechara; Guilherme Rebechi Zuiani; Marcelo Ítalo Risso Neto; Paulo Tadeu Maia Cavali; Ivan Guidolin Veiga; Wagner Pasqualini; Elcio Landim

    2013-01-01

    OBJECTIVE: Assess the clinical improvement of patients undergoing decompression and posterolateral lumbar arthrodesis for degenerative diseases, evaluating the evolution of the scores of Oswestry 2.0 questionnaire and the physical component (PCS) of the SF-36 scale. METHODS: Prospective study of 19 patients with degenerative disc disease (disc herniation, lumbar stenosis or degenerative spondylolisthesis) that underwent decompression and posterolateral arthrodesis. All patients included in ou...

  4. Smartphones vs PCs: Does the Device Affect the Web Survey Experience and the Measurement Error for Sensitive Topics? - A Replication of the Mavletova & Couper’s 2013 Experiment

    Directory of Open Access Journals (Sweden)

    Daniele Toninelli

    2016-08-01

    Full Text Available More and more respondents use mobile devices to complete web surveys. These devices have different characteristics, if compared to PCs (e.g. smaller screen sizes and higher portability. These characteristics can affect the survey responses, mostly when a questionnaire includes sensitive questions. This topic was already studied by Mavletova and Couper (2013, through a two-wave experiment comparing PCs and mobile devices results for the same respondents in a Russian opt-in panel. We replicated this cross-over design, focusing on an opt-in panel for Spain, involving 1,800 panellists and comparing PCs and smartphones. Our results support most of Mavletova and Couper’s (2013 findings (e.g. generally the used device does not significantly affect the reporting of sensitive information, confirming their robustness over the two studied countries. For other results (e.g. trust in data confidentiality, we found differences that can be justified by the diverse context/culture or by the quick changes that are still characterizing the mobile web survey participation.

  5. Bacillus caldolyticus prs gene encoding phosphoribosyldiphosphate synthase

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1996-01-01

    The prs gene, encoding phosphoribosyl-diphosphate (PRPP) synthase, as well as the flanking DNA sequences were cloned and sequenced from the Gram-positive thermophile, Bacillus caldolyticus. Comparison with the homologous sequences from the mesophile, Bacillus subtilis, revealed a gene (gca......D) encoding N-acetylglucosamine-l-phosphate uridyltransferase upstream of prs, and a gene homologous to ctc downstream of prs. cDNA synthesis with a B. caldolyticus gcaD-prs-ctc-specified mRNA as template, followed by amplification utilising the polymerase chain reaction indicated that the three genes are co......-transcribed. Comparison of amino acid sequences revealed a high similarity among PRPP synthases across a wide phylogenetic range. An E. coli strain harbouring the B. caldolyticus prs gene in a multicopy plasmid produced PRPP synthase activity 33-fold over the activity of a haploid B. caldolyticus strain. B. caldolyticus...

  6. Differential response of oxidative stress and thiol metabolism in contrasting rice genotypes for arsenic tolerance.

    Science.gov (United States)

    Tripathi, Preeti; Mishra, Aradhana; Dwivedi, Sanjay; Chakrabarty, Debasis; Trivedi, Prabodh K; Singh, Rana Pratap; Tripathi, Rudra Deo

    2012-05-01

    The mechanism of arsenic (As) tolerance was investigated on two contrasting rice (Oryza sativa L.) genotypes, selected for As tolerance and accumulation. One tolerant (Triguna) and one sensitive (IET-4786) variety were exposed to various arsenate (0-50 μM) levels for 7 d for biochemical analyses. Arsenic induced oxidative stress was more pronounced in IET-4786 than Triguna especially in terms of reactive oxygen species, lipid peroxidation, EC and pro-oxidant enzymes (NADPH oxidase and ascorbate oxidase). However, Triguna tolerated As stress through the enhanced enzymes activities particularly pertaining to thiol metabolism such as serine acetyl transferase (SAT), cysteine synthase (CS), γ-glutamyl cysteine synthase (γ-ECS), γ-glutamyl transpeptidase (γ-GT), and glutathione-S-transferase (GST) as well as arsenate reductase (AR). Besides maintaining the ratio of redox couples GSH/GSSG and ASC/DHA, the level of phytochelatins (PCs) and phytochelatin synthase (PCS) activity were more pronounced in Triguna, in which harmonized responses of thiol metabolism was responsible for As tolerance in contrast to IET-4786 showing its susceptible nature towards As exposure.

  7. Tablet PCs in Elementary Education A Pilot Project at the Practice Primary School of the KPH at Campus Vienna/Krems

    Directory of Open Access Journals (Sweden)

    Anton Reiter

    2015-09-01

    Full Text Available Am Beginn des Schuljahrs 2011/2012 wurde eine Volksschulklasse der im 21. Wiener Gemeindebezirk gelegenen Praxisschule der katholischen Pädagogischen Hochschule Wien/Krems mit Tablet-PCs ausgestattet. Die Geräte wurden von der Institution und den Eltern finanziert und allen SchülerInnen einer dritten Klasse zur Verfügung gestellt. Evaluation und Dokumentation des Projekts wurde vom österreichische Ministerium für Unterricht und Frauen finanziert. Wie aktuelle internationale Studien zeigen, gewöhnen sich neunjährige SchülerInnen schnell an solche Geräte. Das war auch in der hier vorgelegten Studie zu beobachten. Die SchülerInnen waren sehr motiviert und haben verschiedene Anwendungen erprobt. Die Tablets wurden auch außerhalb des Klassenraums verwendet, was als wesentlicher Vorteil mobiler Computer gesehen werden kann. Tablet-PCs ermöglichen individuelles Lernen und können für kooperative, soziale und interaktive Lernformen während des Unterrichts und in der Freizeit verwendet werden. Als persönliche mobile Geräte katalysieren die Tablet-PCs selbstgesteuertes “just-in-time” Lernen. Alle SchülerInnen sollten daher im 21. Jahrhundert solche Geräte zur Verfügung haben. One class of the practice primary school at the church-affilated former pedagogical college (KPH at Campus Vienna/Krems located in the 21st district of the capital of Austria was equipped with Tablet PCs at the beginning of the school year 2011/2012. These devices for all students of a third class were financed by the institution and partly the parents. The Austrian Federal Ministry of Education and Women's Affairs funded the evaluation and documentation of this two-year project. As recent international studies on the use of tablets (mostly iPads in primary and secondary education confirm, also these nine years old kids quickly got familiar with the used Pearl Touchlet tablets. The pupils were highly motivated and tried out different applications. The tablets

  8. The tomato terpene synthase gene family

    NARCIS (Netherlands)

    Falara, V.; Akhtar, T.A.; Nguyen, T.T.H.; Spyropoulou, E.A.; Bleeker, P.M.; Schauvinhold, I.; Matsuba, Y.; Bonini, M.E.; Schilmiller, A.L.; Last, R.L.; Schuurink, R.C.; Pichersky, E.

    2011-01-01

    Compounds of the terpenoid class play many roles in the interactions of plants with their environment, such as attracting pollinators and defending the plant against pests. We show here that the genome of Solanum lycopersicum (cultivated tomato) contains 40 terpene synthase (TPS) genes, including 28

  9. Cloning of parsley flavone synthase I.

    Science.gov (United States)

    Martens, S; Forkmann, G; Matern, U; Lukacin, R

    2001-09-01

    A cDNA encoding flavone synthase I was amplified by RT-PCR from leaflets of Petroselinum crispum cv. Italian Giant seedlings and functionally expressed in yeast cells. The identity of the recombinant, 2-oxoglutarate-dependent enzyme was verified in assays converting (2S)-naringenin to apigenin.

  10. Inducible nitric oxide synthase in renal transplantation

    NARCIS (Netherlands)

    Joles, JA; Vos, IH; Grone, HJ; Rabelink, TJ

    2002-01-01

    The importance of the endothelial isoform of nitric oxide synthase (eNOS) has been well established. Endothelium-derived nitric oxide has been shown to be essential for vascular homeostasis and modulation of eNOS has thus become a target in prevention of cardiovascular disease. The role of the induc

  11. Increased lead and cadmium tolerance of Typha angustifolia from Huaihe River is associated with enhanced phytochelatin synthesis and improved antioxidative capacity.

    Science.gov (United States)

    Liu, Yunlei; Chen, Jian; Lu, Shaonan; Yang, Libo; Qian, Jiazhong; Cao, Shuqing

    2016-11-01

    Heavy metal contamination of water is an increasing environmental problem worldwide, and the use of aquatic plants for phytoremediation of heavy metal pollution has become an important subject of research. One key to successful phytoremediation is the identification of plants that are efficient at sequestering heavy metals. In this study, we examined the growth and heavy metal accumulation of Typha angustifolia and compared growth characteristics and tolerance mechanisms in plants from the Huaihe and Chaohu Rivers irrigated with different concentrations of lead (Pb) and cadmium (Cd). T. angustifolia from Huaihe River showed enhanced tolerance and accumulation of Pb and Cd and had greater biomass and more vigorous growth than the ecotype from Chaohu River. In addition, higher phytochelatin (PC) content and significantly higher superoxide dismutase and catalase activities were detected in T. angustifolia from Huaihe River than in T. angustifolia from Chaohu River. These findings suggest that high Pb and Cd accumulation and tolerance in T. angustifolia from Chaohu River is associated with its higher PC synthesis and better antioxidative capacity, and that the Huaihe ecotype of T. angustifolia might also be an efficient species for phytoremediation of Pb and Cd in water contaminated by heavy metals.

  12. Cytosolic expression of synthetic phytochelatin and bacterial metallothionein genes in Deinococcus radiodurans R1 for enhanced tolerance and bioaccumulation of cadmium.

    Science.gov (United States)

    Chaturvedi, Ruchi; Archana, G

    2014-06-01

    Due to its exemplary resistance to ionising radiation, oxidative stress, desiccation and several DNA damaging agents, Deinococcus radiodurans R1 (DR1) is considered as one of the most appropriate candidates for the bioremediation of the nuclear waste sites. However, the high sensitivity of this bacterium to heavy metals, which are usually preponderant at nuclear waste dump sites, precludes its application for bioremediation. This study deals with the expression two metal binding peptides in DR1 as an attractive strategy for developing metal tolerance in this bacterium. A synthetic gene (EC20) encoding a phytochelatin analogue with twenty repeating units of glutamate and cysteine was constructed by overlap extension and expressed in DR1. The cyanobacterial metallothionein (MT) gene, smtA was cloned for intracellular expression in DR1. Both the genes were expressed under the native groESL promoter. DR1 strain carrying the recombinant EC20 demonstrated 2.5-fold higher tolerance to Cd(2+) and accumulated 1.21-fold greater Cd(2+) as opposed to the control while the heterologous expression of MT SmtA in DR1 imparted the transformant superior tolerance to Cd(2+) amassing 2.5-fold greater Cd(2+) than DR1 expressing EC20.

  13. Trichinella pseudospiralis vs. T. spiralis thymidylate synthase gene structure and T. pseudospiralis thymidylate synthase retrogene sequence.

    Science.gov (United States)

    Jagielska, Elżbieta; Płucienniczak, Andrzej; Dąbrowska, Magdalena; Dowierciał, Anna; Rode, Wojciech

    2014-04-09

    Thymidylate synthase is a housekeeping gene, designated ancient due to its role in DNA synthesis and ubiquitous phyletic distribution. The genomic sequences were characterized coding for thymidylate synthase in two species of the genus Trichinella, an encapsulating T. spiralis and a non-encapsulating T. pseudospiralis. Based on the sequence of parasitic nematode Trichinella spiralis thymidylate synthase cDNA, PCR techniques were employed. Each of the respective gene structures encompassed 6 exons and 5 introns located in conserved sites. Comparison with the corresponding gene structures of other eukaryotic species revealed lack of common introns that would be shared among selected fungi, nematodes, mammals and plants. The two deduced amino acid sequences were 96% identical. In addition to the thymidylate synthase gene, the intron-less retrocopy, i.e. a processed pseudogene, with sequence identical to the T. spiralis gene coding region, was found to be present within the T. pseudospiralis genome. This pseudogene, instead of the gene, was confirmed by RT-PCR to be expressed in the parasite muscle larvae. Intron load, as well as distribution of exon and intron phases in thymidylate synthase genes from various sources, point against the theory of gene assembly by the primordial exon shuffling and support the theory of evolutionary late intron insertion into spliceosomal genes. Thymidylate synthase pseudogene expressed in T. pseudospiralis muscle larvae is designated a retrogene.

  14. Performance of an Optimized Eta Model Code on the Cray T3E and a Network of PCs

    Science.gov (United States)

    Kouatchou, Jules; Rancic, Miodrag; Geiger, Jim

    2000-01-01

    In the year 2001, NASA will launch the satellite TRIANA that will be the first Earth observing mission to provide a continuous, full disk view of the sunlit Earth. As a part of the HPCC Program at NASA GSFC, we have started a project whose objectives are to develop and implement a 3D cloud data assimilation system, by combining TRIANA measurements with model simulation, and to produce accurate statistics of global cloud coverage as an important element of the Earth's climate. For simulation of the atmosphere within this project we are using the NCEP/NOAA operational Eta model. In order to compare TRIANA and the Eta model data on approximately the same grid without significant downscaling, the Eta model will be integrated at a resolution of about 15 km. The integration domain (from -70 to +70 deg in latitude and 150 deg in longitude) will cover most of the sunlit Earth disc and will continuously rotate around the globe following TRIANA. The cloud data assimilation is supposed to run and produce 3D clouds on a near real-time basis. Such a numerical setup and integration design is very ambitious and computationally demanding. Thus, though the Eta model code has been very carefully developed and its computational efficiency has been systematically polished during the years of operational implementation at NCEP, the current MPI version may still have problems with memory and efficiency for the TRIANA simulations. Within this work, we optimize a parallel version of the Eta model code on a Cray T3E and a network of PCs (theHIVE) in order to improve its overall efficiency. Our optimization procedure consists of introducing dynamically allocated arrays to reduce the size of static memory, and optimizing on a single processor by splitting loops to limit the number of streams. All the presented results are derived using an integration domain centered at the equator, with a size of 60 x 60 deg, and with horizontal resolutions of 1/2 and 1/3 deg, respectively. In accompanying

  15. Localization of nitric oxide synthase in human skeletal muscle

    DEFF Research Database (Denmark)

    Frandsen, Ulrik; Lopez-Figueroa, M.; Hellsten, Ylva

    1996-01-01

    The present study investigated the cellular localization of the neuronal type I and endothelial type III nitric oxide synthase in human skeletal muscle. Type I NO synthase immunoreactivity was found in the sarcolemma and the cytoplasm of all muscle fibres. Stronger immunoreactivity was expressed...... I NO synthase immunoreactivity and NADPH diaphorase activity. Type III NO synthase immunoreactivity was observed both in the endothelium of larger vessels and of microvessels. The results establish that human skeletal muscle expresses two different constitutive isoforms of NO synthase in different...... endothelium is consistent with a role for NO in the control of blood flow in human skeletal muscle....

  16. Using PWE/FE method to calculate the band structures of the semi-infinite beam-like PCs: Periodic in z-direction and finite in x–y plane

    Energy Technology Data Exchange (ETDEWEB)

    Qian, Denghui, E-mail: qdhsd318@163.com; Shi, Zhiyu, E-mail: zyshi@nuaa.edu.cn

    2017-05-03

    This paper couples the plane wave expansion (PWE) and finite element (FE) methods to calculate the band structures of the semi-infinite beam-like phononic crystals (PCs) with the infinite periodicity in z-direction and finiteness in x–y plane. Explicit matrix formulations are developed for the calculation of band structures. In order to illustrate the applicability and accuracy of the proposed coupled plane wave expansion and finite element (PWE/FE) method to beam-like PCs, several examples are displayed. At first, PWE/FE method is applied to calculate the band structures of the Pb/rubber beam-like PCs with circular and rectangular cross sections, respectively. Then, it is used to calculate the band structures of steel/epoxy and steel/aluminum beam-like PCs with the same geometric parameters. Last, the band structure of the three-component beam-like PC is also calculated by the proposed method. Moreover, all the results calculated by PWE/FE method are compared with those calculated by finite element (FE) method, and the corresponding results are in good agreement. - Highlights: • The concept of the semi-infinite beam-like phononic crystals (PCs) is proposed. • The PWE/FE method is proposed and formulized to calculate the band structures of the semi-infinite beam-like PCs. • The strong applicability and high accuracy of PWE/FE method are verified.

  17. Cellulose Synthases and Synthesis in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Anne Endler; Staffan Persson

    2011-01-01

    Plant cell walls are complex structures composed of high-molecular-weight polysaccharides,proteins,and lignins. Among the wall polysaccharides,cellulose,a hydrogen-bonded β-1,4-linked glucan microfibril,is the main load-bearing wall component and a key precursor for industrial applications. Cellulose is synthesized by large multi-meric cellulose synthase (CesA) complexes,tracking along cortical microtubules at the plasma membrane. The only known components of these complexes are the cellulose synthase proteins. Recent studies have identified tentative interaction partners for the CesAs and shown that the migratory patterns of the CesA complexes depend on phosphorylation status. These advances may become good platforms for expanding our knowledge about cellulose synthesis in the near future. In addition,our current understanding of cellulose chain polymerization in the context of the CesA complex is discussed.

  18. Caffeine synthase and related methyltransferases in plants.

    Science.gov (United States)

    Misako, Kato; Kouichi, Mizuno

    2004-05-01

    Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in high concentrations in tea and coffee and it is also found in a number of beverages such as coca cola. It is necessary to elucidate the caffeine biosynthetic pathway and to clone the genes related to the production of caffeine not only to determine the metabolism of the purine alkaloid but also to control the content of caffeine in tea and coffee. The available data support the operation of a xanthosine-->7-methylxanthosine-->7-methylxanthine-->theobromine-->caffeine pathway as the major route to caffeine. Since the caffeine biosynthetic pathway contains three S-adenosyl-L-methionine (SAM) dependent methylation steps, N-methyltransferases play important roles. This review focuses on the enzymes and genes involved in the methylation of purine ring. Caffeine synthase, the SAM-dependent methyltransferase involved in the last two steps of caffeine biosynthesis, was originally purified from young tea leaves (Camellia sinensis). The isolated cDNA, termed TCS1, consists of 1,483 base pairs and encodes a protein of 369 amino acids. Subsequently, the homologous genes that encode caffeine biosynthetic enzymes from coffee (Coffea arabica) were isolated. The recombinant proteins are classified into the three types on the basis of their substrate specificity i.e. 7-methylxanthosine synthase, theobromine synthase and caffeine synthase. The predicted amino acid sequences of caffeine biosynthetic enzymes derived from C. arabica exhibit more than 80% homology with those of the clones and but show only 40% homology with TCS1 derived from C. sinensis. In addition, they share 40% homology with the amino acid sequences of salicylic carboxyl methyltransferase, benzoic acid carboxyl methyltransferase and jasmonic acid carboxyl methyltransferase which belong to a family of motif B' methyltransferases which are novel plant methyltransferases with motif B' instead of motif B as the conserved region.

  19. Building-block selectivity of polyketide synthases.

    Science.gov (United States)

    Liou, Grace F; Khosla, Chaitan

    2003-04-01

    For the past decade, polyketide synthases have presented an exciting paradigm for the controlled manipulation of complex natural product structure. These multifunctional enzymes catalyze the biosynthesis of polyketide natural products by stepwise condensation and modification of metabolically derived building blocks. In particular, regioselective modification of polyketide structure is possible by alterations in either intracellular acyl-CoA pools or, more commonly, by manipulation of acyl transferases that act as the primary gatekeepers for building blocks.

  20. Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemol synthase activity.

    Science.gov (United States)

    Yang, Ting; Gao, Liping; Hu, Hao; Stoopen, Geert; Wang, Caiyun; Jongsma, Maarten A

    2014-12-26

    Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1'-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP). Three proteins are known to catalyze this cyclopropanation reaction of terpene precursors. Two of them, phytoene and squalene synthase, are bifunctional enzymes with both prenyltransferase and terpene synthase activity. CDS, the other member, has been reported to perform only the prenyltransferase step. Here we show that the NDXXD catalytic motif of CDS, under the lower substrate conditions prevalent in plants, also catalyzes the next step, converting CPP into chrysanthemol by hydrolyzing the diphosphate moiety. The enzymatic hydrolysis reaction followed conventional Michaelis-Menten kinetics, with a Km value for CPP of 196 μm. For the chrysanthemol synthase activity, DMAPP competed with CPP as substrate. The DMAPP concentration required for half-maximal activity to produce chrysanthemol was ∼100 μm, and significant substrate inhibition was observed at elevated DMAPP concentrations. The N-terminal peptide of CDS was identified as a plastid-targeting peptide. Transgenic tobacco plants overexpressing CDS emitted chrysanthemol at a rate of 0.12-0.16 μg h(-1) g(-1) fresh weight. We propose that CDS should be renamed a chrysanthemol synthase utilizing DMAPP as substrate.

  1. Monoterpene synthases from grand fir (Abies grandis). cDNA isolation, characterization, and functional expression of myrcene synthase, (-)-(4S)-limonene synthase, and (-)-(1S,5S)-pinene synthase.

    Science.gov (United States)

    Bohlmann, J; Steele, C L; Croteau, R

    1997-08-29

    Grand fir (Abies grandis) has been developed as a model system for studying defensive oleoresin formation in conifers in response to insect attack or other injury. The turpentine fraction of the oleoresin is a complex mixture of monoterpene (C10) olefins in which (-)-limonene and (-)-alpha- and (-)-beta-pinene are prominent components; (-)-limonene and (-)-pinene synthase activities are also induced upon stem wounding. A similarity based cloning strategy yielded three new cDNA species from a wounded stem cDNA library that appeared to encode three distinct monoterpene synthases. After expression in Escherichia coli and enzyme assay with geranyl diphosphate as substrate, subsequent analysis of the terpene products by chiral phase gas chromatography and mass spectrometry showed that these sequences encoded a (-)-limonene synthase, a myrcene synthase, and a (-)-pinene synthase that produces both alpha-pinene and beta-pinene. In properties and reaction stereochemistry, the recombinant enzymes resemble the corresponding native monoterpene synthases of wound-induced grand fir stem. The deduced amino acid sequences indicated the limonene synthase to be 637 residues in length (73.5 kDa), the myrcene synthase to be 627 residues in length (72.5 kDa), and the pinene synthase to be 628 residues in length (71.5 kDa); all of these monoterpene synthases appear to be translated as preproteins bearing an amino-terminal plastid targeting sequence. Sequence comparison revealed that these monoterpene synthases from grand fir resemble sesquiterpene (C15) synthases and diterpene (C20) synthases from conifers more closely than other monoterpene synthases from angiosperm species. This similarity between extant monoterpene, sesquiterpene, and diterpene synthases of gymnosperms is surprising since functional diversification of this enzyme class is assumed to have occurred over 300 million years ago. Wound-induced accumulation of transcripts for monoterpene synthases was demonstrated by RNA

  2. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    Energy Technology Data Exchange (ETDEWEB)

    Gou, Ke-Mian [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193 (China); Chang, Chia-Chun [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Shen, Qing-Ji [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); Sung, Li-Ying, E-mail: liyingsung@ntu.edu.tw [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan, ROC (China); Liu, Ji-Long, E-mail: jilong.liu@dpag.ox.ac.uk [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom)

    2014-04-15

    CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus.

  3. Mutational analysis of a monoterpene synthase reaction: altered catalysis through directed mutagenesis of (-)-pinene synthase from Abies grandis.

    Science.gov (United States)

    Hyatt, David C; Croteau, Rodney

    2005-07-15

    Two monoterpene synthases, (-)-pinene synthase and (-)-camphene synthase, from grand fir (Abies grandis) produce different product mixtures despite having highly homologous amino acid sequences and, presumably, very similar three-dimensional structures. The major product of (-)-camphene synthase, (-)-camphene, and the major products of (-)-pinene synthase, (-)-alpha-pinene, and (-)-beta-pinene, arise through distinct mechanistic variations of the electrophilic reaction cascade that is common to terpenoid synthases. Structural modeling followed by directed mutagenesis in (-)-pinene synthase was used to replace selected amino acid residues with the corresponding residues from (-)-camphene synthase in an effort to identify the amino acids responsible for the catalytic differences. This approach produced an enzyme in which more than half of the product was channeled through an alternative pathway. It was also shown that several (-)-pinene synthase to (-)-camphene synthase amino acid substitutions were necessary before catalysis was significantly altered. The data support a model in which the collective action of many key amino acids, located both in and distant from the active site pocket, regulate the course of the electrophilic reaction cascade.

  4. Biochemical characterization and homology modeling of methylbutenol synthase and implications for understanding hemiterpene synthase evolution in plants.

    Science.gov (United States)

    Gray, Dennis W; Breneman, Steven R; Topper, Lauren A; Sharkey, Thomas D

    2011-06-10

    2-Methyl-3-buten-2-ol (MBO) is a five-carbon alcohol produced and emitted in large quantities by many species of pine native to western North America. MBO is structurally and biosynthetically related to isoprene and can have an important impact on regional atmospheric chemistry. The gene for MBO synthase was identified from Pinus sabiniana, and the protein encoded was functionally characterized. MBO synthase is a bifunctional enzyme that produces both MBO and isoprene in a ratio of ~90:1. Divalent cations are required for activity, whereas monovalent cations are not. MBO production is enhanced by K(+), whereas isoprene production is inhibited by K(+) such that, at physiologically relevant [K(+)], little or no isoprene emission should be detected from MBO-emitting trees. The K(m) of MBO synthase for dimethylallyl diphosphate (20 mm) is comparable with that observed for angiosperm isoprene synthases and 3 orders of magnitude higher than that observed for monoterpene and sesquiterpene synthases. Phylogenetic analysis showed that MBO synthase falls into the TPS-d1 group (gymnosperm monoterpene synthases) and is most closely related to linalool synthase from Picea abies. Structural modeling showed that up to three phenylalanine residues restrict the size of the active site and may be responsible for making this a hemiterpene synthase rather than a monoterpene synthase. One of these residues is homologous to a Phe residue found in the active site of isoprene synthases. The remaining two Phe residues do not have homologs in isoprene synthases but occupy the same space as a second Phe residue that closes off the isoprene synthase active site.

  5. Geranyl diphosphate synthase molecules, and nucleic acid molecules encoding same

    Science.gov (United States)

    Croteau, Rodney Bruce; Burke, Charles Cullen

    2008-06-24

    In one aspect, the present invention provides isolated nucleic acid molecules that each encode a geranyl diphosphate synthase protein, wherein each isolated nucleic acid molecule hybridizes to a nucleic acid molecule consisting of the sequence set forth in SEQ ID NO:1 under conditions of 5.times.SSC at 45.degree. C. for one hour. The present invention also provides isolated geranyl diphosphate synthase proteins, and methods for altering the level of expression of geranyl diphosphate synthase protein in a host cell.

  6. Functional and evolutionary relationships between terpene synthases from Australian Myrtaceae.

    Science.gov (United States)

    Keszei, Andras; Brubaker, Curt L; Carter, Richard; Köllner, Tobias; Degenhardt, Jörg; Foley, William J

    2010-06-01

    Myrtaceae is one of the chemically most variable and most significant essential oil yielding plant families. Despite an abundance of chemical information, very little work has focussed on the biochemistry of terpene production in these plants. We describe 70 unique partial terpene synthase transcripts and eight full-length cDNA clones from 21 myrtaceous species, and compare phylogenetic relationships and leaf oil composition to reveal clades defined by common function. We provide further support for the correlation between function and phylogenetic relationships by the first functional characterisation of terpene synthases from Myrtaceae: a 1,8-cineole synthase from Eucalyptus sideroxylon and a caryophyllene synthase from Eucalyptusdives.

  7. Benzophenone Synthase and Chalcone Synthase Accumulate in the Mesophyll of Hypericum perforatum Leaves at Different Developmental Stages

    OpenAIRE

    Belkheir, Asma K.; Gaid, Mariam; Liu, Benye; Hänsch, Robert; Beerhues, Ludger

    2016-01-01

    The active medicinal constituents in Hypericum perforatum, used to treat depression and skin irritation, include flavonoids and xanthones. The carbon skeletons of these compounds are formed by chalcone synthase (CHS) and benzophenone synthase (BPS), respectively. Polyclonal antisera were raised against the polyketide synthases from Hypericum androsaemum and their IgG fractions were isolated. Immunoblotting and immunotitration were used to test the IgGs for crossreactivity and monospecificity ...

  8. Genomic Analysis of Terpene Synthase Family and Functional Characterization of Seven Sesquiterpene Synthases from Citrus sinensis

    Science.gov (United States)

    Alquézar, Berta; Rodríguez, Ana; de la Peña, Marcos; Peña, Leandro

    2017-01-01

    Citrus aroma and flavor, chief traits of fruit quality, are derived from their high content in essential oils of most plant tissues, including leaves, stems, flowers, and fruits. Accumulated in secretory cavities, most components of these oils are volatile terpenes. They contribute to defense against herbivores and pathogens, and perhaps also protect tissues against abiotic stress. In spite of their importance, our understanding of the physiological, biochemical, and genetic regulation of citrus terpene volatiles is still limited. The availability of the sweet orange (Citrus sinensis L. Osbeck) genome sequence allowed us to characterize for the first time the terpene synthase (TPS) family in a citrus type. CsTPS is one of the largest angiosperm TPS families characterized so far, formed by 95 loci from which just 55 encode for putative functional TPSs. All TPS angiosperm families, TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g were represented in the sweet orange genome, with 28, 18, 2, 2, and 5 putative full length genes each. Additionally, sweet orange β-farnesene synthase, (Z)-β-cubebene/α-copaene synthase, two β-caryophyllene synthases, and three multiproduct enzymes yielding β-cadinene/α-copaene, β-elemene, and β-cadinene/ledene/allo-aromandendrene as major products were identified, and functionally characterized via in vivo recombinant Escherichia coli assays. PMID:28883829

  9. Genomic Analysis of Terpene Synthase Family and Functional Characterization of Seven Sesquiterpene Synthases from Citrus sinensis

    Directory of Open Access Journals (Sweden)

    Berta Alquézar

    2017-08-01

    Full Text Available Citrus aroma and flavor, chief traits of fruit quality, are derived from their high content in essential oils of most plant tissues, including leaves, stems, flowers, and fruits. Accumulated in secretory cavities, most components of these oils are volatile terpenes. They contribute to defense against herbivores and pathogens, and perhaps also protect tissues against abiotic stress. In spite of their importance, our understanding of the physiological, biochemical, and genetic regulation of citrus terpene volatiles is still limited. The availability of the sweet orange (Citrus sinensis L. Osbeck genome sequence allowed us to characterize for the first time the terpene synthase (TPS family in a citrus type. CsTPS is one of the largest angiosperm TPS families characterized so far, formed by 95 loci from which just 55 encode for putative functional TPSs. All TPS angiosperm families, TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g were represented in the sweet orange genome, with 28, 18, 2, 2, and 5 putative full length genes each. Additionally, sweet orange β-farnesene synthase, (Z-β-cubebene/α-copaene synthase, two β-caryophyllene synthases, and three multiproduct enzymes yielding β-cadinene/α-copaene, β-elemene, and β-cadinene/ledene/allo-aromandendrene as major products were identified, and functionally characterized via in vivo recombinant Escherichia coli assays.

  10. Torque generation mechanism of ATP synthase

    Science.gov (United States)

    Miller, John; Maric, Sladjana; Scoppa, M.; Cheung, M.

    2010-03-01

    ATP synthase is a rotary motor that produces adenosine triphosphate (ATP), the chemical currency of life. Our proposed electric field driven torque (EFT) model of FoF1-ATP synthase describes how torque, which scales with the number of c-ring proton binding sites, is generated by the proton motive force (pmf) across the mitochondrial inner membrane. When Fo is coupled to F1, the model predicts a critical pmf to drive ATP production. In order to fully understand how the electric field resulting from the pmf drives the c-ring to rotate, it is important to examine the charge distributions in the protonated c-ring and a-subunit containing the proton channels. Our calculations use a self-consistent field approach based on a refinement of reported structural data. The results reveal changes in pKa for key residues on the a-subunit and c-ring, as well as titration curves and protonation state energy diagrams. Health implications will be briefly discussed.

  11. [Four cases of aldosterone synthase deficiency in childhood].

    Science.gov (United States)

    Collinet, E; Pelissier, P; Richard, O; Gay, C; Pugeat, M; Morel, Y; Stephan, J-L

    2012-11-01

    Neonatal salt-wasting syndromes are rare but potentially serious conditions. Isolated hypoaldosteronism is an autosomal recessive inherited disorder of terminal aldosterone synthesis, leading to selective aldosterone deficiency. Two different biochemical forms of this disease have been described, called aldosterone synthase deficiency or corticosterone methyl oxydase, types I and II. In type I, there is no aldosterone synthase activity and the 18 hydroxycorticosterone (18 OHB) level is low, whereas in type II, a residual activity of aldosterone synthase persists and 18 OHB is overproduced. We report on four patients with isolated hypoaldosteronism. In 2 of them, who were recently diagnosed with aldosterone synthase deficit, we discuss the symptoms and treatment. The 2 other patients are now adults. We discuss the long-term outcome, the quality of adult life, aldosterone synthase deficits, as well as the pathophysiology and molecular analysis.

  12. Pseudouridines and pseudouridine synthases of the ribosome.

    Science.gov (United States)

    Ofengand, J; Malhotra, A; Remme, J; Gutgsell, N S; Del Campo, M; Jean-Charles, S; Peil, L; Kaya, Y

    2001-01-01

    psi are ubiquitous in ribosomal RNA. Eubacteria, Archaea, and eukaryotes all contain psi, although their number varies widely, with eukaryotes having the most. The small ribosomal subunit can apparently do without psi in some organisms, even though others have as many as 40 or more. Large subunits appear to need at least one psi but can have up to 50-60. psi is made by a set of site-specific enzymes in eubacteria, and in eukaryotes by a single enzyme complexed with auxiliary proteins and specificity-conferring guide RNAs. The mechanism is not known in Archaea, but based on an analysis of the kinds of psi synthases found in sequenced archaeal genomes, it is likely to involve use of guide RNAs. All psi synthases can be classified into one of four related groups, virtually all of which have a conserved aspartate residue in a conserved sequence motif. The aspartate is essential for psi formation in all twelve synthases examined so far. When the need for psi in E. coli was examined, the only synthase whose absence caused a major decrease in growth rate under normal conditions was RluD, the synthase that makes psi 1911, psi 1915, and psi 1917 in the helix 69 end-loop. This growth defect was the result of a major failure in assembly of the large ribosomal subunit. The defect could be prevented by supplying the rluD structural gene in trans, and also by providing a point mutant gene that made a synthase unable to make psi. Therefore, the RluD synthase protein appears to be directly involved in 50S subunit assembly, possibly as an RNA chaperone, and this activity is independent of its ability to form psi. This result is not without precedent. Depletion of PET56, a 2'-O-methyltransferase specific for G2251 (E. coli numbering) in yeast mitochondria virtually blocks 50S subunit assembly and mitochondrial function (Sirum-Connolly et al. 1995), but the methylation activity of the enzyme is not required (T. Mason, pers. comm.). The absence of FtsJ, a heat shock protein that makes

  13. A Comparison of the Effects of Neuronal Nitric Oxide Synthase and Inducible Nitric Oxide Synthase Inhibition on Cartilage Damage

    Directory of Open Access Journals (Sweden)

    Nevzat Selim Gokay

    2016-01-01

    Full Text Available The objective of this study was to investigate the effects of selective inducible nitric oxide synthase and neuronal nitric oxide synthase inhibitors on cartilage regeneration. The study involved 27 Wistar rats that were divided into five groups. On Day 1, both knees of 3 rats were resected and placed in a formalin solution as a control group. The remaining 24 rats were separated into 4 groups, and their right knees were surgically damaged. Depending on the groups, the rats were injected with intra-articular normal saline solution, neuronal nitric oxide synthase inhibitor 7-nitroindazole (50 mg/kg, inducible nitric oxide synthase inhibitor amino-guanidine (30 mg/kg, or nitric oxide precursor L-arginine (200 mg/kg. After 21 days, the right and left knees of the rats were resected and placed in formalin solution. The samples were histopathologically examined by a blinded evaluator and scored on 8 parameters. Although selective neuronal nitric oxide synthase inhibition exhibited significant (P=0.044 positive effects on cartilage regeneration following cartilage damage, it was determined that inducible nitric oxide synthase inhibition had no statistically significant effect on cartilage regeneration. It was observed that the nitric oxide synthase activation triggered advanced arthrosis symptoms, such as osteophyte formation. The fact that selective neuronal nitric oxide synthase inhibitors were observed to have mitigating effects on the severity of the damage may, in the future, influence the development of new agents to be used in the treatment of cartilage disorders.

  14. Characterization of olivetol synthase, a polyketide synthase putatively involved in cannabinoid biosynthetic pathway.

    Science.gov (United States)

    Taura, Futoshi; Tanaka, Shinji; Taguchi, Chiho; Fukamizu, Tomohide; Tanaka, Hiroyuki; Shoyama, Yukihiro; Morimoto, Satoshi

    2009-06-18

    Alkylresorcinol moieties of cannabinoids are derived from olivetolic acid (OLA), a polyketide metabolite. However, the polyketide synthase (PKS) responsible for OLA biosynthesis has not been identified. In the present study, a cDNA encoding a novel PKS, olivetol synthase (OLS), was cloned from Cannabis sativa. Recombinant OLS did not produce OLA, but synthesized olivetol, the decarboxylated form of OLA, as the major reaction product. Interestingly, it was also confirmed that the crude enzyme extracts from flowers and rapidly expanding leaves, the cannabinoid-producing tissues of C. sativa, also exhibited olivetol-producing activity, suggesting that the native OLS is functionally expressed in these tissues. The possibility that OLS could be involved in OLA biosynthesis was discussed based on its catalytic properties and expression profile.

  15. Transfer RNA pseudouridine synthases in Saccharomyces cerevisiae.

    Science.gov (United States)

    Samuelsson, T; Olsson, M

    1990-05-25

    A transfer RNA lacking modified nucleosides was produced by transcription in vitro of a cloned gene that encodes a Saccharomyces cerevisiae glycine tRNA. At least three different uridines (in nucleotide positions 13, 32, and 55) of this transcript tRNA are modified to pseudouridine by an extract of S. cerevisiae. Variants of the RNA substrate were also constructed that each had only one of these sites, thus allowing specific monitoring of pseudouridylation at different nucleotide positions. Using such RNAs to assay pseudouridine synthesis, enzymes producing this nucleoside were purified from an extract of S. cerevisiae. The activities corresponding to positions 13, 32, and 55 in the tRNA substrate could all be separated chromatographically, indicating that there is a separate enzyme for each of these sites. The enzyme specific for position 55 (denoted pseudouridine synthase 55) was purified approximately 4000-fold using a combination of DEAE-Sepharose, heparin-Sepharose, and hydroxylapatite.

  16. The nitric oxide synthase of mouse spermatozoa.

    Science.gov (United States)

    Herrero, M B; Goin, J C; Boquet, M; Canteros, M G; Franchi, A M; Perez Martinez, S; Polak, J M; Viggiano, J M; Gimeno, M A

    1997-07-01

    Nitric oxide synthase (NOS) was evidenced in mature mouse spermatozoa by means of biochemical techniques and Western blot. During 120 min of incubation, 10(7) spermatozoa synthesized 7 +/- 2 pmol of L-[14C]citrulline. Besides, L-citrulline formation depended on the incubation time and on the concentration of L-arginine present in the incubation medium. Different concentrations of N(G)-nitro-L-arginine methyl ester (L-NAME) but not aminoguanidine, inhibited L-[14C]citrulline formation. Western-blot analysis of solubilized sperm proteins revealed a unique band of M(r)=140 kDa with the neural, endothelial and inducible NOS antisera tested. These results provide evidence that mature mouse sperm contains a NOS isoform and that spermatozoa have the potential ability to synthesize NO, suggesting a role for endogenous NO on mammalian sperm function.

  17. Endothelial nitric oxide synthase in the microcirculation.

    Science.gov (United States)

    Shu, Xiaohong; Keller, T C Stevenson; Begandt, Daniela; Butcher, Joshua T; Biwer, Lauren; Keller, Alexander S; Columbus, Linda; Isakson, Brant E

    2015-12-01

    Endothelial nitric oxide synthase (eNOS, NOS3) is responsible for producing nitric oxide (NO)--a key molecule that can directly (or indirectly) act as a vasodilator and anti-inflammatory mediator. In this review, we examine the structural effects of regulation of the eNOS enzyme, including post-translational modifications and subcellular localization. After production, NO diffuses to surrounding cells with a variety of effects. We focus on the physiological role of NO and NO-derived molecules, including microvascular effects on vessel tone and immune response. Regulation of eNOS and NO action is complicated; we address endogenous and exogenous mechanisms of NO regulation with a discussion of pharmacological agents used in clinical and laboratory settings and a proposed role for eNOS in circulating red blood cells.

  18. A Single Amino Acid Substitution Converts Benzophenone Synthase into Phenylpyrone Synthase*

    Science.gov (United States)

    Klundt, Tim; Bocola, Marco; Lütge, Maren; Beuerle, Till; Liu, Benye; Beerhues, Ludger

    2009-01-01

    Benzophenone metabolism provides a number of plant natural products with fascinating chemical structures and intriguing pharmacological activities. Formation of the carbon skeleton of benzophenone derivatives from benzoyl-CoA and three molecules of malonyl-CoA is catalyzed by benzophenone synthase (BPS), a member of the superfamily of type III polyketide synthases. A point mutation in the active site cavity (T135L) transformed BPS into a functional phenylpyrone synthase (PPS). The dramatic change in both substrate and product specificities of BPS was rationalized by homology modeling. The mutation may open a new pocket that accommodates the phenyl moiety of the triketide intermediate but limits polyketide elongation to two reactions, resulting in phenylpyrone formation. 3-Hydroxybenzoyl-CoA is the second best starter molecule for BPS but a poor substrate for PPS. The aryl moiety of the triketide intermediate may be trapped in the new pocket by hydrogen bond formation with the backbone, thereby acting as an inhibitor. PPS is a promising biotechnological tool for manipulating benzoate-primed biosynthetic pathways to produce novel compounds. PMID:19710020

  19. Structure and Function of Fusicoccadiene Synthase, a Hexameric Bifunctional Diterpene Synthase.

    Science.gov (United States)

    Chen, Mengbin; Chou, Wayne K W; Toyomasu, Tomonobu; Cane, David E; Christianson, David W

    2016-04-15

    Fusicoccin A is a diterpene glucoside phytotoxin generated by the fungal pathogen Phomopsis amygdali that causes the plant disease constriction canker, first discovered in New Jersey peach orchards in the 1930s. Fusicoccin A is also an emerging new lead in cancer chemotherapy. The hydrocarbon precursor of fusicoccin A is the tricyclic diterpene fusicoccadiene, which is generated by a bifunctional terpenoid synthase. Here, we report X-ray crystal structures of the individual catalytic domains of fusicoccadiene synthase: the C-terminal domain is a chain elongation enzyme that generates geranylgeranyl diphosphate, and the N-terminal domain catalyzes the cyclization of geranylgeranyl diphosphate to form fusicoccadiene. Crystal structures of each domain complexed with bisphosphonate substrate analogues suggest that three metal ions and three positively charged amino acid side chains trigger substrate ionization in each active site. While in vitro incubations reveal that the cyclase domain can utilize farnesyl diphosphate and geranyl diphosphate as surrogate substrates, these shorter isoprenoid diphosphates are mainly converted into acyclic alcohol or hydrocarbon products. Gel filtration chromatography and analytical ultracentrifugation experiments indicate that full-length fusicoccadiene synthase adopts hexameric quaternary structure, and small-angle X-ray scattering data yield a well-defined molecular envelope illustrating a plausible model for hexamer assembly.

  20. Irradiations y Jusqu'a 109 Rads De Fibres Toute Silice Comparaisons Avec Des Fibres Pcs Et Des Echantillons Massifs Entre 0,4 Et 2,5 Microns

    Science.gov (United States)

    Boisde, G.; Bonnejean, C.; Perez, J. J.; Neumann, V.; Wurier, B.; Boucher, D.

    1984-03-01

    The effect of y radiation (Co60 ) on optical fibers is studied for investigations to remote measurement in irradiating environments. The spectral characteristics of PCS and AS silica fibers, PCS "Wet" and AS "wet" and "superwet" are compared. An automatic computerized system for measurement "in situ" is used between 0,4 and 1,1 micron. Radiation-induced effects are examinated at 840 nm on PCS, AS with silicone, AS with epoxy (200 and 400/6004m) up to 5.108 rads.Samples of "dry", "wet", "superwet" and "doped fluor" silica are studied up to 1,3.109 rads between 0,3 and 2,5μm. The photobleaching, the radiation hardening and the breakeage of PCS and AS-silicone after 107 rads are recognized. The spectral characte-ristics of a band at 610-630 nm, a spectral deformation at 945 nm for "wet" fibers and a peak of the radiation-induced attenuation are observed. The kinetic of the 630 nm defect in silica fibers and silica samples is also investigated.

  1. Application of Ti Powder on Fabrication of Multiphase Ceramics by PCS Pyrolysis%钛粉在裂解聚碳硅烷制备陶瓷材料中的应用

    Institute of Scientific and Technical Information of China (English)

    周长江; 魏红; 陈朝辉

    2001-01-01

    Pyrolytic conversion of polycarbosilane(PCS) into ceramic materials can be significantly influenced by active filler(Ti) dispersoids.During pyrolytic decomposition of the PCS matrix,Ti can accelerate PCS pyrolysis and increase ceramic yields of PCS.The linear shrinkage of the preceramic polymers in pyrolyzing and porosity of ceramic products will decrease with the addition of Ti powders,and properties of the ceramic materials can be improved as a result.%以聚碳硅烷为先驱体,Ti粉为活性填料,研究了Ti粉对聚碳硅烷裂解反应及先驱体转化法制备的块体复相陶瓷的性能的影响,并表征了其晶相组成。结果表明,Ti粉可促进PCs的裂解反应,增加先驱体的陶瓷产率,能降低先驱体在裂解过程中的线性收缩率和气孔率,提高陶瓷材料性能。

  2. Role of cysteine residues in pseudouridine synthases of different families.

    Science.gov (United States)

    Ramamurthy, V; Swann, S L; Spedaliere, C J; Mueller, E G

    1999-10-01

    The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine in RNA molecules. An attractive mechanism was proposed based on that of thymidylate synthase, in which the thiol(ate) group of a cysteine side chain serves as the nucleophile in a Michael addition to C6 of the isomerized uridine. Such a role for cysteine in the pseudouridine synthase TruA (also named Psi synthase I) has been discredited by site-directed mutagenesis, but sequence alignments have led to the conclusion that there are four distinct "families" of pseudouridine synthases that share no statistically significant global sequence similarity. It was, therefore, necessary to probe the role of cysteine residues in pseudouridine synthases of the families that do not include TruA. We examined the enzymes RluA and TruB, which are members of different families than TruA and each other. Substitution of cysteine for amino acids with nonnucleophilic side chains did not significantly alter the catalytic activity of either pseudouridine synthase. We conclude, therefore, that neither TruB nor RluA require thiol(ate) groups to effect catalysis, excluding their participation in a Michael addition to C6 of uridine, although not eliminating that mechanism (with an alternate nucleophile) from future consideration.

  3. Subcellular localization of the homocitrate synthase in Penicillium chrysogenum.

    Science.gov (United States)

    Bañuelos, O; Casqueiro, J; Steidl, S; Gutiérrez, S; Brakhage, A; Martín, J F

    2002-01-01

    There are conflicting reports regarding the cellular localization in Saccharomyces cerevisiae and filamentous fungi of homocitrate synthase, the first enzyme in the lysine biosynthetic pathway. The homocitrate synthase (HS) gene (lys1) of Penicillium chrysogenum was disrupted in three transformants (HS(-)) of the Wis 54-1255 pyrG strain. The three mutants named HS1(-), HS2(-) and HS3(-) all lacked homocitrate synthase activity and showed lysine auxotrophy, indicating that there is a single gene for homocitrate synthase in P. chrysogenum. The lys1 ORF was fused in frame to the gene for the green fluorescent protein (GFP) gene of the jellyfish Aequorea victoria. Homocitrate synthase-deficient mutants transformed with a plasmid containing the lys1-GFP fusion recovered prototrophy and showed similar levels of homocitrate synthase activity to the parental strain Wis 54-1255, indicating that the hybrid protein retains the biological function of wild-type homocitrate synthase. Immunoblotting analysis revealed that the HS-GFP fusion protein is maintained intact and does not release the GFP moiety. Fluorescence microscopy analysis of the transformants showed that homocitrate synthase was mainly located in the cytoplasm in P. chrysogenum; in S. cerevisiae the enzyme is targeted to the nucleus. The control nuclear protein StuA was properly targeted to the nucleus when the StuA (targeting domain)-GFP hybrid protein was expressed in P. chrysogenum. The difference in localization of homocitrate synthase between P. chrysogenum and S. cerevisiae suggests that this protein may play a regulatory function, in addition to its catalytic function, in S. cerevisiae but not in P. chrysogenum.

  4. The Pseudouridine Synthases Proceed through a Glycal Intermediate.

    Science.gov (United States)

    Veerareddygari, Govardhan Reddy; Singh, Sanjay K; Mueller, Eugene G

    2016-06-29

    The pseudouridine synthases isomerize (U) in RNA to pseudouridine (Ψ), and the mechanism that they follow has long been a question of interest. The recent elucidation of a product of the mechanistic probe 5-fluorouridine that had been epimerized to the arabino isomer suggested that the Ψ synthases might operate through a glycal intermediate formed by deprotonation of C2'. When that position in substrate U is deuterated, a primary kinetic isotope effect is observed, which indisputably indicates that the proposed deprotonation occurs during the isomerization of U to Ψ and establishes the mechanism followed by the Ψ synthases.

  5. Peroxisomal and mitochondrial citrate synthase in CAM plants.

    Science.gov (United States)

    Zafra, M F; Segovia, J L; Alejandre, M J; García-Peregrín, E

    1981-12-01

    Citrate synthase wa studied for the first time in peroxisomes and mitochondria of crassulacean acid metabolism plants. Cellular organelles were isolated from Agave americana leaves by sucrose density gradient centrifugation and characterized by the use of catalase and cytochrome oxidase as marker enzymes, respectively. 48,000 X g centrifugation caused the breakdown of the cellular organelles. The presence of a glyoxylate cycle enzyme (citrate synthase) and a glycollate pathway enzyme (catalase) in the same organelles, besides the absence of another glyoxalate cycle enzyme (malate synthase) is reported for the first time, suggesting that peroxisomal and glyoxysomal proteins are synthesized at the same time and housed in he same organelle.

  6. PCR method for the rapid detection and discrimination of Legionella spp. based on the amplification of pcs, pmtA, and 16S rRNA genes.

    Science.gov (United States)

    Janczarek, Monika; Palusińska-Szysz, Marta

    2016-05-01

    Legionella bacteria are organisms of public health interest due to their ability to cause pneumonia (Legionnaires' disease) in susceptible humans and their ubiquitous presence in water supply systems. Rapid diagnosis of Legionnaires' disease allows the use of therapy specific for the disease. L. pneumophila serogroup 1 is the most common cause of infection acquired in community and hospital environments. The non-L. pneumophila infections are likely under-detected because of a lack of effective diagnosis. In this work, simplex and duplex PCR assays with the use of new molecular markers pcs and pmtA involved in phosphatidylcholine synthesis were specified for rapid and cost-efficient identification and distinguishing Legionella species. The sets of primers developed were found to be sensitive and specific for reliable detection of Legionella belonging to the eight most clinically relevant species. Among these, four primer sets I, II, VI, and VII used for duplex-PCRs proved to have the highest identification power and reliability in the detection of the bacteria. Application of this PCR-based method should improve detection of Legionella spp. in both clinical and environmental settings and facilitate molecular typing of these organisms.

  7. A 3 to 5 GHz low-phase-noise fractional-N frequency synthesizer with adaptive frequency calibration for GSM/PCS/DCS/WCDMA transceivers

    Institute of Scientific and Technical Information of China (English)

    Pan Yaohua; Mei Niansong; Chen Hu; Huang Yumei; Hong Zhiliang

    2012-01-01

    A low-phase-noise ∑-△ fractional-N frequency synthesizer for GSM/PCS/DCS/WCDMA transceivers is presented.The voltage controlled oscillator is designed with a modified digital controlled capacitor array to extend the tuning range and minimize phase noise.A high-resolution adaptive frequency calibration technique is introduced to automatically choose frequency bands and increase phase-noise immunity.A prototype is implemented in 0.13 μm CMOS technology.The experimental results show that the designed 1.2 V wideband frequency synthesizer is locked from 3.05 to 5.17 GHz within 30μs,which covers all five required frequency bands.The measured in-band phase noise are -89,-95.5 and -101 dBc/Hz for 3.8 GHz,2 GHz and 948 MHz carriers,respectively,and accordingly the out-of-band phase noise are -121,-123 and -132 dBc/Hz at 1 MHz offset,which meet the phase-noise-mask requirements of the above-mentioned standards.

  8. Prostaglandin H synthase immunoreactivity in human gut. An immunohistochemical study

    DEFF Research Database (Denmark)

    Mikkelsen, H B; Rumessen, J J; Qvortrup, Klaus

    1991-01-01

    Prostaglandins exhibit a variety of actions on intestinal smooth muscle depending upon the type, dose and muscle layer studied. As the cellular origin of prostaglandin H (PGH) synthase has not been established with certainty in the human gut wall, we studied the localization of PGH synthase...... in the human duodenum, jejunum, ileum and colon by immunohistochemistry. PGH synthase immunoreactivity appeared to be similar in all segments of the intestine. Most smooth muscle cells seemed to contain PGH synthase; however, the reaction in the lamina muscularis mucosae was much stronger than...... in the longitudinal and circular muscle layers. Endothelial cells in capillaries and larger vessels showed a positive reaction. In addition, unidentified cells in subserosa, at the level of Auerbach's plexus and in the submucosa were stained. We concluded that the smooth muscle cells of the human gut has a rather...

  9. Cooperativity of peptidoglycan synthases active in bacterial cell elongation.

    NARCIS (Netherlands)

    Banzhaf, M.; van den Berg van Saparoea, B.; Terrak, M.; Fraipont, C.; Egan, A.; Philippe, J.; Zapun, A.; Breukink, E.; Nguyen-Distèche, M.; den Blaauwen, T.; Vollmer, W.

    2012-01-01

    Growth of the bacterial cell wall peptidoglycan sacculus requires the co-ordinated activities of peptidoglycan synthases, hydrolases and cell morphogenesis proteins, but the details of these interactions are largely unknown. We now show that the Escherichia coli peptidoglycan

  10. Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth.

    Directory of Open Access Journals (Sweden)

    Zulfiqar Ahmad

    Full Text Available We examined the thymoquinone induced inhibition of purified F1 or membrane bound F1FO E. coli ATP synthase. Both purified F1 and membrane bound F1FO were completely inhibited by thymoquinone with no residual ATPase activity. The process of inhibition was fully reversible and identical in both membrane bound F1Fo and purified F1 preparations. Moreover, thymoquinone induced inhibition of ATP synthase expressing wild-type E. coli cell growth and non-inhibition of ATPase gene deleted null control cells demonstrates that ATP synthase is a molecular target for thymoquinone. This also links the beneficial dietary based antimicrobial and anticancer effects of thymoquinone to its inhibitory action on ATP synthase.

  11. Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth.

    Science.gov (United States)

    Ahmad, Zulfiqar; Laughlin, Thomas F; Kady, Ismail O

    2015-01-01

    We examined the thymoquinone induced inhibition of purified F1 or membrane bound F1FO E. coli ATP synthase. Both purified F1 and membrane bound F1FO were completely inhibited by thymoquinone with no residual ATPase activity. The process of inhibition was fully reversible and identical in both membrane bound F1Fo and purified F1 preparations. Moreover, thymoquinone induced inhibition of ATP synthase expressing wild-type E. coli cell growth and non-inhibition of ATPase gene deleted null control cells demonstrates that ATP synthase is a molecular target for thymoquinone. This also links the beneficial dietary based antimicrobial and anticancer effects of thymoquinone to its inhibitory action on ATP synthase.

  12. Sequence analysis of cereal sucrose synthase genes and isolation ...

    African Journals Online (AJOL)

    SERVER

    2007-10-18

    Oct 18, 2007 ... 1Department of Environmental Biotechnology, Bharathidasan University, ... script and UA cloning vector (QIAGEN PCR Cloning Kit) was used to clone ..... Expression of a Arabidopsis sucrose synthase gene indicates a role.

  13. Insulin transcriptionally regulates argininosuccinate synthase to maintain vascular endothelial function

    OpenAIRE

    Haines, Ricci J.; Corbin, Karen D.; Pendleton, Laura C; Meininger, Cynthia J; Eichler, Duane C.

    2012-01-01

    Diminished vascular endothelial cell nitric oxide (NO) production is a major factor in the complex pathogenesis of diabetes mellitus. In this report, we demonstrate that insulin not only maintains endothelial NO production through regulation of endothelial nitric oxide synthase (eNOS), but also via the regulation of argininosuccinate synthase (AS), which is the rate-limiting step of the citrulline-NO cycle. Using serum starved, cultured vascular endothelial cells, we show that insulin up-regu...

  14. Understanding plant cellulose synthases through a comprehensive investigation of the cellulose synthase family sequences.

    Directory of Open Access Journals (Sweden)

    Andrew eCarroll

    2011-03-01

    Full Text Available The development of cellulose as an organizing structure in the plant cell wall was a key event in both the initial colonization and the subsequent domination of the terrestrial ecosystem by vascular plants. A wealth of experimental data has demonstrated the complicated genetic interactions required to form the large synthetic complex that synthesizes cellulose. However, these results are lacking an extensive analysis of the evolution, specialization, and regulation of the proteins that compose this complex. Here we perform an in-depth analysis of the sequences in the cellulose synthase (CesA family. We investigate the phylogeny of the CesA family, with emphasis on evolutionary specialization. We define specialized subfamilies and identify the class-specific regions within the CesA sequence that may explain this specialization. We investigate changes in regulation of CesAs by looking at the conservation of proposed phosphorylation sites. We investigate the conservation of sites where mutations have been documented that impair cellulose synthase function, and compare these sites to those observed in the closest cellulose synthase-like (Csl families to better understand what regions may separate the CesAs from other Csls. Finally we identify two positions with strong conservation of the aromatic trait, but lacking conservation of amino acid identity, which may represent residues important for positioning the sugar substrate for catalysis. These analyses provide useful tools for understanding characterized mutations and post-translational modifications, and for informing further experiments to probe CesA assembly, regulation, and function through site-directed mutagenesis or domain swapping experiments.

  15. Homocystinuria due to cystathionine beta synthase deficiency

    Directory of Open Access Journals (Sweden)

    Rao T

    2008-01-01

    Full Text Available A two year-old male child presented with cutis marmorata congenita universalis, brittle hair, mild mental retardation, and finger spasms. Biochemical findings include increased levels of homocysteine in the blood-106.62 µmol/L (normal levels: 5.90-16µmol/L. Biochemical tests such as the silver nitroprusside and nitroprusside tests were positive suggesting homocystinuria. The patient was treated with oral pyridoxine therapy for three months. The child responded well to this therapy and the muscle spasms as well as skin manifestations such as cutis marmorata subsided. The treatment is being continued; the case is reported here because of its rarity. Homocysteinuria arising due to cystathionine beta-synthase (CBS deficiency is an autosomal recessive disorder of methionine metabolism that produces increased levels of urinary homocysteine and methionine It manifests itself in vascular, central nervous system, cutaneous, and connective tissue disturbances and phenotypically resembles Marfan′s syndrome. Skin manifestations include malar flush, thin hair, and cutis reticulata / marmorata.

  16. Nitric Oxide Synthases in Heart Failure

    Science.gov (United States)

    Carnicer, Ricardo; Crabtree, Mark J.; Sivakumaran, Vidhya

    2013-01-01

    Abstract Significance: The regulation of myocardial function by constitutive nitric oxide synthases (NOS) is important for the maintenance of myocardial Ca2+ homeostasis, relaxation and distensibility, and protection from arrhythmia and abnormal stress stimuli. However, sustained insults such as diabetes, hypertension, hemodynamic overload, and atrial fibrillation lead to dysfunctional NOS activity with superoxide produced instead of NO and worse pathophysiology. Recent Advances: Major strides in understanding the role of normal and abnormal constitutive NOS in the heart have revealed molecular targets by which NO modulates myocyte function and morphology, the role and nature of post-translational modifications of NOS, and factors controlling nitroso-redox balance. Localized and differential signaling from NOS1 (neuronal) versus NOS3 (endothelial) isoforms are being identified, as are methods to restore NOS function in heart disease. Critical Issues: Abnormal NOS signaling plays a key role in many cardiac disorders, while targeted modulation may potentially reverse this pathogenic source of oxidative stress. Future Directions: Improvements in the clinical translation of potent modulators of NOS function/dysfunction may ultimately provide a powerful new treatment for many hearts diseases that are fueled by nitroso-redox imbalance. Antioxid. Redox Signal. 18, 1078–1099. PMID:22871241

  17. Understanding structure, function, and mutations in the mitochondrial ATP synthase

    Directory of Open Access Journals (Sweden)

    Ting Xu

    2015-03-01

    Full Text Available The mitochondrial ATP synthase is a multimeric enzyme complex with an overall molecular weight of about 600,000 Da. The ATP synthase is a molecular motor composed of two separable parts: F1 and Fo. The F1 portion contains the catalytic sites for ATP synthesis and protrudes into the mitochondrial matrix. Fo forms a proton turbine that is embedded in the inner membrane and connected to the rotor of F1. The flux of protons flowing down a potential gradient powers the rotation of the rotor driving the synthesis of ATP. Thus, the flow of protons though Fo is coupled to the synthesis of ATP. This review will discuss the structure/function relationship in the ATP synthase as determined by biochemical, crystallographic, and genetic studies. An emphasis will be placed on linking the structure/function relationship with understanding how disease causing mutations or putative single nucleotide polymorphisms (SNPs in genes encoding the subunits of the ATP synthase, will affect the function of the enzyme and the health of the individual. The review will start by summarizing the current understanding of the subunit composition of the enzyme and the role of the subunits followed by a discussion on known mutations and their effect on the activity of the ATP synthase. The review will conclude with a summary of mutations in genes encoding subunits of the ATP synthase that are known to be responsible for human disease, and a brief discussion on SNPs.

  18. Linking pseudouridine synthases to growth, development and cell competition.

    Science.gov (United States)

    Tortoriello, Giuseppe; de Celis, José F; Furia, Maria

    2010-08-01

    Eukaryotic pseudouridine synthases direct RNA pseudouridylation and bind H/ACA small nucleolar RNA (snoRNAs), which, in turn, may act as precursors of microRNA-like molecules. In humans, loss of pseudouridine synthase activity causes dyskeratosis congenita (DC), a complex systemic disorder characterized by cancer susceptibility, failures in ribosome biogenesis and telomere stability, and defects in stem cell formation. Considering the significant interest in deciphering the various molecular consequences of pseudouridine synthase failure, we performed a loss of function analysis of minifly (mfl), the pseudouridine synthase gene of Drosophila, in the wing disc, an advantageous model system for studies of cell growth and differentiation. In this organ, depletion of the mfl-encoded pseudouridine synthase causes a severe reduction in size by decreasing both the number and the size of wing cells. Reduction of cell number was mainly attributable to cell death rather than reduced proliferation, establishing that apoptosis plays a key role in the development of the loss of function mutant phenotype. Depletion of Mfl also causes a proliferative disadvantage in mosaic tissues that leads to the elimination of mutant cells by cell competition. Intriguingly, mfl silencing also triggered unexpected effects on wing patterning and cell differentiation, including deviations from normal lineage boundaries, mingling of cells of different compartments, and defects in the formation of the wing margin that closely mimic the phenotype of reduced Notch activity. These results suggest that a component of the pseudouridine synthase loss of function phenotype is caused by defects in Notch signalling.

  19. Alendronate is a specific, nanomolar inhibitor of farnesyl diphosphate synthase.

    Science.gov (United States)

    Bergstrom, J D; Bostedor, R G; Masarachia, P J; Reszka, A A; Rodan, G

    2000-01-01

    Alendronate, a nitrogen-containing bisphosphonate, is a potent inhibitor of bone resorption used for the treatment and prevention of osteoporosis. Recent findings suggest that alendronate and other N-containing bisphosphonates inhibit the isoprenoid biosynthesis pathway and interfere with protein prenylation, as a result of reduced geranylgeranyl diphosphate levels. This study identified farnesyl disphosphate synthase as the mevalonate pathway enzyme inhibited by bisphosphonates. HPLC analysis of products from a liver cytosolic extract narrowed the potential targets for alendronate inhibition (IC(50) = 1700 nM) to isopentenyl diphosphate isomerase and farnesyl diphosphate synthase. Recombinant human farnesyl diphosphate synthase was inhibited by alendronate with an IC(50) of 460 nM (following 15 min preincubation). Alendronate did not inhibit isopentenyl diphosphate isomerase or GGPP synthase, partially purified from liver cytosol. Recombinant farnesyl diphosphate synthase was also inhibited by pamidronate (IC(50) = 500 nM) and risedronate (IC(50) = 3.9 nM), negligibly by etidronate (IC50 = 80 microM), and not at all by clodronate. In osteoclasts, alendronate inhibited the incorporation of [(3)H]mevalonolactone into proteins of 18-25 kDa and into nonsaponifiable lipids, including sterols. These findings (i) identify farnesyl diphosphate synthase as the selective target of alendronate in the mevalonate pathway, (ii) show that this enzyme is inhibited by other N-containing bisphosphonates, such as risendronate, but not by clodronate, supporting a different mechanism of action for different bisphosphonates, and (iii) document in purified osteoclasts alendronate inhibition of prenylation and sterol biosynthesis.

  20. Dehydration induces expression of GALACTINOL SYNTHASE and RAFFINOSE SYNTHASE in seedlings of pea (Pisum sativum L.).

    Science.gov (United States)

    Lahuta, Lesław B; Pluskota, Wioletta E; Stelmaszewska, Joanna; Szablińska, Joanna

    2014-09-01

    The exposition of 7-day-old pea seedlings to dehydration induced sudden changes in the concentration of monosaccharides and sucrose in epicotyl and roots tissues. During 24h of dehydration, the concentration of glucose and, to a lesser extent, fructose in seedling tissues decreased. The accumulation of sucrose was observed in roots after 4h and in epicotyls after 8h of stress. Epicotyls and roots also began to accumulate galactinol and raffinose after 8h of stress, when small changes in the water content of tissues occurred. The accumulation of galactinol and raffinose progressed parallel to water withdrawal from tissues, but after seedling rehydration both galactosides disappeared. The synthesis of galactinol and raffinose by an early induction (during the first hour of treatment) of galactinol synthase (PsGolS) and raffinose synthase (PsRS) gene expression as well as a later increase in the activity of both enzymes was noted. Signals possibly triggering the induction of PsGolS and PsRS gene expression and accumulation of galactinol and raffinose in seedlings are discussed.

  1. Cloning and characterization of squalene synthase and cycloartenol synthase from Siraitia grosvenorii

    Directory of Open Access Journals (Sweden)

    Huan Zhao

    2017-03-01

    Full Text Available Mogrosides and steroid saponins are tetracyclic triterpenoids found in Siraitia grosvenorii. Squalene synthase (SQS and cycloartenol synthase (CAS are key enzymes in triterpenoid and steroid biosynthesis. In this study, full-length cDNAs of SgSQS and SgCAS were cloned by a rapid amplification of cDNA-ends with polymerase chain reaction (RACE-PCR approach. The SgSQS cDNA has a 1254 bp open reading frame (ORF encoding 417 amino acids, and the SgCAS cDNA contains a 2298 bp ORF encoding 765 amino acids. Bioinformatic analysis showed that the deduced SgSQS protein has two transmembrane regions in the C-terminal. Both SgSQS and SgCAS have significantly higher levels in fruits than in other tissues, suggesting that steroids and mogrosides are competitors for the same precursors in fruits. Combined in silico prediction and subcellular localization, experiments in tobacco indicated that SgSQS was probably in the cytoplasm or on the cytoskeleton, and SgCAS was likely located in the nucleus or cytosol. These results will provide a foundation for further study of SgSQS and SgCAS gene functions in S. grosvenorii, and may facilitate improvements in mogroside content in fruit by regulating gene expression.

  2. Characterisation of the tryptophan synthase alpha subunit in maize

    Directory of Open Access Journals (Sweden)

    Gierl Alfons

    2008-04-01

    Full Text Available Abstract Background In bacteria, such as Salmonella typhimurium, tryptophan is synthesized from indole-3-glycerole phosphate (IGP by a tryptophan synthase αββα heterotetramer. Plants have evolved multiple α (TSA and β (TSB homologs, which have probably diverged in biological function and their ability of subunit interaction. There is some evidence for a tryptophan synthase (TS complex in Arabidopsis. On the other hand maize (Zea mays expresses the TSA-homologs BX1 and IGL that efficiently cleave IGP, independent of interaction with TSB. Results In order to clarify, how tryptophan is synthesized in maize, two TSA homologs, hitherto uncharacterized ZmTSA and ZmTSAlike, were functionally analyzed. ZmTSA is localized in plastids, the major site of tryptophan biosynthesis in plants. It catalyzes the tryptophan synthase α-reaction (cleavage of IGP, and forms a tryptophan synthase complex with ZmTSB1 in vitro. The catalytic efficiency of the α-reaction is strongly enhanced upon complex formation. A 160 kD tryptophan synthase complex was partially purified from maize leaves and ZmTSA was identified as native α-subunit of this complex by mass spectrometry. ZmTSAlike, for which no in vitro activity was detected, is localized in the cytosol. ZmTSAlike, BX1, and IGL were not detectable in the native tryptophan synthase complex in leaves. Conclusion It was demonstrated in vivo and in vitro that maize forms a tryptophan synthase complex and ZmTSA functions as α-subunit in this complex.

  3. Administrative simplification: change to the compliance date for the International Classification of Diseases, 10th Revision (ICD-10-CM and ICD-10-PCS) medical data code sets. Final rule.

    Science.gov (United States)

    2014-08-04

    This final rule implements section 212 of the Protecting Access to Medicare Act of 2014 by changing the compliance date for the International Classification of Diseases, 10th Revision, Clinical Modification (ICD-10-CM) for diagnosis coding, including the Official ICD-10-CM Guidelines for Coding and Reporting, and the International Classification of Diseases, 10th Revision, Procedure Coding System (ICD-10-PCS) for inpatient hospital procedure coding, including the Official ICD-10-PCS Guidelines for Coding and Reporting, from October 1, 2014 to October 1, 2015. It also requires the continued use of the International Classification of Diseases, 9th Revision, Clinical Modification, Volumes 1 and 2 (diagnoses), and 3 (procedures) (ICD-9-CM), including the Official ICD-9-CM Guidelines for Coding and Reporting, through September 30, 2015.

  4. Bacillus caldolyticus prs gene encoding phosphoribosyl-diphosphate synthase

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1996-01-01

    The prs gene, encoding phosphoribosyl-diphosphate (PRPP) synthase, as well as the flanking DNA sequences were cloned and sequenced from the Gram-positive thermophile, Bacillus caldolyticus. Comparison with the homologous sequences from the mesophile, Bacillus subtilis, revealed a gene (gca......D) encoding N-acetylglucosamine-1-phosphate uridyltransferase upstream of prs, and a gene homologous to ctc downstream of prs. cDNA synthesis with a B. caldolyticus gcaD-prs-ctc-specified mRNA as template, followed by amplification utilising the polymerase chain reaction indicated that the three genes are co......-transcribed. Comparison of amino acid sequences revealed a high similarity among PRPP synthases across a wide phylogenetic range. An E. coli strain harbouring the B. caldolyticus prs gene in a multicopy plasmid produced PRPP synthase activity 33-fold over the activity of a haploid B. caldolyticus strain. B. caldolyticus...

  5. Properties of peroxisomal and mitochondrial citrate synthase from Agave americana.

    Science.gov (United States)

    Segovia, J L; Zafra, M F; Alejandre, M J; García-Peregrín, E

    1982-09-01

    Adenine nucleotides were tested as effectors of peroxisomal and mitochondrial citrate synthase from Agave americana leaves in the presence of different concentrations of acetyl-CoA and oxalacetate substrates. ATP inhibited both enzyme activities but with a different inhibition profile. 1.0-7.5 mM ADP did not inhibit the peroxisomal citrate synthase in the presence of high substrate concentrations, while the mitochondrial enzyme was strongly inhibited by 1.0 mM ADP in the same conditions. Likewise, a different pattern was obtained with AMP on both peroxisomal and mitochondrial activities. The rate of citrate formation as function of acetyl-CoA and oxalacetate concentration was also studied in both fractions. Maximal velocity was highest in the peroxisomal fraction, whether acetyl-CoA or oxalacetate were the variable substrates. These differences indicate that peroxisomal and mitochondrial citrate synthases seem to be two different isoenzymes.

  6. Solubilization of microsomal-associated phosphatidylinositol synthase from germinating soybeans.

    Science.gov (United States)

    Robinson, M L; Carman, G M

    1982-01-01

    CDP-1,2-diacyl-sn-glycerol (CDP-diacylglycerol):myo-inositol phosphatidyltransferase (EC 2.7.8.11, phosphatidylinositol synthase) catalyzes the final step in the de novo synthesis of phosphatidylinositol in the endoplasmic reticulum fraction of germinating soybeans (Glycine max L. var Cutler 71). A variety of solubilization agents were examined for their ability to release phosphatidylinositol synthase activity from the microsome fraction. The most effective agent to solubilize the enzyme was the nonionic detergent Brij W-1. A 2.1-fold increase in specific activity was achieved using 1% Brij W-1 with 69% activity solubilized.Maximal solubilization of phosphatidylinositol synthase was completely dependent on Brij W-1 (1%), potassium ions (0.3 m), and manganese ions (0.5 mm). Solubilization of the enzyme was not affected by the protein concentration of microsomes between 3 to 20 milligrams per milliliter. Solubilization was not affected by the pH of solubilization buffer between 6.5 to 8.5. To our knowledge, this is the first phospholipid biosynthetic enzyme solubilized from plant membranes. The Brij W-1-solubilized phosphatidylinositol synthase remained at the top of a glycerol gradient, whereas the membrane-associated enzyme sedimented to the bottom of the gradient. Maximal activity of the Brij W-1-solubilized phosphatidylinositol synthase was dependent on manganese (5 mm) or magnesium (30 mm) ions, and Triton X-100 (3.6 mm) at pH 8.0 with Tris-HCl buffer. The apparent K(m) values for CDP-diacylglycerol and myo-inositol for the solubilized enzyme was 0.1 mm and 46 mum, respectively. Solubilized phosphatidylinositol synthase activity was thermally inactivated at temperatures above 30 degrees C.

  7. Exploiting the Biosynthetic Potential of Type III Polyketide Synthases

    Directory of Open Access Journals (Sweden)

    Yan Ping Lim

    2016-06-01

    Full Text Available Polyketides are structurally and functionally diverse secondary metabolites that are biosynthesized by polyketide synthases (PKSs using acyl-CoA precursors. Recent studies in the engineering and structural characterization of PKSs have facilitated the use of target enzymes as biocatalysts to produce novel functionally optimized polyketides. These compounds may serve as potential drug leads. This review summarizes the insights gained from research on type III PKSs, from the discovery of chalcone synthase in plants to novel PKSs in bacteria and fungi. To date, at least 15 families of type III PKSs have been characterized, highlighting the utility of PKSs in the development of natural product libraries for therapeutic development.

  8. Inhibition of Escherichia coli ATP synthase by amphibian antimicrobial peptides

    OpenAIRE

    2010-01-01

    Previously melittin, the α-helical basic honey bee venom peptide, was shown to inhibit F1-ATPase by binding at the β-subunit DELSEED motif of F1Fo ATP synthase. Herein, we present the inhibitory effects of the basic α-helical amphibian antimicrobial peptides, ascaphin-8, aurein 2.2, aurein 2.3, carein 1.8, carein 1.9, citropin 1.1, dermaseptin, maculatin 1.1, maganin II, MRP, or XT-7, on purified F1 and membrane bound F1Fo E. coli ATP synthase. We found that the extent of inhibition by amphib...

  9. Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth

    OpenAIRE

    2015-01-01

    We examined the thymoquinone induced inhibition of purified F1 or membrane bound F1FO E. coli ATP synthase. Both purified F1 and membrane bound F1FO were completely inhibited by thymoquinone with no residual ATPase activity. The process of inhibition was fully reversible and identical in both membrane bound F1Fo and purified F1 preparations. Moreover, thymoquinone induced inhibition of ATP synthase expressing wild-type E. coli cell growth and non-inhibition of ATPase gene deleted null control...

  10. Structure of dimeric, recombinant Sulfolobus solfataricus phosphoribosyl diphosphate synthase

    DEFF Research Database (Denmark)

    Andersen, Rune W.; Lo Leggio, Leila; Hove-Jensen, Bjarne

    2015-01-01

    The enzyme 5-phosphoribosyl-1-α-diphosphate (PRPP) synthase (EC 2.7.6.1) catalyses the Mg2+-dependent transfer of a diphosphoryl group from ATP to the C1 hydroxyl group of ribose 5-phosphate resulting in the production of PRPP and AMP. A nucleotide sequence specifying Sulfolobus solfataricus PRPP...... PRPP synthase as a search model. The two amino acid sequences share 35 % identity. The resulting asymmetric unit consists of three separated dimers. The protein was co-crystallised in the presence of AMP and ribose 5-phosphate, but in the electron density map of the active site only AMP and a sulphate...

  11. Nitric oxide synthase expression and enzymatic activity in multiple sclerosis

    DEFF Research Database (Denmark)

    Broholm, H; Andersen, B; Wanscher, B

    2004-01-01

    and endothelial nitric oxide synthase (NOS)], and enzymatic NO synthase activity. MRI guided biopsies documented more active plaques than macroscopic examination, and histological examination revealed further lesions. Inducible NOS (iNOS) was the dominant IR isoform, while reactive astrocytes were the dominant i......NOS expressing cells in active lesions. NOS IR expressing cells were widely distributed in plaques, in white and gray matter that appeared normal macroscopically, and on MR. Endothelial NOS (eNOS) was highly expressed in intraparenchymal vascular endothelial cells of MS patients. A control group matched for age...

  12. Benzophenone Synthase and Chalcone Synthase Accumulate in the Mesophyll of Hypericum perforatum Leaves at Different Developmental Stages.

    Science.gov (United States)

    Belkheir, Asma K; Gaid, Mariam; Liu, Benye; Hänsch, Robert; Beerhues, Ludger

    2016-01-01

    The active medicinal constituents in Hypericum perforatum, used to treat depression and skin irritation, include flavonoids and xanthones. The carbon skeletons of these compounds are formed by chalcone synthase (CHS) and benzophenone synthase (BPS), respectively. Polyclonal antisera were raised against the polyketide synthases from Hypericum androsaemum and their IgG fractions were isolated. Immunoblotting and immunotitration were used to test the IgGs for crossreactivity and monospecificity in H. perforatum leaf protein extract. Immunofluorescence localization revealed that both CHS and BPS are located in the mesophyll. The maximum fluorescence levels were observed in approx. 0.5 and 1 cm long leaves, respectively. The fluorescence intensity observed for CHS significantly exceeded that for BPS. Using histochemical staining, flavonoids were detected in the mesophyll, indicating that the sites of biosynthesis and accumulation coincide. Our results help understand the biosynthesis and underlying regulation of active H. perforatum constituents.

  13. Benzophenone Synthase and Chalcone Synthase Accumulate in the Mesophyll of Hypericum perforatum Leaves at Different Developmental Stages

    Science.gov (United States)

    Belkheir, Asma K.; Gaid, Mariam; Liu, Benye; Hänsch, Robert; Beerhues, Ludger

    2016-01-01

    The active medicinal constituents in Hypericum perforatum, used to treat depression and skin irritation, include flavonoids and xanthones. The carbon skeletons of these compounds are formed by chalcone synthase (CHS) and benzophenone synthase (BPS), respectively. Polyclonal antisera were raised against the polyketide synthases from Hypericum androsaemum and their IgG fractions were isolated. Immunoblotting and immunotitration were used to test the IgGs for crossreactivity and monospecificity in H. perforatum leaf protein extract. Immunofluorescence localization revealed that both CHS and BPS are located in the mesophyll. The maximum fluorescence levels were observed in approx. 0.5 and 1 cm long leaves, respectively. The fluorescence intensity observed for CHS significantly exceeded that for BPS. Using histochemical staining, flavonoids were detected in the mesophyll, indicating that the sites of biosynthesis and accumulation coincide. Our results help understand the biosynthesis and underlying regulation of active H. perforatum constituents. PMID:27446151

  14. An Unusual Chimeric Diterpene Synthase from Emericella variecolor and Its Functional Conversion into a Sesterterpene Synthase by Domain Swapping.

    Science.gov (United States)

    Qin, Bin; Matsuda, Yudai; Mori, Takahiro; Okada, Masahiro; Quan, Zhiyang; Mitsuhashi, Takaaki; Wakimoto, Toshiyuki; Abe, Ikuro

    2016-01-26

    Di- and sesterterpene synthases produce C20 and C25 isoprenoid scaffolds from geranylgeranyl pyrophosphate (GGPP) and geranylfarnesyl pyrophosphate (GFPP), respectively. By genome mining of the fungus Emericella variecolor, we identified a multitasking chimeric terpene synthase, EvVS, which has terpene cyclase (TC) and prenyltransferase (PT) domains. Heterologous gene expression in Aspergillus oryzae led to the isolation of variediene (1), a novel tricyclic diterpene hydrocarbon. Intriguingly, in vitro reaction with the enzyme afforded the new macrocyclic sesterterpene 2 as a minor product from dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). The TC domain thus produces the diterpene 1 and the sesterterpene 2 from GGPP and GFPP, respectively. Notably, a domain swap of the PT domain of EvVS with that of another chimeric sesterterpene synthase, EvSS, successfully resulted in the production of 2 in vivo as well. Cyclization mechanisms for the production of these two compounds are proposed.

  15. The Remarkable Character of Porphobilinogen Synthase.

    Science.gov (United States)

    Jaffe, Eileen K

    2016-11-15

    Porphobilinogen synthase (PBGS), also known as 5-aminolevulinate dehydratase, is an essential enzyme in the biosynthesis of all tetrapyrroles, which function in respiration, photosynthesis, and methanogenesis. Throughout evolution, PBGS adapted to a diversity of cellular niches and evolved to use an unusual variety of metal ions both for catalytic function and to control protein multimerization. With regard to the active site, some PBGSs require Zn(2+); a subset of those, including human PBGS, contain a constellation of cysteine residues that acts as a sink for the environmental toxin Pb(2+). PBGSs that do not require the soft metal ion Zn(2+) at the active site instead are suspected of using the hard metal Mg(2+). The most unexpected property of the PBGS family of enzymes is a dissociative allosteric mechanism that utilizes an equilibrium of architecturally and functionally distinct protein assemblies. The high-activity assembly is an octamer in which intersubunit interactions modulate active-site lid motion. This octamer can dissociate to dimer, the dimer can undergo a hinge twist, and the twisted dimer can assemble to a low-activity hexamer. The hexamer does not have the intersubunit interactions required to stabilize a closed conformation of the active site lid. PBGS active site chemistry benefits from a closed lid because porphobilinogen biosynthesis includes Schiff base formation, which requires deprotonated lysine amino groups. N-terminal and C-terminal sequence extensions dictate whether a specific species of PBGS can sample the hexameric assembly. The bulk of species (nearly all except animals and yeasts) use Mg(2+) as an allosteric activator. Mg(2+) functions allosterically by binding to an intersubunit interface that is present in the octamer but absent in the hexamer. This conformational selection allosteric mechanism is purported to be essential to avoid the untimely accumulation of phototoxic chlorophyll precursors in plants. For those PBGSs that do

  16. Structure and mechanism of the diterpene cyclase ent-copalyl diphosphate synthase

    Energy Technology Data Exchange (ETDEWEB)

    Köksal, Mustafa; Hu, Huayou; Coates, Robert M.; Peters, Reuben J.; Christianson, David W. (UIUC); (Iowa State); (Penn)

    2011-09-20

    The structure of ent-copalyl diphosphate synthase reveals three {alpha}-helical domains ({alpha}, {beta} and {gamma}), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the {beta}{gamma} domains in ent-copalyl diphosphate synthase but exclusively in the {alpha} domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions.

  17. Isolation and expression of the Pneumocystis carinii thymidylate synthase gene

    DEFF Research Database (Denmark)

    Edman, U; Edman, J C; Lundgren, B;

    1989-01-01

    The thymidylate synthase (TS) gene from Pneumocystis carinii has been isolated from complementary and genomic DNA libraries and expressed in Escherichia coli. The coding sequence of TS is 891 nucleotides, encoding a 297-amino acid protein of Mr 34,269. The deduced amino acid sequence is similar...

  18. Characterising the cellulose synthase complexes of cell walls

    NARCIS (Netherlands)

    Mansoori Zangir, N.

    2012-01-01

    One of the characteristics of the plant kingdom is the presence of a structural cell wall. Cellulose is a major component in both the primary and secondary cell walls of plants. In higher plants cellulose is synthesized by so called rosette protein complexes with cellulose synthases (CESAs) as the c

  19. Biosynthesis of polyketides by trans-AT polyketide synthases.

    Science.gov (United States)

    Piel, Jörn

    2010-07-01

    This review discusses the biosynthesis of natural products that are generated by trans-AT polyketide synthases, a family of catalytically versatile enzymes that have recently been recognized as one of the major group of proteins involved in the production of bioactive polyketides. 436 references are cited.

  20. Polyhydroyxalkanoate Synthase Fusions as a Strategy for Oriented Enzyme Immobilisation

    Directory of Open Access Journals (Sweden)

    David O. Hooks

    2014-06-01

    Full Text Available Polyhydroxyalkanoate (PHA is a carbon storage polymer produced by certain bacteria in unbalanced nutrient conditions. The PHA forms spherical inclusions surrounded by granule associate proteins including the PHA synthase (PhaC. Recently, the intracellular formation of PHA granules with covalently attached synthase from Ralstonia eutropha has been exploited as a novel strategy for oriented enzyme immobilisation. Fusing the enzyme of interest to PHA synthase results in a bifunctional protein able to produce PHA granules and immobilise the active enzyme of choice to the granule surface. Functionalised PHA granules can be isolated from the bacterial hosts, such as Escherichia coli, and maintain enzymatic activity in a wide variety of assay conditions. This approach to oriented enzyme immobilisation has produced higher enzyme activities and product levels than non-oriented immobilisation techniques such as protein inclusion based particles. Here, enzyme immobilisation via PHA synthase fusion is reviewed in terms of the genetic designs, the choices of enzymes, the control of enzyme orientations, as well as their current and potential applications.

  1. Functional Characterization of Sesquiterpene Synthase from Polygonum minus

    Directory of Open Access Journals (Sweden)

    Su-Fang Ee

    2014-01-01

    Full Text Available Polygonum minus is an aromatic plant, which contains high abundance of terpenoids, especially the sesquiterpenes C15H24. Sesquiterpenes were believed to contribute to the many useful biological properties in plants. This study aimed to functionally characterize a full length sesquiterpene synthase gene from P. minus. P. minus sesquiterpene synthase (PmSTS has a complete open reading frame (ORF of 1689 base pairs encoding a 562 amino acid protein. Similar to other sesquiterpene synthases, PmSTS has two large domains: the N-terminal domain and the C-terminal metal-binding domain. It also consists of three conserved motifs: the DDXXD, NSE/DTE, and RXR. A three-dimensional protein model for PmSTS built clearly distinguished the two main domains, where conserved motifs were highlighted. We also constructed a phylogenetic tree, which showed that PmSTS belongs to the angiosperm sesquiterpene synthase subfamily Tps-a. To examine the function of PmSTS, we expressed this gene in Arabidopsis thaliana. Two transgenic lines, designated as OE3 and OE7, were further characterized, both molecularly and functionally. The transgenic plants demonstrated smaller basal rosette leaves, shorter and fewer flowering stems, and fewer seeds compared to wild type plants. Gas chromatography-mass spectrometry analysis of the transgenic plants showed that PmSTS was responsible for the production of β-sesquiphellandrene.

  2. Insight into Biochemical Characterization of Plant Sesquiterpene Synthases

    Science.gov (United States)

    Manczak, Tom; Simonsen, Henrik Toft

    2016-01-01

    A fast and reproducible protocol was established for enzymatic characterization of plant sesquiterpene synthases that can incorporate radioactivity in their products. The method utilizes the 96-well format in conjunction with cluster tubes and enables processing of >200 samples a day. Along with reduced reagent usage, it allows further reduction in the use of radioactive isotopes and flammable organic solvents. The sesquiterpene synthases previously characterized were expressed in yeast, and the plant-derived Thapsia garganica kunzeaol synthase TgTPS2 was tested in this method. KM for TgTPS2 was found to be 0.55 μM; the turnover number, kcat, was found to be 0.29 s−1, kcat for TgTPS2 is in agreement with that of terpene synthases of other plants, and kcat/KM was found to be 0.53 s−1 μM−1 for TgTPS2. The kinetic parameters were in agreement with previously published data. PMID:27721652

  3. Highly Divergent Mitochondrial ATP Synthase Complexes in Tetrahymena thermophila

    NARCIS (Netherlands)

    Nina, Praveen Balabaskaran; Dudkina, Natalya V.; Kane, Lesley A.; van Eyk, Jennifer E.; Boekema, Egbert J.; Mather, Michael W.; Vaidya, Akhil B.; Eisen, Jonathan A.

    2010-01-01

    The F-type ATP synthase complex is a rotary nano-motor driven by proton motive force to synthesize ATP. Its F(1) sector catalyzes ATP synthesis, whereas the F(o) sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F(1) and F(o) sectors are highl

  4. Absence of Pneumocystis dihydropteroate synthase mutants in Brittany, France.

    Science.gov (United States)

    Le Gal, Solène; Robert-Gangneux, Florence; Perrot, Maëla; Rouillé, Amélie; Virmaux, Michèle; Damiani, Céline; Totet, Anne; Gangneux, Jean-Pierre; Nevez, Gilles

    2013-05-01

    Archival Pneumocystis jirovecii specimens from 84 patients monitored at Rennes University Hospital (Rennes, France) were assayed at the dihydropteroate synthase (DHPS) locus. No patient was infected with mutants. The results provide additional data showing that P. jirovecii infections involving DHPS mutants do not represent a public health issue in Brittany, western France.

  5. Cloning and expression pattern of chitin synthase (CHS) gene in ...

    African Journals Online (AJOL)

    USER

    2010-08-16

    Aug 16, 2010 ... African Journal of Biotechnology Vol. 9(33), pp. 5297-5308, 16 ... Chitin synthase (CHS) plays an important role in biosynthesis of chitin .... strand cDNA Synthesis kit, 5'/3' RACE kit and pMD18-T vector were purchased from ...

  6. Detailed characterization of the substrate specificity of mouse wax synthase.

    Science.gov (United States)

    Miklaszewska, Magdalena; Kawiński, Adam; Banaś, Antoni

    2013-01-01

    Wax synthases are membrane-associated enzymes catalysing the esterification reaction between fatty acyl-CoA and a long chain fatty alcohol. In living organisms, wax esters function as storage materials or provide protection against harmful environmental influences. In industry, they are used as ingredients for the production of lubricants, pharmaceuticals, and cosmetics. Currently the biological sources of wax esters are limited to jojoba oil. In order to establish a large-scale production of desired wax esters in transgenic high-yielding oilseed plants, enzymes involved in wax esters synthesis from different biological resources should be characterized in detail taking into consideration their substrate specificity. Therefore, this study aims at determining the substrate specificity of one of such enzymes -- the mouse wax synthase. The gene encoding this enzyme was expressed heterologously in Saccharomyces cerevisiae. In the in vitro assays (using microsomal fraction from transgenic yeast), we evaluated the preferences of mouse wax synthase towards a set of combinations of 11 acyl-CoAs with 17 fatty alcohols. The highest activity was observed for 14:0-CoA, 12:0-CoA, and 16:0-CoA in combination with medium chain alcohols (up to 5.2, 3.4, and 3.3 nmol wax esters/min/mg microsomal protein, respectively). Unsaturated alcohols longer than 18°C were better utilized by the enzyme in comparison to the saturated ones. Combinations of all tested alcohols with 20:0-CoA, 22:1-CoA, or Ric-CoA were poorly utilized by the enzyme, and conjugated acyl-CoAs were not utilized at all. Apart from the wax synthase activity, mouse wax synthase also exhibited a very low acyl-CoA:diacylglycerol acyltransferase activity. However, it displayed neither acyl-CoA:monoacylglycerol acyltransferase, nor acyl-CoA:sterol acyltransferase activity.

  7. Significance of nitric oxide synthases: Lessons from triple nitric oxide synthases null mice.

    Science.gov (United States)

    Tsutsui, Masato; Tanimoto, Akihide; Tamura, Masahito; Mukae, Hiroshi; Yanagihara, Nobuyuki; Shimokawa, Hiroaki; Otsuji, Yutaka

    2015-01-01

    Nitric oxide (NO) is synthesized by three distinct NO synthases (neuronal, inducible, and endothelial NOSs), all of which are expressed in almost all tissues and organs in humans. The regulatory roles of NOSs in vivo have been investigated in pharmacological studies with non-selective NOS inhibitors. However, the specificity of the inhibitors continues to be an issue of debate, and the authentic significance of NOSs is still poorly understood. To address this issue, we generated mice in which all three NOS genes are completely disrupted. The triple NOSs null mice exhibited cardiovascular abnormalities, including hypertension, arteriosclerosis, myocardial infarction, cardiac hypertrophy, diastolic heart failure, and reduced EDHF responses, with a shorter survival. The triple NOSs null mice also displayed metabolic abnormalities, including metabolic syndrome and high-fat diet-induced severe dyslipidemia. Furthermore, the triple NOSs null mice showed renal abnormalities (nephrogenic diabetes insipidus and pathological renal remodeling), lung abnormalities (accelerated pulmonary fibrosis), and bone abnormalities (increased bone mineral density and bone turnover). These results provide evidence that NOSs play pivotal roles in the pathogenesis of a wide variety of disorders. This review summarizes the latest knowledge on the significance of NOSs in vivo, based on lessons learned from experiments with our triple mutant model.

  8. Significance of nitric oxide synthases: Lessons from triple nitric oxide synthases null mice

    Directory of Open Access Journals (Sweden)

    Masato Tsutsui

    2015-01-01

    Full Text Available Nitric oxide (NO is synthesized by three distinct NO synthases (neuronal, inducible, and endothelial NOSs, all of which are expressed in almost all tissues and organs in humans. The regulatory roles of NOSs in vivo have been investigated in pharmacological studies with non-selective NOS inhibitors. However, the specificity of the inhibitors continues to be an issue of debate, and the authentic significance of NOSs is still poorly understood. To address this issue, we generated mice in which all three NOS genes are completely disrupted. The triple NOSs null mice exhibited cardiovascular abnormalities, including hypertension, arteriosclerosis, myocardial infarction, cardiac hypertrophy, diastolic heart failure, and reduced EDHF responses, with a shorter survival. The triple NOSs null mice also displayed metabolic abnormalities, including metabolic syndrome and high-fat diet-induced severe dyslipidemia. Furthermore, the triple NOSs null mice showed renal abnormalities (nephrogenic diabetes insipidus and pathological renal remodeling, lung abnormalities (accelerated pulmonary fibrosis, and bone abnormalities (increased bone mineral density and bone turnover. These results provide evidence that NOSs play pivotal roles in the pathogenesis of a wide variety of disorders. This review summarizes the latest knowledge on the significance of NOSs in vivo, based on lessons learned from experiments with our triple mutant model.

  9. The pCS20 PCR assay for Ehrlichia ruminantium does not cross-react with the novel deer ehrlichial agent found in white-tailed deer in the United States of America

    Directory of Open Access Journals (Sweden)

    S.M. Mahan

    2004-11-01

    Full Text Available White-tailed deer are susceptible to heartwater (Ehrlichia [Cowdria] ruminantium infection and are likely to suffer high mortality if the disease spreads to the United States. It is vital, therefore, to validate a highly specific and sensitive detection method for E. ruminantium infection that can be reliably used in testing white-tailed deer, which are reservoirs of antigenically or genetically related agents such as Ehrlichia chaffeensis, Anaplasma (Ehrlichia phagocytophilum (HGE agent and Ehrlichia ewingii. Recently, a novel but as yet unnamed ehrlichial species, the white-tailed deer ehrlichia (WTDE, has been discovered in deer populations in the United States. Although the significance of WTDE as a pathogen is unknown at present, it can be distinguished from other Ehrlichia spp. based on 16S rRNA gene sequence analysis. In this study it was differentiated from E. ruminantium by the use of the pCS20 PCR assay which has high specificity and sensitivity for the detection of E. ruminantium. This assay did not amplify DNA from the WTDE DNA samples isolated from deer resident in Florida, Georgia and Missouri, but amplified the specific 279 bp fragment from E. ruminantium DNA. The specificity of the pCS20 PCR assay for E. ruminantium was confirmed by Southern hybridization. Similarly, the 16S PCR primers (nested that amplify a specific 405-412 bp fragment from the WTDE DNA samples, did not amplify any product from E. ruminantium DNA. This result demonstrates that it would be possible to differentiate between E. ruminantium and the novel WTDE agent found in white tailed deer by applying the two respective PCR assays followed by Southern hybridizations. Since the pCS20 PCR assay also does not amplify any DNA products from E. chaffeensis or Ehrlichia canis DNA, it is therefore the method of choice for the detection of E. ruminantium in these deer and other animal hosts.

  10. Identifying the catalytic components of cellulose synthase and the maize mixed-linkage beta-glucan synthase

    Energy Technology Data Exchange (ETDEWEB)

    Nicholas C Carpita

    2009-04-20

    Five specific objectives of this project are to develop strategies to identify the genes that encode the catalytic components of "mixed-linkage" (1→3),(1→4)-beta-D-glucans in grasses, to determine the protein components of the synthase complex, and determine the biochemical mechanism of synthesis. We have used proteomic approaches to define intrinsic and extrinsic polypeptides of Golgi membranes that are associated with polysaccharide synthesis and trafficking. We were successful in producing recombinant catalytic domains of cellulose synthase genes and discovered that they dimerize upon concentration, indicating that two CesA proteins form the catalytic unit. We characterized a brittle stalk2 mutant as a defect in a COBRA-like protein that results in compromised lignin-cellulose interactions that decrease tissue flexibility. We used virus-induced gene silencing of barley cell wall polysaccharide synthesis by BSMV in an attempt to silence specific members of the cellulose synthase-like gene family. However, we unexpectedly found that regardless of the specificity of the target gene, whole gene interaction networks were silenced. We discovered the cause to be an antisense transcript of the cellulose synthase gene initiated small interfering RNAs that spread silencing to related genes.

  11. The role of glutathione in mercury tolerance resembles its function under cadmium stress in Arabidopsis.

    Science.gov (United States)

    Sobrino-Plata, Juan; Carrasco-Gil, Sandra; Abadía, Javier; Escobar, Carolina; Álvarez-Fernández, Ana; Hernández, Luis E

    2014-02-01

    Recent research efforts have highlighted the importance of glutathione (GSH) as a key antioxidant metabolite for metal tolerance in plants. Little is known about the mechanisms involved in stress due to mercury (Hg), one of the most hazardous metals to the environment and human health. To understand the implication of GSH metabolism for Hg tolerance, we used two γ-glutamylcysteine synthetase (γECS) Arabidopsis thaliana allele mutants (rax1-1 and cad2-1) and a phytochelatin synthase (PCS) mutant (cad1-3). The leaves of these mutants and of wild type (Col-0) were infiltrated with a solution containing Cd or Hg (0, 3 and 30 μM) and incubated for 24 and 48 h. The formation of phytochelatins (PCs) in the leaf extracts was followed by two different HPLC-based methods and occurred in Col-0, cad2-1 and rax1-1 plants exposed to Cd, whereas in the Hg treatments, PCs accumulated mainly in Col-0 and rax1-1, where Hg-PC complexes were also detected. ASA and GSH/GSSG levels increased under moderate metal stress conditions, accompanied by increased GSH reductase (GR) activity and expression. However, higher metal doses led to a decrease in the analysed parameters, and stronger toxic effects appeared with 30 μM Hg. The GSH concentration was significantly higher in rax1-1 (70% of Col-0) than in cad2-1 (40% of Col-0). The leaves of rax1-1 were less sensitive than cad2-1, in accordance with the greater expression of γECS in rax1-1. Our results underline the existence of a minimal GSH concentration threshold needed to minimise the toxic effects exerted by Hg.

  12. Selenium ameliorates arsenic induced oxidative stress through modulation of antioxidant enzymes and thiols in rice (Oryza sativa L.).

    Science.gov (United States)

    Kumar, Amit; Singh, Rana Pratap; Singh, Pradyumna Kumar; Awasthi, Surabhi; Chakrabarty, Debasis; Trivedi, Prabodh Kumar; Tripathi, Rudra Deo

    2014-09-01

    Arsenic (As) contamination of rice is a major problem for South-East Asia. In the present study, the effect of selenium (Se) on rice (Oryza sativa L.) plants exposed to As was studied in hydroponic culture. Arsenic accumulation, plant growth, thiolic ligands and antioxidative enzyme activities were assayed after single (As and Se) and simultaneous supplementations (As + Se). The results indicated that the presence of Se (25 µM) decreased As accumulation by threefold in roots and twofold in shoots as compared to single As (25 µM) exposed plants. Arsenic induced oxidative stress in roots and shoots was significantly ameliorated by Se supplementation. The observed positive response was found associated with the increased activities of ascorbate peroxidase (APX; EC 1.11.1.11), catalase (CAT; EC 1.11.1.6) and glutathione peroxidase (GPx; EC 1.11.1.9) and induced levels of non-protein thiols (NPTs), glutathione (GSH) and phytochelatins (PCs) in As + Se exposed plants as compared to single As treatment. Selenium supplementation modulated the thiol metabolism enzymes viz., γ-glutamylcysteine synthetase (γ-ECS; EC 6.3.2.2), glutathione-S-transferase (GST; EC 2.5.1.18) and phytochelatin synthase (PCS; EC 2.3.2.15). Gene expression analysis of several metalloid responsive genes (LOX, SOD and MATE) showed upregulation during As stress, however, significant downregulation during As + Se exposure as compared to single As treatment. Gene expressions of enzymes of antioxidant and GSH and PC biosynthetic systems, such as APX, CAT, GPx, γ-ECS and PCS were found to be significantly positively correlated with their enzyme activities. The findings suggested that Se supplementation could be an effective strategy to reduce As accumulation and toxicity in rice plants.

  13. Mechanism of Germacradien-4-ol Synthase-Controlled Water Capture

    Science.gov (United States)

    2016-01-01

    The sesquiterpene synthase germacradiene-4-ol synthase (GdolS) from Streptomyces citricolor is one of only a few known high-fidelity terpene synthases that convert farnesyl diphosphate (FDP) into a single hydroxylated product. Crystals of unliganded GdolS-E248A diffracted to 1.50 Å and revealed a typical class 1 sesquiterpene synthase fold with the active site in an open conformation. The metal binding motifs were identified as D80DQFD and N218DVRSFAQE. Some bound water molecules were evident in the X-ray crystal structure, but none were obviously positioned to quench a putative final carbocation intermediate. Incubations in H218O generated labeled product, confirming that the alcohol functionality arises from nucleophilic capture of the final carbocation by water originating from solution. Site-directed mutagenesis of amino acid residues from both within the metal binding motifs and without identified by sequence alignment with aristolochene synthase from Aspergillus terreus generated mostly functional germacradien-4-ol synthases. Only GdolS-N218Q generated radically different products (∼50% germacrene A), but no direct evidence of the mechanism of incorporation of water into the active site was obtained. Fluorinated FDP analogues 2F-FDP and 15,15,15-F3-FDP were potent noncompetitive inhibitors of GdolS. 12,13-DiF-FDP generated 12,13-(E)-β-farnesene upon being incubated with GdolS, suggesting stepwise formation of the germacryl cation during the catalytic cycle. Incubation of GdolS with [1-2H2]FDP and (R)-[1-2H]FDP demonstrated that following germacryl cation formation a [1,3]-hydride shift generates the final carbocation prior to nucleophilic capture. The stereochemistry of this shift is not defined, and the deuteron in the final product was scrambled. Because no clear candidate residue for binding of a nucleophilic water molecule in the active site and no significant perturbation of product distribution from the replacement of active site residues were

  14. Sulfur assimilation and glutathione metabolism under cadmium stress in yeast, protists and plants.

    Science.gov (United States)

    Mendoza-Cózatl, David; Loza-Tavera, Herminia; Hernández-Navarro, Andrea; Moreno-Sánchez, Rafael

    2005-09-01

    Glutathione (gamma-glu-cys-gly; GSH) is usually present at high concentrations in most living cells, being the major reservoir of non-protein reduced sulfur. Because of its unique redox and nucleophilic properties, GSH serves in bio-reductive reactions as an important line of defense against reactive oxygen species, xenobiotics and heavy metals. GSH is synthesized from its constituent amino acids by two ATP-dependent reactions catalyzed by gamma-glutamylcysteine synthetase and glutathione synthetase. In yeast, these enzymes are found in the cytosol, whereas in plants they are located in the cytosol and chloroplast. In protists, their location is not well established. In turn, the sulfur assimilation pathway, which leads to cysteine biosynthesis, involves high and low affinity sulfate transporters, and the enzymes ATP sulfurylase, APS kinase, PAPS reductase or APS reductase, sulfite reductase, serine acetyl transferase, O-acetylserine/O-acetylhomoserine sulfhydrylase and, in some organisms, also cystathionine beta-synthase and cystathionine gamma-lyase. The biochemical and genetic regulation of these pathways is affected by oxidative stress, sulfur deficiency and heavy metal exposure. Cells cope with heavy metal stress using different mechanisms, such as complexation and compartmentation. One of these mechanisms in some yeast, plants and protists is the enhanced synthesis of the heavy metal-chelating molecules GSH and phytochelatins, which are formed from GSH by phytochelatin synthase (PCS) in a heavy metal-dependent reaction; Cd(2+) is the most potent activator of PCS. In this work, we review the biochemical and genetic mechanisms involved in the regulation of sulfate assimilation-reduction and GSH metabolism when yeast, plants and protists are challenged by Cd(2+).

  15. Sucrose Synthase Expression during Cold Acclimation in Wheat 1

    Science.gov (United States)

    Crespi, Martin D.; Zabaleta, Eduardo J.; Pontis, Horacio G.; Salerno, Graciela L.

    1991-01-01

    When wheat (Triticum aestivum) seedlings are exposed to a cold temperature (2-4°C) above 0°C, sucrose accumulates and sucrose synthase activity increases. The effect of a cold period on the level of sucrose synthase (SS) was investigated. Using antibodies against wheat germ SS, Western blots studies showed that the amount of the SS peptide increased during 14 days in the cold, when plants were moved from 23°C to 4°C. The level of SS diminished when plants were moved back to 23°C. Northern blots of poly(A)+ RNA, confirmed a five- to sixfold induction of SS in wheat leaves during cold acclimation. These results indicate that SS is involved in the plant response to a chilling stress. ImagesFigure 1Figure 2Figure 3 PMID:16668270

  16. In vitro biochemical characterization of all barley endosperm starch synthases

    DEFF Research Database (Denmark)

    Cuesta-Seijo, Jose A.; Nielsen, Morten M.; Ruzanski, Christian;

    2016-01-01

    Starch is the main storage polysaccharide in cereals and the major source of calories in the human diet. It is synthesized by a panel of enzymes including five classes of starch synthases (SSs). While the overall starch synthase (SS) reaction is known, the functional differences between the five SS...... classes are poorly understood. Much of our knowledge comes from analyzing mutant plants with altered SS activities, but the resulting data are often difficult to interpret as a result of pleitropic effects, competition between enzymes, overlaps in enzyme activity and disruption of multi-enzyme complexes....... Here we provide a detailed biochemical study of the activity of all five classes of SSs in barley endosperm. Each enzyme was produced recombinantly in E. coli and the properties and modes of action in vitro were studied in isolation from other SSs and other substrate modifying activities. Our results...

  17. Insight into Biochemical Characterization of Plant Sesquiterpene Synthases

    DEFF Research Database (Denmark)

    Manczak, Tom; Simonsen, Henrik Toft

    2016-01-01

    A fast and reproducible protocol was established for enzymatic characterization of plant sesquiterpene synthases that can incorporate radioactivity in their products. The method utilizes the 96-well format in conjunction with cluster tubes and enables processing of >200 samples a day. Along with ...... was found to be 0.55 μM; the turnover number, kcat, was found to be 0.29 s-1, kcat for TgTPS2 is in agreement with that of terpene synthases of other plants, and kcat/KM was found to be 0.53 s-1 μM-1 for TgTPS2. The kinetic parameters were in agreement with previously published data....

  18. Differential transcytosis and toxicity of the hNGAL receptor ligands cadmium-metallothionein and cadmium-phytochelatin in colon-like Caco-2 cells: implications for in vivo cadmium toxicity.

    Science.gov (United States)

    Langelueddecke, Christian; Lee, Wing-Kee; Thévenod, Frank

    2014-04-21

    The environmental toxicant cadmium (Cd) enters the food chain. A substantial proportion of Cd in nutrients of plant origin is present as Cd-metallothionein (CdMT) and Cd-phytochelatin (CdPC) complexes, which may be absorbed and transcytosed intact by colonic enterocytes following bacterial fermentation and contribute to systemic Cd toxicity, e.g. in liver and kidneys. We have recently demonstrated that the receptor for human neutrophil gelatinase-associated lipocalin (hNGAL) is expressed in human colon and colon-like Caco-2 BBE cells where it mediates transcytosis of MT and PC. Here we show in colon-like Caco-2 BBE cells that hNGAL receptor (hNGAL-R) dependent toxicity is significantly higher with CdMT than with CdPC3 (2.5-50μM Cd(2+) complexed to MT or PC3 for ≤24h), using MTT assay. Fluorescence-labelled A546-MT, but not A488-PC3 (both 700nM), co-localizes with the lysosomal marker cathepsin-B, as determined by confocal microscopy. In transwell experiments with confluent monolayers, transcytosis efficiency (i.e. the ratio of basal delivery to apical decrease) of A546-MT is decreased compared to A488-PC3 (both 700nM). The tubulin polymerization disruptor nocodazole (16.7μM) almost abolished CdMT and CdPC3 toxicity, reduced apical uptake of both A546-MT and A488-PC3, but increased transcytosis efficiency of A546-MT compared to that of A488-PC3 by preventing trafficking of A546-MT to lysosomes. Hence, following hNGAL-R dependent endocytosis of CdMT/CdPC3 in colonic epithelia, a nocodazole-sensitive trafficking pathway may preferentially target CdMT, but not CdPC3, to lysosomes, causing increased colonic epithelial toxicity but reduced systemic toxicity.

  19. Isolation and characterization of terpene synthases in cotton (Gossypium hirsutum).

    Science.gov (United States)

    Yang, Chang-Qing; Wu, Xiu-Ming; Ruan, Ju-Xin; Hu, Wen-Li; Mao, Yin-Bo; Chen, Xiao-Ya; Wang, Ling-Jian

    2013-12-01

    Cotton plants accumulate gossypol and related sesquiterpene aldehydes, which function as phytoalexins against pathogens and feeding deterrents to herbivorous insects. However, to date little is known about the biosynthesis of volatile terpenes in this crop. Herein is reported that 5 monoterpenes and 11 sesquiterpenes from extracts of a glanded cotton cultivar, Gossypium hirsutum cv. CCRI12, were detected by gas chromatography-mass spectrometry (GC-MS). By EST data mining combined with Rapid Amplification of cDNA Ends (RACE), full-length cDNAs of three terpene synthases (TPSs), GhTPS1, GhTPS2 and GhTPS3 were isolated. By in vitro assays of the recombinant proteins, it was found that GhTPS1 and GhTPS2 are sesquiterpene synthases: the former converted farnesyl pyrophosphate (FPP) into β-caryophyllene and α-humulene in a ratio of 2:1, whereas the latter produced several sesquiterpenes with guaia-1(10),11-diene as the major product. By contrast, GhTPS3 is a monoterpene synthase, which produced α-pinene, β-pinene, β-phellandrene and trace amounts of other monoterpenes from geranyl pyrophosphate (GPP). The TPS activities were also supported by Virus Induced Gene Silencing (VIGS) in the cotton plant. GhTPS1 and GhTPS3 were highly expressed in the cotton plant overall, whereas GhTPS2 was expressed only in leaves. When stimulated by mechanical wounding, Verticillium dahliae (Vde) elicitor or methyl jasmonate (MeJA), production of terpenes and expression of the corresponding synthase genes were induced. These data demonstrate that the three genes account for the biosynthesis of volatile terpenes of cotton, at least of this Upland cotton.

  20. Dihydrodipicolinate synthase in opaque and floury maize mutants

    NARCIS (Netherlands)

    Varisi, V.A.; Medici, L.O.; Meer, van der I.M.; Lea, P.J.; Azevedo, J.L.

    2007-01-01

    Dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) was isolated and studied in four high-lysine maize mutants (Oh43o1, Oh43o2, Oh43fl1 and Oh43fl2). The activity of DHDPS was analyzed at 16, 20, and 24 DAP and characterized in the presence of the amino acids, lysine, S-(2-aminoethyl)-l-cysteine (AEC)

  1. Impaired glycogen synthase activity and mitochondrial dysfunction in skeletal muscle

    DEFF Research Database (Denmark)

    Højlund, Kurt; Beck-Nielsen, Henning

    2006-01-01

    expression analysis and proteomics have pointed to abnormalities in mitochondrial oxidative phosphorylation and cellular stress in muscle of type 2 diabetic subjects, and recent work suggests that impaired mitochondrial activity is another early defect in the pathogenesis of type 2 diabetes. This review...... will discuss the latest advances in the understanding of the molecular mechanisms underlying insulin resistance in human skeletal muscle in type 2 diabetes with focus on possible links between impaired glycogen synthase activity and mitochondrial dysfunction....

  2. Structure and Mechanistic Implications of a Tryptophan Synthase Quinonoid Intermediate

    Energy Technology Data Exchange (ETDEWEB)

    Barends,T.; Domratcheva, T.; Kulik, V.; Blumenstein, L.; Niks, D.; Dunn, M.; Schlichting, I.

    2008-01-01

    Quinonoid intermediates play a key role in the catalytic mechanism of pyridoxal 5'-phosphate (PLP)-dependent enzymes. Whereas structures of other PLP-bound reaction intermediates have been determined, a high-quality structure of a quinonoid species has not been reported. We present the crystal structure of the indoline quinonoid intermediate of tryptophan synthase (see figure) and discuss its implications for the enzymatic mechanism and allosteric regulation.

  3. Reduced Expression of Lipoic Acid Synthase Accelerates Diabetic Nephropathy

    OpenAIRE

    Yi, Xianwen; Xu, Longquan; Hiller, Sylvia; Kim, Hyung-Suk; Nickeleit, Volker; James, Leighton R; Maeda, Nobuyo

    2011-01-01

    Oxidative stress contributes to the pathogenesis of diabetic nephropathy. In mitochondria, lipoic acid synthase produces α-lipoic acid, an antioxidant and an essential cofactor in α-ketoacid dehydrogenase complexes, which participate in glucose oxidation and ATP generation. Administration of lipoic acid abrogates diabetic nephropathy in animal models, but whether lower production of endogenous lipoic acid promotes diabetic nephropathy is unknown. Here, we crossed mice heterozygous for lipoic ...

  4. The cellulose synthase superfamily in fully sequenced plants and algae

    Directory of Open Access Journals (Sweden)

    Xu Ying

    2009-07-01

    Full Text Available Abstract Background The cellulose synthase superfamily has been classified into nine cellulose synthase-like (Csl families and one cellulose synthase (CesA family. The Csl families have been proposed to be involved in the synthesis of the backbones of hemicelluloses of plant cell walls. With 17 plant and algal genomes fully sequenced, we sought to conduct a genome-wide and systematic investigation of this superfamily through in-depth phylogenetic analyses. Results A single-copy gene is found in the six chlorophyte green algae, which is most closely related to the CslA and CslC families that are present in the seven land plants investigated in our analyses. Six proteins from poplar, grape and sorghum form a distinct family (CslJ, providing further support for the conclusions from two recent studies. CslB/E/G/H/J families have evolved significantly more rapidly than their widely distributed relatives, and tend to have intragenomic duplications, in particular in the grape genome. Conclusion Our data suggest that the CslA and CslC families originated through an ancient gene duplication event in land plants. We speculate that the single-copy Csl gene in green algae may encode a mannan synthase. We confirm that the rest of the Csl families have a different evolutionary origin than CslA and CslC, and have proposed a model for the divergence order among them. Our study provides new insights about the evolution of this important gene family in plants.

  5. From bacterial to human dihydrouridine synthase: automated structure determination

    Energy Technology Data Exchange (ETDEWEB)

    Whelan, Fiona, E-mail: fiona.whelan@york.ac.uk; Jenkins, Huw T., E-mail: fiona.whelan@york.ac.uk [The University of York, Heslington, York YO10 5DD (United Kingdom); Griffiths, Samuel C. [University of Oxford, Headington, Oxford OX3 7BN (United Kingdom); Byrne, Robert T. [Ludwig-Maximilians-University Munich, Feodor-Lynen-Strasse 25, 81377 Munich (Germany); Dodson, Eleanor J.; Antson, Alfred A., E-mail: fiona.whelan@york.ac.uk [The University of York, Heslington, York YO10 5DD (United Kingdom)

    2015-06-30

    The crystal structure of a human dihydrouridine synthase, an enzyme associated with lung cancer, with 18% sequence identity to a T. maritima enzyme, has been determined at 1.9 Å resolution by molecular replacement after extensive molecular remodelling of the template. The reduction of uridine to dihydrouridine at specific positions in tRNA is catalysed by dihydrouridine synthase (Dus) enzymes. Increased expression of human dihydrouridine synthase 2 (hDus2) has been linked to pulmonary carcinogenesis, while its knockdown decreased cancer cell line viability, suggesting that it may serve as a valuable target for therapeutic intervention. Here, the X-ray crystal structure of a construct of hDus2 encompassing the catalytic and tRNA-recognition domains (residues 1–340) determined at 1.9 Å resolution is presented. It is shown that the structure can be determined automatically by phenix.mr-rosetta starting from a bacterial Dus enzyme with only 18% sequence identity and a significantly divergent structure. The overall fold of the human Dus2 is similar to that of bacterial enzymes, but has a larger recognition domain and a unique three-stranded antiparallel β-sheet insertion into the catalytic domain that packs next to the recognition domain, contributing to domain–domain interactions. The structure may inform the development of novel therapeutic approaches in the fight against lung cancer.

  6. Mechanism of Action and Inhibition of dehydrosqualene Synthase

    Energy Technology Data Exchange (ETDEWEB)

    F Lin; C Liu; Y Liu; Y Zhang; K Wang; W Jeng; T Ko; R Cao; A Wang; E Oldfield

    2011-12-31

    'Head-to-head' terpene synthases catalyze the first committed steps in sterol and carotenoid biosynthesis: the condensation of two isoprenoid diphosphates to form cyclopropylcarbinyl diphosphates, followed by ring opening. Here, we report the structures of Staphylococcus aureus dehydrosqualene synthase (CrtM) complexed with its reaction intermediate, presqualene diphosphate (PSPP), the dehydrosqualene (DHS) product, as well as a series of inhibitors. The results indicate that, on initial diphosphate loss, the primary carbocation so formed bends down into the interior of the protein to react with C2,3 double bond in the prenyl acceptor to form PSPP, with the lower two-thirds of both PSPP chains occupying essentially the same positions as found in the two farnesyl chains in the substrates. The second-half reaction is then initiated by the PSPP diphosphate returning back to the Mg{sup 2+} cluster for ionization, with the resultant DHS so formed being trapped in a surface pocket. This mechanism is supported by the observation that cationic inhibitors (of interest as antiinfectives) bind with their positive charge located in the same region as the cyclopropyl carbinyl group; that S-thiolo-diphosphates only inhibit when in the allylic site; activity results on 11 mutants show that both DXXXD conserved domains are essential for PSPP ionization; and the observation that head-to-tail isoprenoid synthases as well as terpene cyclases have ionization and alkene-donor sites which spatially overlap those found in CrtM.

  7. Rotation and structure of FoF1-ATP synthase.

    Science.gov (United States)

    Okuno, Daichi; Iino, Ryota; Noji, Hiroyuki

    2011-06-01

    F(o)F(1)-ATP synthase is one of the most ubiquitous enzymes; it is found widely in the biological world, including the plasma membrane of bacteria, inner membrane of mitochondria and thylakoid membrane of chloroplasts. However, this enzyme has a unique mechanism of action: it is composed of two mechanical rotary motors, each driven by ATP hydrolysis or proton flux down the membrane potential of protons. The two molecular motors interconvert the chemical energy of ATP hydrolysis and proton electrochemical potential via the mechanical rotation of the rotary shaft. This unique energy transmission mechanism is not found in other biological systems. Although there are other similar man-made systems like hydroelectric generators, F(o)F(1)-ATP synthase operates on the nanometre scale and works with extremely high efficiency. Therefore, this enzyme has attracted significant attention in a wide variety of fields from bioenergetics and biophysics to chemistry, physics and nanoscience. This review summarizes the latest findings about the two motors of F(o)F(1)-ATP synthase as well as a brief historical background.

  8. The pseudouridine synthases: revisiting a mechanism that seemed settled.

    Science.gov (United States)

    Spedaliere, Christopher J; Ginter, Joy M; Johnston, Murray V; Mueller, Eugene G

    2004-10-13

    RNA containing 5-fluorouridine, [f 5U]RNA, has been used as a mechanistic probe for the pseudouridine synthases, which convert uridine in RNA to its C-glycoside isomer, pseudouridine. Hydrated products of f 5U were attributed to ester hydrolysis of a covalent complex between an essential aspartic acid residue and f 5U, and the results were construed as strong support for a mechanism involving Michael addition by the aspartic acid residue. Labeling studies with [18O]water are now reported that rule out such ester hydrolysis in one pseudouridine synthase, TruB. The aspartic acid residue does not become labeled, and the hydroxyl group in the hydrated product of f 5U derives directly from solvent. The hydrated product, therefore, cannot be construed to support Michael addition during the conversion of uridine to pseudouridine, but the results do not rule out such a mechanism. A hypothesis is offered for the seemingly disparate behavior of different pseudouridine synthases toward [f 5U]RNA.

  9. Cellulose Microfibril Formation by Surface-Tethered Cellulose Synthase Enzymes.

    Science.gov (United States)

    Basu, Snehasish; Omadjela, Okako; Gaddes, David; Tadigadapa, Srinivas; Zimmer, Jochen; Catchmark, Jeffrey M

    2016-02-23

    Cellulose microfibrils are pseudocrystalline arrays of cellulose chains that are synthesized by cellulose synthases. The enzymes are organized into large membrane-embedded complexes in which each enzyme likely synthesizes and secretes a β-(1→4) glucan. The relationship between the organization of the enzymes in these complexes and cellulose crystallization has not been explored. To better understand this relationship, we used atomic force microscopy to visualize cellulose microfibril formation from nickel-film-immobilized bacterial cellulose synthase enzymes (BcsA-Bs), which in standard solution only form amorphous cellulose from monomeric BcsA-B complexes. Fourier transform infrared spectroscopy and X-ray diffraction techniques show that surface-tethered BcsA-Bs synthesize highly crystalline cellulose II in the presence of UDP-Glc, the allosteric activator cyclic-di-GMP, as well as magnesium. The cellulose II cross section/diameter and the crystal size and crystallinity depend on the surface density of tethered enzymes as well as the overall concentration of substrates. Our results provide the correlation between cellulose microfibril formation and the spatial organization of cellulose synthases.

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

    Science.gov (United States)

    Pazouki, Leila; Niinemets, Ülo

    2016-01-01

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

  11. Phosphatidate phosphatase regulates membrane phospholipid synthesis via phosphatidylserine synthase.

    Science.gov (United States)

    Carman, George M; Han, Gil-Soo

    2017-08-16

    The yeast Saccharomyces cerevisiae serves as a model eukaryote to elucidate the regulation of lipid metabolism. In exponentially growing yeast, a diverse set of membrane lipids are synthesized from the precursor phosphatidate via the liponucleotide intermediate CDP-diacylglycerol. As cells exhaust nutrients and progress into the stationary phase, phosphatidate is channeled via diacylglycerol to the synthesis of triacylglycerol. The CHO1-encoded phosphatidylserine synthase, which catalyzes the committed step in membrane phospholipid synthesis via CDP-diacylglycerol, and the PAH1-encoded phosphatidate phosphatase, which catalyzes the committed step in triacylglycerol synthesis are regulated throughout cell growth by genetic and biochemical mechanisms to control the balanced synthesis of membrane phospholipids and triacylglycerol. The loss of phosphatidate phosphatase activity (e.g., pah1Δ mutation) increases the level of phosphatidate and its conversion to membrane phospholipids by inducing Cho1 expression and phosphatidylserine synthase activity. The regulation of the CHO1 expression is mediated through the inositol-sensitive upstream activation sequence (UASINO), a cis-acting element for the phosphatidate-controlled Henry (Ino2-Ino4/Opi1) regulatory circuit. Consequently, phosphatidate phosphatase activity regulates phospholipid synthesis through the transcriptional regulation of the phosphatidylserine synthase enzyme. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. The structural basis of Erwinia rhapontici isomaltulose synthase.

    Science.gov (United States)

    Xu, Zheng; Li, Sha; Li, Jie; Li, Yan; Feng, Xiaohai; Wang, Renxiao; Xu, Hong; Zhou, Jiahai

    2013-01-01

    Sucrose isomerase NX-5 from Erwiniarhapontici efficiently catalyzes the isomerization of sucrose to isomaltulose (main product) and trehalulose (by-product). To investigate the molecular mechanism controlling sucrose isomer formation, we determined the crystal structures of native NX-5 and its mutant complexes E295Q/sucrose and D241A/glucose at 1.70 Å, 1.70 Å and 2.00 Å, respectively. The overall structure and active site architecture of NX-5 resemble those of other reported sucrose isomerases. Strikingly, the substrate binding mode of NX-5 is also similar to that of trehalulose synthase from Pseudomonasmesoacidophila MX-45 (MutB). Detailed structural analysis revealed the catalytic RXDRX motif and the adjacent 10-residue loop of NX-5 and isomaltulose synthase PalI from Klebsiella sp. LX3 adopt a distinct orientation from those of trehalulose synthases. Mutations of the loop region of NX-5 resulted in significant changes of the product ratio between isomaltulose and trehalulose. The molecular dynamics simulation data supported the product specificity of NX-5 towards isomaltulose and the role of the loop(330-339) in NX-5 catalysis. This work should prove useful for the engineering of sucrose isomerase for industrial carbohydrate biotransformations.

  13. Multi-substrate terpene synthases: their occurrence and physiological significance

    Directory of Open Access Journals (Sweden)

    Leila Pazouki

    2016-07-01

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

  14. Site-directed mutagenesis of bacterial cellulose synthase highlights sulfur–arene interaction as key to catalysis

    OpenAIRE

    Sun, Shi-jing; Horikawa, Yoshiki; Wada, Masahisa; SUGIYAMA, Junji; Imai, Tomoya

    2016-01-01

    Cellulose is one of the most abundant biological polymers on Earth, and is synthesized by the cellulose synthase complex in cell membranes. Although many cellulose synthase genes have been identified over the past 25 years, functional studies of cellulose synthase using recombinant proteins have rarely been conducted. In this study, we conducted a functional analysis of cellulose synthase with site-directed mutagenesis, by using recombinant cellulose synthase reconstituted in living Escherich...

  15. Suites of terpene synthases explain differential terpenoid production in ginger and turmeric tissues.

    Directory of Open Access Journals (Sweden)

    Hyun Jo Koo

    Full Text Available The essential oils of ginger (Zingiber officinale and turmeric (Curcuma longa contain a large variety of terpenoids, some of which possess anticancer, antiulcer, and antioxidant properties. Despite their importance, only four terpene synthases have been identified from the Zingiberaceae family: (+-germacrene D synthase and (S-β-bisabolene synthase from ginger rhizome, and α-humulene synthase and β-eudesmol synthase from shampoo ginger (Zingiber zerumbet rhizome. We report the identification of 25 mono- and 18 sesquiterpene synthases from ginger and turmeric, with 13 and 11, respectively, being functionally characterized. Novel terpene synthases, (--caryolan-1-ol synthase and α-zingiberene/β-sesquiphellandrene synthase, which is responsible for formation of the major sesquiterpenoids in ginger and turmeric rhizomes, were also discovered. These suites of enzymes are responsible for formation of the majority of the terpenoids present in these two plants. Structures of several were modeled, and a comparison of sets of paralogs suggests how the terpene synthases in ginger and turmeric evolved. The most abundant and most important sesquiterpenoids in turmeric rhizomes, (+-α-turmerone and (+-β-turmerone, are produced from (--α-zingiberene and (--β-sesquiphellandrene, respectively, via α-zingiberene/β-sesquiphellandrene oxidase and a still unidentified dehydrogenase.

  16. Two branches of the lupeol synthase gene in the molecular evolution of plant oxidosqualene cyclases.

    Science.gov (United States)

    Shibuya, M; Zhang, H; Endo, A; Shishikura, K; Kushiro, T; Ebizuka, Y

    1999-11-01

    Two new triterpene synthase cDNAs, named as OEW and TRW, were cloned from olive leaves (Olea europaea) and from dandelion roots (Taraxacum officinale), respectively, by the PCR method with primers designed from the conserved sequences found in the known oxidosqualene cyclases. Their ORFs consisted of 2274 bp nucleotides and coded for 758 amino acid long polypeptides. They shared high sequence identity (78%) to each other, while they showed only about 60% identities to the known triterpene synthases LUPI (lupeol synthase clone from Arabidopsis thaliana) and PNY (beta-amyrin synthase clone from Panax ginseng) at amino acid level. To determine the enzyme functions of the translates, they were expressed in an ERG7 deficient yeast mutant. Accumulation of lupeol in the cells of yeast transformants proved both of these clones code for lupeol synthase proteins. An EST (expression sequence tag) clone isolated from Medicago truncatula roots as a homologue of cycloartenol synthase gene, exhibits high sequence identity (75-77%) to these two lupeol synthase cDNAs, suggesting it to be another lupeol synthase clone. Comparatively low identity (approximately 57%) of LUP1 from Arabidopsis thaliana to either one of these clones leaves LUP1 as a distinct clone among lupeol synthases. From these sequence comparisons, now we propose that two branches of lupeol synthase gene have been generated in higher plants during the course of evolution.

  17. Structure of the human beta-ketoacyl [ACP] synthase from the mitochondrial type II fatty acid synthase

    DEFF Research Database (Denmark)

    Christensen, Caspar Elo; Kragelund, Birthe Brandt; Von Wettstein-Knowles, Penny

    2007-01-01

    Two distinct ways of organizing fatty acid biosynthesis exist: the multifunctional type I fatty acid synthase (FAS) of mammals, fungi, and lower eukaryotes with activities residing on one or two polypeptides; and the dissociated type II FAS of prokaryotes, plastids, and mitochondria with individual...... activities encoded by discrete genes. The beta-ketoacyl [ACP] synthase (KAS) moiety of the mitochondrial FAS (mtKAS) is targeted by the antibiotic cerulenin and possibly by the other antibiotics inhibiting prokaryotic KASes: thiolactomycin, platensimycin, and the alpha-methylene butyrolactone, C75. The high...... degree of structural similarity between mitochondrial and prokaryotic KASes complicates development of novel antibiotics targeting prokaryotic KAS without affecting KAS domains of cytoplasmic FAS. KASes catalyze the C(2) fatty acid elongation reaction using either a Cys-His-His or Cys-His-Asn catalytic...

  18. Role of neuronal nitric oxide synthase and inducible nitric oxide synthase in intestinal injury in neonatal rats

    Institute of Scientific and Technical Information of China (English)

    Hui LU; Bing Zhu; Xin-Dong Xue

    2006-01-01

    AIM: To investigate the dynamic change and role of neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) in neonatal rat with intestinal injury and to define whether necrotizing enterocolitis (NEC) is associated with the levels of nitric oxide synthase (NOS) in the mucosa of the affected intestine tissue.METHODS: Wistar rats less than 24 h in age received an intraperitoneal injection with 5 mg/kg lipopolysaccharide (LPS). Ileum tissues were collected at 1, 3, 6, 12 and 24 h following LPS challenge for histological evaluation of NEC and for measurements of nNOS and iNOS. The correlation between the degree of intestinal injury and levels of NOS was determined.RESULTS: The LPS-injected pups showed a significant increase in injury scores versus the control. The expression of nNOS protein and mRNA was diminished after LPS injection. There was a negative significant correlation between the nNOS protein and the grade of median intestinal injury within 24 h. The expression of iNOS protein and mRNA was significantly increased in the peak of intestinal injury.CONCLUSION: nNOS and iNOS play different roles in LPS-induced intestinal injury. Caution should be exerted concerning potential therapeutic uses of NOS inhibitors in NEC.

  19. 非能动安全壳冷却系统空气流道自然循环的比例分析与设计%Scaling Analysis and Design for PCS Air Flow Path Natural Circulation

    Institute of Scientific and Technical Information of China (English)

    刘卓; 常华健; 阳祥; 杨燕宁

    2016-01-01

    在考虑建设试验台架经济性的前提下,缩小比例的单项和整体效应试验台架对研究和开发大型先进压水堆核电站及其分析验证程序都具有重要意义。非能动安全壳冷却系统(PCS )壳外空气流道内的自然循环在安全壳非能动冷却性能中发挥着重要的作用。本文从自然循环的数学模型出发,推导出了单项和整体效应试验台架的比例设计方法。在给定壳内热流密度的条件下,通过PCCSAP‐3D程序对C A P1400非能动安全壳的2/5比例单项效应试验理想比例台架(IS F )进行模拟。结果表明,本比例分析与设计方法以及在降低高度台架上模拟自然循环是可行的。%The scale‐down separate and integral effect test facilities are of vital signifi‐cance not only for the research and development of large‐scale advanced pressurized w ater reactor nuclear pow er plants ,but also for validation and verification of the related analysis codes . The passive containment cooling system (PCS ) air flow path system plays an important role in containment passive cooling .Based on the natural circulation model ,the scaling and design methods for both the separate and integral effect test facil‐ities were proposed .Under given heat flux from the inner side of the shell ,simulation of an ideal scaled facility (ISF) design of a 2/5 length scaled separate effect test facility for CAP1400 PCS was preliminarily performed by the PCCSAP‐3D code .The simulation results show that the design method and the modeling of natural circulation on a reduced height test facility are feasible .

  20. 不同药物用于病人自控镇静的疗效%Intraoperative patient-controlled sed at ion:Comparison of 4 drug combinations us ed for PCS during epidural anesthesia

    Institute of Scientific and Technical Information of China (English)

    徐庆; 汤建高; 黄宇光

    2001-01-01

    目的比较四种不同药物配方用于下腹部或下肢手术硬膜外麻醉中病人自控镇静 (PCS)的临床效果.方法 60例下腹部或下肢择期手术病人随机分为四组,每组15 例,分别接受丙泊酚(每次10mg,P组)、咪唑安定(每次1mg,M组)、D+ P(每次哌替啶12.5mg+异丙嗪6.25mg,D组)、Innovor(每次氟哌利多1. 25mg+芬太尼0.025mg,I组)行PCS,观察生命体征、镇静效果、用药情况及副反应.结果四组病人中,镇静程度满意的(镇静评分2~4分 ),P组为100 %(15/15),M组为93.3%(14/15),D组及Ⅰ组均为10 0%(15/15),各组间无显著差异(P>0.05).病人术中合作、术后满意 ,无明显副作用.四组病人使用PCS 5分钟后,BP、HR及焦虑程度均有所下降.P组的起效时间最短、清醒过程最快,而D组和I组清醒所需时间较长.M组病人存在着明显的药物顺行性遗忘作用.结论 PCS是一种合理有效的镇静方法.本研究病例的98.3%可通过PCS达到临床理想的镇静效果.其中,丙泊酚镇静效果好,恢复迅速,最适合用于P CS.而对于防止术中知晓方面,咪唑安定是较为理想的选择.虽然本研究中D+P和In novor用于术中PCS效果亦尚属满意,但可能引起病人镇静恢复时间延迟.

  1. 14-3-3 protein is a regulator of the mitochondrial and chloroplast ATP synthase

    OpenAIRE

    Bunney, Tom D.; van Walraven, Hendrika S.; de Boer, Albertus H.

    2001-01-01

    Mitochondrial and chloroplast ATP synthases are key enzymes in plant metabolism, providing cells with ATP, the universal energy currency. ATP synthases use a transmembrane electrochemical proton gradient to drive synthesis of ATP. The enzyme complexes function as miniature rotary engines, ensuring energy coupling with very high efficiency. Although our understanding of the structure and functioning of the synthase has made enormous progress in recent years, our und...

  2. Structure and Mechanism of the Diterpene Cyclase ent-Copalyl Diphosphate Synthase

    Science.gov (United States)

    Köksal, Mustafa; Hu, Huayou; Coates, Robert M.; Peters, Reuben J.; Christianson, David W.

    2011-01-01

    The structure of ent-copalyl diphosphate synthase (CPS) reveals three α-helical domains (α, β, γ), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the βγ domains in CPS but exclusively in the α domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions. PMID:21602811

  3. Stabilization and enhanced reactivity of actinorhodin polyketide synthase minimal complex in polymer-nucleotide coacervate droplets.

    Science.gov (United States)

    Crosby, John; Treadwell, Tom; Hammerton, Michelle; Vasilakis, Konstantinos; Crump, Matthew P; Williams, David S; Mann, Stephen

    2012-12-18

    Compartmentalization of the minimal complex of actinorhodin polyketide synthase in coacervate liquid droplets produces enhanced yields of shunt polyketides under conditions of low and high ionic strength.

  4. Outsourcing e geração de valor na indústria de computadores pessoais (PCs: estudo de múltiplos casos The disruption of the Brazilian Personal Computer (PC industry: a multiple case study

    Directory of Open Access Journals (Sweden)

    Fernando José Barbin Laurindo

    2003-12-01

    Full Text Available Apesar de o conceito de computador pessoal (personal computer - PC ter surgido nos anos 70, essa indústria realmente deslanchou somente após o lançamento do IBM PC, em 1981. Por meio de uma agressiva estratégia de outsourcing, a IBM rapidamente conquistou o mercado, mas esta estratégia também causou perda de competências internas para seus principais parceiros, Intel e Microsoft, bem como outros revendedores autônomos. No Brasil, atualmente, a indústria de PCs está relegada à montagem de kits importados e também à manufatura das etapas finais do processo de produção de componentes, com pouco valor agregado localmente. Após o término da reserva de mercado, marcado pela súbita abertura aos concorrentes externos, houve desmantelamento da indústria brasileira de PCs e de semicondutores, nos elos de maior valor agregado dessa cadeia. Para obter inserção nas cadeias globais, as empresas brasileiras precisam atingir qualificação nos critérios de desempenho, a fim de participar dos processos de outsourcing. Este artigo visa, por meio de um aparato teórico que relaciona aspectos de outsourcing, inovação, valor e evolução da indústria de PCs, a investigar os impactos desses aspectos na competitividade da indústria de PCs do Brasil e as repercussões para seu crescimento e inserção no mercado global. A abordagem metodológica adotada foi estudo de múltiplos casos, com os seguintes critérios de seleção: diferentes papéis na cadeia de valor, grau de verticalização, principal mercado visado e tamanho da empresa.Although the personal computer (PC appeared in the seventies, this industry really boomed only after IBM launched its PC, in 1981. Through an aggressive outsourcing strategy, IBM rapidly conquered the market, but this strategy also caused lost of internal competences to its main partners, Intel and Microsoft and also to autonomous vendors. Many PC manufacturers have appeared since then. In Brazil, nowadays, PC

  5. Fatty acid synthase inhibitors isolated from Punica granatum L

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, He-Zhong [School of Life Science and Engineering, Southwest Jiaotong University, Chengdu, (China); Ma, Qing-Yun; Liang, Wen-Juan; Huang, Sheng-Zhuo; Dai, Hao-Fu; Wang, Peng-Cheng; Zhao, You-Xing, E-mail: zhaoyx1011@163.com [Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou (China); Fan, Hui-Jin; Ma, Xiao-Feng, E-mail: maxiaofeng@gucas.ac.cn [College of Life Sciences, Graduate University of Chinese Academy of Sciences, Beijing (China)

    2012-05-15

    The aim of this work is the isolation of fatty acid synthase (FAS) inhibitors from the ethyl acetate extracts of fruit peels of Punica granatum L. Bioassay-guided chemical investigation of the fruit peels resulted in the isolation of seventeen compounds mainly including triterpenoids and phenolic compounds, from which one new oleanane-type triterpene (punicaone) along with fourteen known compounds were isolated for the first time from this plant. Seven isolates were evaluated for inhibitory activities of FAS and two compounds showed to be active. Particularly, flavogallonic acid exhibited strong FAS inhibitory activity with IC{sub 50} value of 10.3 {mu}mol L{sup -1}. (author)

  6. CTP limitation increases expression of CTP synthase in Lactococcus lactis

    DEFF Research Database (Denmark)

    Jørgensen, C.M.; Hammer, Karin; Martinussen, Jan

    2003-01-01

    for regulation of the pyrG gene. It is possible to fold the pyrG leader in an alternative structure that would prevent the formation of the terminator. We suggest a model for pyrG regulation in L. lactis, and probably in other gram-positive bacteria as well, in which pyrG expression is directly dependent...... on the CTP concentration through an attenuator mechanism. At normal CTP concentrations a terminator is preferentially formed in the pyrG leader, thereby reducing expression of CTP synthase. At low CTP concentrations the RNA polymerase pauses at a stretch of C residues in the pyrG leader, thereby allowing...

  7. Structures of citrate synthase and malate dehydrogenase of Mycobacterium tuberculosis.

    Science.gov (United States)

    Ferraris, Davide M; Spallek, Ralf; Oehlmann, Wulf; Singh, Mahavir; Rizzi, Menico

    2015-02-01

    The tricarboxylic acid (TCA) cycle is a central metabolic pathway of all aerobic organisms and is responsible for the synthesis of many important precursors and molecules. TCA cycle plays a key role in the metabolism of Mycobacterium tuberculosis and is involved in the adaptation process of the bacteria to the host immune response. We present here the first crystal structures of M. tuberculosis malate dehydrogenase and citrate synthase, two consecutive enzymes of the TCA, at 2.6 Å and 1.5 Å resolution, respectively. General analogies and local differences with the previously reported homologous protein structures are described. © 2014 Wiley Periodicals, Inc.

  8. Argininosuccinate synthase as a novel biomarker for inflammatory conditions.

    Science.gov (United States)

    Cao, Mengde; George, Thomas J; Prima, Victor; Nelson, David; Svetlov, Stanislav

    2013-05-01

    Argininosuccinate synthase (ASS) plays an important role in regulating metabolic functions in mammals. We previously reported that hepatic ASS is released into circulation at very high concentrations in response to endotoxin and acute liver injury. We propose that ASS may serve as a novel biomarker for various inflammatory conditions. Our data showed that ASS accumulated in serum and urine of septic, obese or tumor mice in a condition-dependent fashion. Moreover, ASS significantly increased in urine within the first week after tumor cell implantation in mice which subsequently develop tumors. These results suggest that ASS is a novel biomarker increased upon diverse inflammatory conditions.

  9. Structural and functional characterization of Staphylococcus aureus dihydrodipicolinate synthase.

    Science.gov (United States)

    Girish, Tavarekere S; Sharma, Eshita; Gopal, B

    2008-08-20

    Lysine biosynthesis is crucial for cell-wall formation in bacteria. Enzymes involved in lysine biosynthesis are thus potential targets for anti-microbial therapeutics. Dihydrodipicolinate synthase (DHDPS) catalyzes the first step of this pathway. Unlike its homologues, Staphylococcus aureus DHDPS is a dimer both in solution and in the crystal and is not feedback inhibited by lysine. The crystal structure of S. aureus DHDPS in the free and substrate bound forms provides a structural rationale for its catalytic mechanism. The structure also reveals unique conformational features of the S. aureus enzyme that could be crucial for the design of specific non-competitive inhibitors.

  10. Microsomal prostaglandin E synthase-1 in rheumatic diseases

    Directory of Open Access Journals (Sweden)

    Marina eKorotkova

    2011-01-01

    Full Text Available Microsomal prostaglandin E synthase-1 (mPGES-1 is a well recognized target for the development of novel anti-inflammatory drugs that can reduce symptoms of inflammation in rheumatic diseases and other inflammatory conditions. In this review, we focus on mPGES-1 in rheumatic diseases with the aim to cover the most recent advances in the understanding of mPGES-1 in rheumatoid arthritis, osteoarthritis and inflammatory myopathies. Novel findings regarding regulation of mPGES1 cell expression as well as enzyme inhibitors are also summarized.

  11. Chemical synthesis of yeast mitochondrial ATP synthase membranous subunit 8.

    Science.gov (United States)

    Goetz, M; Schmitter, J M; Geoffre, S; Dufourc, E J

    1999-06-01

    Chemical synthesis of highly hydrophobic peptides and proteins remains a challenging problem. Strong interchain associations within the peptide-resin matrix have to be overcome. A synthetic strategy for solid phase peptide synthesis is proposed, mainly based on prolonged coupling time using aprotic polar solvent mixtures. A tailored chromatographic purification was required to obtain a sample sufficiently pure for structural analysis. In this work, the total chemical synthesis of the membrane-embedded yeast mitochondrial ATP synthase subunit 8 is described. The quality of the synthetic protein was checked by electrospray mass spectrometry, its tendency to adopt alpha-helical secondary structure is evidenced by circular dichroism spectroscopy.

  12. Nitric Oxide Synthase-3 Promotes Embryonic Development of Atrioventricular Valves

    OpenAIRE

    Yin Liu; Xiangru Lu; Fu-Li Xiang; Man Lu; Qingping Feng

    2013-01-01

    Nitric oxide synthase-3 (NOS3) has recently been shown to promote endothelial-to-mesenchymal transition (EndMT) in the developing atrioventricular (AV) canal. The present study was aimed to investigate the role of NOS3 in embryonic development of AV valves. We hypothesized that NOS3 promotes embryonic development of AV valves via EndMT. To test this hypothesis, morphological and functional analysis of AV valves were performed in wild-type (WT) and NOS3(-/-) mice at postnatal day 0. Our data s...

  13. Molecular cloning and functional expression of geranylgeranyl pyrophosphate synthase from Coleus forskohlii Briq

    Directory of Open Access Journals (Sweden)

    Kawamukai Makoto

    2004-11-01

    Full Text Available Abstract Background Isopentenyl diphosphate (IPP, a common biosynthetic precursor to the labdane diterpene forskolin, has been biosynthesised via a non-mevalonate pathway. Geranylgeranyl diphosphate (GGPP synthase is an important branch point enzyme in terpenoid biosynthesis. Therefore, GGPP synthase is thought to be a key enzyme in biosynthesis of forskolin. Herein we report the first confirmation of the GGPP synthase gene in Coleus forskohlii Briq. Results The open reading frame for full-length GGPP synthase encodes a protein of 359 amino acids, in which 1,077 nucleotides long with calculated molecular mass of 39.3 kDa. Alignments of C. forskohlii GGPP synthase amino acid sequences revealed high homologies with other plant GGPP synthases. Several highly conserved regions, including two aspartate-rich motifs were identified. Transient expression of the N-terminal region of C. forskohlii GGPP synthase-GFP fusion protein in tobacco cells demonstrated subcellular localization in the chloroplast. Carotenoid production was observed in Escherichia coli harboring pACCAR25ΔcrtE from Erwinia uredovora and plasmid carrying C. forskohlii GGPP synthase. These results suggested that cDNA encoded functional GGPP synthase. Furthermore, C. forskohlii GGPP synthase expression was strong in leaves, decreased in stems and very little expression was observed in roots. Conclusion This investigation proposed that forskolin was synthesised via a non-mevalonate pathway. GGPP synthase is thought to be involved in the biosynthesis of forskolin, which is primarily synthesised in the leaves and subsequently accumulates in the stems and roots.

  14. Glycogen synthase from the parabasalian parasite Trichomonas vaginalis: An unusual member of the starch/glycogen synthase family.

    Science.gov (United States)

    Wilson, Wayne A; Pradhan, Prajakta; Madhan, Nayasha; Gist, Galen C; Brittingham, Andrew

    2017-07-01

    Trichomonas vaginalis, a parasitic protist, is the causative agent of the common sexually-transmitted infection trichomoniasis. The organism has long been known to synthesize substantial glycogen as a storage polysaccharide, presumably mobilizing this compound during periods of carbohydrate limitation, such as might be encountered during transmission between hosts. However, little is known regarding the enzymes of glycogen metabolism in T. vaginalis. We had previously described the identification and characterization of two forms of glycogen phosphorylase in the organism. Here, we measure UDP-glucose-dependent glycogen synthase activity in cell-free extracts of T. vaginalis. We then demonstrate that the TVAG_258220 open reading frame encodes a glycosyltransferase that is presumably responsible for this synthetic activity. We show that expression of TVAG_258220 in a yeast strain lacking endogenous glycogen synthase activity is sufficient to restore glycogen accumulation. Furthermore, when TVAG_258220 is expressed in bacteria, the resulting recombinant protein has glycogen synthase activity in vitro, transferring glucose from either UDP-glucose or ADP-glucose to glycogen and using both substrates with similar affinity. This protein is also able to transfer glucose from UDP-glucose or ADP-glucose to maltose and longer oligomers of glucose but not to glucose itself. However, with these substrates, there is no evidence of processivity and sugar transfer is limited to between one and three glucose residues. Taken together with our earlier work on glycogen phosphorylase, we are now well positioned to define both how T. vaginalis synthesizes and utilizes glycogen, and how these processes are regulated. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  15. In Vitro Biochemical Characterization of All Barley Endosperm Starch Synthases

    Directory of Open Access Journals (Sweden)

    Jose Antonio Cuesta-Seijo

    2016-01-01

    Full Text Available Starch is the main storage polysaccharide in cereals and the major source of calories in the human diet. It is synthesized by a panel of enzymes including five classes of starch synthases (SSs. While the overall starch synthase (SS reaction is known, the functional differences between the five SS classes are poorly understood. Much of our knowledge comes from analyzing mutant plants with altered SS activities, but the resulting data are often difficult to interpret as a result of pleitropic effects, competition between enzymes, overlaps in enzyme activity and disruption of multi-enzyme complexes. Here we provide a detailed biochemical study of the activity of all five classes of SSs in barley endosperm. Each enzyme was produced recombinantly in E. coli and the properties and modes of action in vitro were studied in isolation from other SSs and other substrate modifying activities. Our results define the mode of action of each SS class in unprecedented detail; we analyze their substrate selection, temperature dependence and stability, substrate affinity and temporal abundance during barley development. Our results are at variance with some generally accepted ideas about starch biosynthesis and might lead to the reinterpretation of results obtained in planta. In particular, they indicate that granule bound SS is capable of processive action even in the absence of a starch matrix, that SSI has no elongation limit, and that SSIV, believed to be critical for the initiation of starch granules, has maltoligosaccharides and not polysaccharides as its preferred substrates.

  16. Inhibitors of polyhydroxyalkanoate (PHA) synthases: synthesis, molecular docking, and implications.

    Science.gov (United States)

    Zhang, Wei; Chen, Chao; Cao, Ruikai; Maurmann, Leila; Li, Ping

    2015-01-01

    Polyhydroxyalkanoate (PHA) synthases (PhaCs) catalyze the formation of biodegradable PHAs that are considered to be ideal alternatives to non-biodegradable synthetic plastics. However, study of PhaCs has been challenging because the rate of PHA chain elongation is much faster than that of initiation. This difficulty, along with lack of a crystal structure, has become the main hurdle to understanding and engineering PhaCs for economical PHA production. Here we report the synthesis of two carbadethia CoA analogues--sT-CH2-CoA (26 a) and sTet-CH2-CoA (26 b)--as well as sT-aldehyde (saturated trimer aldehyde, 29), as new PhaC inhibitors. Study of these analogues with PhaECAv revealed that 26 a/b and 29 are competitive and mixed inhibitors, respectively. Both the CoA moiety and extension of PHA chain will increase binding affinity; this is consistent with our docking study. Estimation of the Kic values of 26 a and 26 b predicts that a CoA analogue incorporating an octameric hydroxybutanoate (HB) chain might facilitate the formation of a kinetically well-behaved synthase.

  17. Insulin transcriptionally regulates argininosuccinate synthase to maintain vascular endothelial function.

    Science.gov (United States)

    Haines, Ricci J; Corbin, Karen D; Pendleton, Laura C; Meininger, Cynthia J; Eichler, Duane C

    2012-04-27

    Diminished vascular endothelial cell nitric oxide (NO) production is a major factor in the complex pathogenesis of diabetes mellitus. In this report, we demonstrate that insulin not only maintains endothelial NO production through regulation of endothelial nitric oxide synthase (eNOS), but also via the regulation of argininosuccinate synthase (AS), which is the rate-limiting step of the citrulline-NO cycle. Using serum starved, cultured vascular endothelial cells, we show that insulin up-regulates AS and eNOS transcription to support NO production. Moreover, we show that insulin enhances NO production in response to physiological cues such as bradykinin. To translate these results to an in vivo model, we show that AS transcription is diminished in coronary endothelial cells isolated from rats with streptozotocin (STZ)-induced diabetes. Importantly, we demonstrate restoration of AS and eNOS transcription by insulin treatment in STZ-diabetic rats, and show that this restoration was accompanied by improved endothelial function as measured by endothelium-dependent vasorelaxation. Overall, this report demonstrates, both in cell culture and whole animal studies, that insulin maintains vascular function, in part, through the maintenance of AS transcription, thus ensuring an adequate supply of arginine to maintain vascular endothelial response to physiological cues. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. Chromosomal localization of the human and mouse hyaluronan synthase genes

    Energy Technology Data Exchange (ETDEWEB)

    Spicer, A.P.; McDonald, J.A. [Mayo Clinic Scottsdale, AZ (United States); Seldin, M.F. [Univ. of California Davis, CA (United States)] [and others

    1997-05-01

    We have recently identified a new vertebrate gene family encoding putative hyaluronan (HA) synthases. Three highly conserved related genes have been identified, designated HAS1, HAS2, and HAS3 in humans and Has1, Has2, and Has3 in the mouse. All three genes encode predicted plasma membrane proteins with multiple transmembrane domains and approximately 25% amino acid sequence identity to the Streptococcus pyogenes HA synthase, HasA. Furthermore, expression of any one HAS gene in transfected mammalian cells leads to high levels of HA biosynthesis. We now report the chromosomal localization of the three HAS genes in human and in mouse. The genes localized to three different positions within both the human and the mouse genomes. HAS1 was localized to the human chromosome 19q13.3-q13.4 boundary and Has1 to mouse Chr 17. HAS2 was localized to human chromosome 8q24.12 and Has2 to mouse Chr 15. HAS3 was localized to human chromosome 16q22.1 and Has3 to mouse Chr 8. The map position for HAS1 reinforces the recently reported relationship between a small region of human chromosome 19q and proximal mouse chromosome 17. HAS2 mapped outside the predicted critical region delineated for the Langer-Giedion syndrome and can thus be excluded as a candidate gene for this genetic syndrome. 33 refs., 2 figs.

  19. CTP limitation increases expression of CTP synthase in Lactococcus lactis

    DEFF Research Database (Denmark)

    Jørgensen, C.M.; Hammer, Karin; Martinussen, Jan

    2003-01-01

    on the CTP concentration through an attenuator mechanism. At normal CTP concentrations a terminator is preferentially formed in the pyrG leader, thereby reducing expression of CTP synthase. At low CTP concentrations the RNA polymerase pauses at a stretch of C residues in the pyrG leader, thereby allowing......CTP synthase is encoded by the pyrG gene and catalyzes the conversion of UTP to CTP. A Lactococcus lactis pyrG mutant with a cytidine requirement was constructed, in which beta-galactosidase activity in a pyrG-lacLM transcriptional fusion was used to monitor gene expression of pyrG. A 10-fold...... decrease in the CTP pool induced by cytidine limitation was found to immediately increase expression of the L. lactis pyrG gene. The final level of expression of pyrG is 37-fold higher than the uninduced level. CTP limitation has pronounced effects on central cellular metabolism, and both RNA and protein...

  20. Aldosterone synthase inhibitors in hypertension: current status and future possibilities

    Directory of Open Access Journals (Sweden)

    Milan Hargovan

    2014-02-01

    Full Text Available The renin-angiotensin aldosterone system is a critical mechanism for controlling blood pressure, and exerts most of its physiological effects through the action of angiotensin II. In addition to increasing blood pressure by increasing vascular resistance, angiotensin II also stimulates aldosterone secretion from the adrenal gland. Aldosterone acts to cause an increase in sodium and water reabsorption, thus elevating blood pressure. Although treatment with angiotensin converting enzyme inhibitors initially lowers circulating aldosterone, with chronic treatment aldosterone levels increase back to baseline, a phenomenon termed aldosterone escape; aldosterone blockade may therefore give added value in the treatment of hypertension. The first mineralocorticoid receptor antagonist developed was spironolactone, but its use has been severely hampered by adverse (notably oestrogenic effects. The more recently developed mineralocorticoid receptor antagonist eplerenone exhibits a better adverse effect profile, although it is not devoid of effects similar to spironolactone. In addition, aldosterone activates non-genomic receptors that are not inhibited by either eplerenone or spironolactone. It is believed that deleterious organ remodelling is mediated by aldosterone via such non-genomic pathways. A new class of drugs, the aldosterone synthase inhibitors, is currently under development. These may offer a novel therapeutic approach for both lowering blood pressure and preventing the non-genomic effects of aldosterone. Here, we will review the cardiovascular effects of aldosterone and review the drugs available that target this hormone, with a particular focus on the aldosterone synthase inhibitors.

  1. Aldosterone synthase inhibitors in hypertension: current status and future possibilities.

    Science.gov (United States)

    Hargovan, Milan; Ferro, Albert

    2014-01-01

    The renin-angiotensin aldosterone system is a critical mechanism for controlling blood pressure, and exerts most of its physiological effects through the action of angiotensin II. In addition to increasing blood pressure by increasing vascular resistance, angiotensin II also stimulates aldosterone secretion from the adrenal gland. Aldosterone acts to cause an increase in sodium and water reabsorption, thus elevating blood pressure. Although treatment with angiotensin converting enzyme inhibitors initially lowers circulating aldosterone, with chronic treatment aldosterone levels increase back to baseline, a phenomenon termed aldosterone escape; aldosterone blockade may therefore give added value in the treatment of hypertension. The first mineralocorticoid receptor antagonist developed was spironolactone, but its use has been severely hampered by adverse (notably oestrogenic) effects. The more recently developed mineralocorticoid receptor antagonist eplerenone exhibits a better adverse effect profile, although it is not devoid of effects similar to spironolactone. In addition, aldosterone activates non-genomic receptors that are not inhibited by either eplerenone or spironolactone. It is believed that deleterious organ remodelling is mediated by aldosterone via such non-genomic pathways. A new class of drugs, the aldosterone synthase inhibitors, is currently under development. These may offer a novel therapeutic approach for both lowering blood pressure and preventing the non-genomic effects of aldosterone. Here, we will review the cardiovascular effects of aldosterone and review the drugs available that target this hormone, with a particular focus on the aldosterone synthase inhibitors.

  2. Cryptic Polyketide Synthase Genes in Non-Pathogenic Clostridium SPP

    Science.gov (United States)

    Behnken, Swantje; Hertweck, Christian

    2012-01-01

    Modular type I polyketide synthases (PKS) produce a vast array of bacterial metabolites with highly diverse biological functions. Notably, all known polyketides were isolated from aerobic bacteria, and yet no example has been reported for strict anaerobes. In this study we explored the diversity and distribution of PKS genes in the genus Clostridium. In addition to comparative genomic analyses combined with predictions of modular type I polyketide synthase (PKS) gene clusters in sequenced genomes of Clostridium spp., a representative selection of other species inhabiting a variety of ecological niches was investigated by PCR screening for PKS genes. Our data reveal that all studied pathogenic Clostridium spp. are devoid of putative PKS genes. In stark contrast, cryptic PKS genes are widespread in genomes of non-pathogenic Clostridium species. According to phylogenetic analyses, the Clostridium PKS genes have unusual and diverse origins. However, reverse transcription quantitative PCR demonstrates that these genes are silent under standard cultivation conditions, explaining why the related metabolites have been overlooked until now. This study presents clostridia as a putative source for novel bioactive polyketides. PMID:22235310

  3. IPC synthase as a useful target for antifungal drugs.

    Science.gov (United States)

    Sugimoto, Yuichi; Sakoh, Hiroki; Yamada, Koji

    2004-12-01

    Inositol phosphorylceramide (IPC) synthase is a common and essential enzyme in fungi and plants, which catalyzes the transfer of phosphoinositol to the C-1 hydroxy of ceramide to produce IPC. This reaction is a key step in fungal sphingolipid biosynthesis, therefore the enzyme is a potential target for the development of nontoxic therapeutic antifungal agents. Natural products with a desired biological activity, aureobasidin A (AbA), khafrefungin, and galbonolide A, have been reported. AbA, a cyclic depsipeptide containing 8 amino acids and a hydroxyl acid, is a broad spectrum antifungal with strong activity against many pathogenic fungi such as Candida spp., Cryptococcus neoformans, and some Aspergillus spp. Khafrefungin, an aldonic acid ester with a C22 long alkyl chain, has antifungal activity against C. albicans, Cr. Neoformans, and Saccharomyces cerevisiae. Galbonolide A is a 14-membered macrolide with fungicidal activity against clinically important strains, and is especially potent against Cr. neoformans. These classes of natural products are potent and specific antifungal agents. We review current progress in the development of IPC synthase inhibitors with antifungal activities, and present structure-activity relationships (SAR), physicochemical and structural properties, and synthetic methodology for chemical modification.

  4. Eugenol synthase genes in floral scent variation in Gymnadenia species.

    Science.gov (United States)

    Gupta, Alok K; Schauvinhold, Ines; Pichersky, Eran; Schiestl, Florian P

    2014-12-01

    Floral signaling, especially through floral scent, is often highly complex, and little is known about the molecular mechanisms and evolutionary causes of this complexity. In this study, we focused on the evolution of "floral scent genes" and the associated changes in their functions in three closely related orchid species of the genus Gymnadenia. We developed a benchmark repertoire of 2,571 expressed sequence tags (ESTs) in Gymnadenia odoratissima. For the functional characterization and evolutionary analysis, we focused on eugenol synthase, as eugenol is a widespread and important scent compound. We obtained complete coding complementary DNAs (cDNAs) of two copies of putative eugenol synthase genes in each of the three species. The proteins encoded by these cDNAs were characterized by expression and testing for activity in Escherichia coli. While G. odoratissima and Gymnadenia conopsea enzymes were found to catalyze the formation of eugenol only, the Gymnadenia densiflora proteins synthesize eugenol, as well as a smaller amount of isoeugenol. Finally, we showed that the eugenol and isoeugenol producing gene copies of G. densiflora are evolutionarily derived from the ancestral genes of the other species producing only eugenol. The evolutionary switch from production of one to two compounds evolved under relaxed purifying selection. In conclusion, our study shows the molecular bases of eugenol and isoeugenol production and suggests that an evolutionary transition in a single gene can lead to an increased complexity in floral scent emitted by plants.

  5. Cloning and Identification of Methionine Synthase Gene from Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    Lan HUANG; Dong-Yang LI; Shao-Xiao WANG; Shi-Ming ZHANG; Jun-Hui CHEN; Xiang-Fu WU

    2005-01-01

    Methionine synthase (MS) is grouped into two classes. Class One MS (MetH) and Class Two MS (MetE) share no homology and differ in their catalytic model. Based on the conserved sequences of metE genes from different organisms, a segment of the metE gene was first cloned from Pichia pastoris genomic DNA by PCR, and its 5' and 3' regions were further cloned by 5'- and 3'-rapid amplification of cDNA ends (RACE), respectively. The assembled sequence reveals an open reading frame encoding a polypeptide of 768 residues, and the deduced product shares 76% identity with MetE of Saccharomyces cerevisiae. P. pastoris methionine synthase (PpMetE) consists of two domains common to MetEs. The active site is located in the C-terminal domain, in which the residues involved in the interaction of zinc with substrates are conserved. Homologous expression of PpMetE in P. pastoris was achieved, and the heterologous expression of PpMetE in the S. cerevisiae strain XJB3-1D that is MetE-defective restored the growth of the mutant on methionine-free minimal media. The gene sequence has been submitted to GenBank/EMBL/DDBJ under accession No. AY601648.

  6. Tryptophan synthase of Phaeophyceae originated from the secondary host nucleus

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yalan; CHI Shan; WU Shuangxiu; LIU Cui; YU Jun; WANG Xumin; CHEN Shengping; LIU Tao

    2014-01-01

    Tryptophan synthase (TS, EC 4.2.1.20) catalyzes the last two steps of L-tryptophan biosynthesis. In pro-karyotes, tryptophan synthase is a multi-enzyme complex, and it consists ofαandβsubunit which forms anα-ββ-αcomplex. In fungi and diatoms, TS is a bifunctional enzyme. Because of the limited genomic and transcriptomic data of algae, there are few studies on TS evolution of algae. Here we analyzed the data of the 1000 Plants Project (1KP), and focused on red algae and brown algae. We found out that the TS of Phaeophy-ceae were fusion genes, which probably originated from the secondary host nucleus, and that the TS of Rho-dophyta contained two genes, TSA and TSB, which both display a possible cyanobacterial origin at the time of primary endosymbiosis. In addition, there were two types of TSB genes (TSB1 and TSB2). Through the multiple sequence alignment of TSB proteins, we found several residues conserved in TSB1 but variable in TSB2 which connect withαsubunit. The phenomenon may suggest that the TSB2 sequences of Rhodophyta cannot form stable complex with TSA.

  7. Phylogenetic analysis of uroporphyrinogen III synthase (UROS) gene.

    Science.gov (United States)

    Shaik, Abjal Pasha; Alsaeed, Abbas H; Sultana, Asma

    2012-01-01

    The uroporphyrinogen III synthase (UROS) enzyme (also known as hydroxymethylbilane hydrolyase) catalyzes the cyclization of hydroxymethylbilane to uroporphyrinogen III during heme biosynthesis. A deficiency of this enzyme is associated with the very rare Gunther's disease or congenital erythropoietic porphyria, an autosomal recessive inborn error of metabolism. The current study investigated the possible role of UROS (Homo sapiens [EC: 4.2.1.75; 265 aa; 1371 bp mRNA; Entrez Pubmed ref NP_000366.1, NM_000375.2]) in evolution by studying the phylogenetic relationship and divergence of this gene using computational methods. The UROS protein sequences from various taxa were retrieved from GenBank database and were compared using Clustal-W (multiple sequence alignment) with defaults and a first-pass phylogenetic tree was built using neighbor-joining method as in DELTA BLAST 2.2.27+ version. A total of 163 BLAST hits were found for the uroporphyrinogen III synthase query sequence and these hits showed putative conserved domain, HemD superfamily (as on 14(th) Nov 2012). We then narrowed down the search by manually deleting the proteins which were not UROS sequences and sequences belonging to phyla other than Chordata were deleted. A repeat phylogenetic analysis of 39 taxa was performed using PhyML and TreeDyn software to confirm that UROS is a highly conserved protein with approximately 85% conserved sequences in almost all chordate taxons emphasizing its importance in heme synthesis.

  8. The crystal structure of human GDP-L-fucose synthase.

    Science.gov (United States)

    Zhou, Huan; Sun, Lihua; Li, Jian; Xu, Chunyan; Yu, Feng; Liu, Yahui; Ji, Chaoneng; He, Jianhua

    2013-09-01

    Human GDP-l-fucose synthase, also known as FX protein, synthesizes GDP-l-fucose from its substrate GDP-4-keto-6-deoxy-d-mannose. The reaction involves epimerization at both C-3 and C-5 followed by an NADPH-dependent reduction of the carbonyl at C-4. In this paper, the first crystal structure of human FX protein was determined at 2.37 Å resolution. The asymmetric unit of the crystal structure contains four molecules which form two homodimers. Each molecule consists of two domains, a Rossmann-fold NADPH-binding motif and a carboxyl terminal domain. Compared with the Escherichia coli GDP-l-fucose synthase, the overall structures of these two enzymes have four major differences. There are four loops in the structure of human FX protein corresponding to two α-helices and two β-sheets in that of the E. coli enzyme. Besides, there are seven different amino acid residues binding with NAPDH comparing human FX protein with that from E. coli. The structure of human FX reveals the key catalytic residues and could be useful for the design of drugs for the treatment of inflammation, auto-immune diseases, and possibly certain types of cancer.

  9. Selectivity of the surface binding site (SBS) on barley starch synthase I

    DEFF Research Database (Denmark)

    Wilkens, Casper; Cuesta-Seijo, Jose A.; Palcic, Monica

    2014-01-01

    Starch synthase I (SSI) from various sources has been shown to preferentially elongate branch chains of degree of polymerisation (DP) from 6–7 to produce chains of DP 8–12. In the recently determined crystal structure of barley starch synthase I (HvSSI) a so-called surface binding site (SBS) was ...

  10. Identification and site of action of the remaining four putative pseudouridine synthases in Escherichia coli.

    Science.gov (United States)

    Del Campo, M; Kaya, Y; Ofengand, J

    2001-11-01

    There are 10 known putative pseudouridine synthase genes in Escherichia coli. The products of six have been previously assigned, one to formation of the single pseudouridine in 16S RNA, three to the formation of seven pseudouridines in 23S RNA, and three to the formation of three pseudouridines in tRNA (one synthase makes pseudouridine in 23S RNA and tRNA). Here we show that the remaining four putative synthase genes make bona fide pseudouridine synthases and identify which pseudouridines they make. RluB (formerly YciL) and RluE (formerly YmfC) make pseudouridine2605 and pseudouridine2457, respectively, in 23S RNA. RluF (formerly YjbC) makes the newly discovered pseudouridine2604 in 23S RNA, and TruC (formerly YqcB) makes pseudouridine65 in tRNA(Ile1) and tRNA(Asp). Deletion of each of these synthase genes individually had no effect on exponential growth in rich media at 25 degrees C, 37 degrees C, or 42 degrees C. A strain lacking RluB and RluF also showed no growth defect under these conditions. Mutation of a conserved aspartate in a common sequence motif, previously shown to be essential for the other six E. coli pseudouridine synthases and several yeast pseudouridine synthases, also caused a loss of in vivo activity in all four of the synthases studied in this work.

  11. Insights into the subunit in-teractions of the chloroplast ATP synthase

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Subunit interactions of the chloroplast F0F1- ATP synthase were studied using the yeast two-hybrid system. The coding sequences of all the nine subunits of spinach chloroplast ATP synthase were cloned in two-hybrid vectors. The vectors were transformed into the yeast strains HF7c and SFY526 by various pairwise combinations, and the protein interactions were analyzed by measuring the yeast growth on minimal SD medium without serine, lucine and histidine. Interactions of γ Subunit with wild type or two truncated mutants of γ sununit, △εN21 and △εC45, which lose their abilities to inhibit the ATP hydrolysis, were also detected by in vitro and in vivo binding assay. The present results are largely accordant to the common structure model of F0F1-ATP synthase. Different from that in the E. Coli F0F1-ATP synthase, the δ subunit of chloroplast ATP syn- thase could interact with β,γ,ε and all the CF0 subunits in the two-hybrid system. These results suggested that though the chloroplast ATP synthase shares the similar structure and composition of subunits with the enzyme from E. Coli, it may be different in the subunit interactions and con- formational change during catalysis between these two sources of ATP synthase. Based on the present results and our knowledge of structure model of E. Coli ATP synthase, a deduced structure model of chloroplast ATP synthase was proposed.

  12. Domain swapping of Citrus limon monoterpene synthases: impact on enzymatic activity and product specifity.

    NARCIS (Netherlands)

    Tamer, el M.K.; Lucker, J.; Bosch, D.; Verhoeven, H.A.; Verstappen, F.W.A.; Schwab, W.; Tunen, van A.J.; Voragen, A.G.J.; Maagd, de R.A.; Bouwmeester, H.J.

    2003-01-01

    Monoterpene cyclases are the key enzymes in the monoterpene biosynthetic pathway, as they catalyze the cyclization of the ubiquitous geranyl diphosphate (GDP) to the specific monoterpene skeletons. From Citrus limon, four monoterpene synthase-encoding cDNAs for a P-pinene synthase named

  13. KORRIGAN1 Interacts Specifically with Integral Components of the Cellulose Synthase Machinery

    NARCIS (Netherlands)

    Mansoori Zangir, N.; Timmers, J.F.P.; Desprez, T.; Lessa Alvim Kamei, C.; Dees, D.C.T.; Vincken, J.P.; Visser, R.G.F.; Höfte, H.; Vernhettes, S.; Trindade, L.M.

    2014-01-01

    Cellulose is synthesized by the so called rosette protein complex and the catalytic subunits of this complex are the cellulose synthases (CESAs). It is thought that the rosette complexes in the primary and secondary cell walls each contains at least three different non-redundant cellulose synthases.

  14. Domain swapping of Citrus limon monoterpene synthases: impact on enzymatic activity and product specifity.

    NARCIS (Netherlands)

    Tamer, el M.K.; Lucker, J.; Bosch, D.; Verhoeven, H.A.; Verstappen, F.W.A.; Schwab, W.; Tunen, van A.J.; Voragen, A.G.J.; Maagd, de R.A.; Bouwmeester, H.J.

    2003-01-01

    Monoterpene cyclases are the key enzymes in the monoterpene biosynthetic pathway, as they catalyze the cyclization of the ubiquitous geranyl diphosphate (GDP) to the specific monoterpene skeletons. From Citrus limon, four monoterpene synthase-encoding cDNAs for a P-pinene synthase named Cl(-)betaPIN

  15. Structure of the dimeric form of CTP synthase from Sulfolobus solfataricus

    DEFF Research Database (Denmark)

    Lauritsen, Iben; Willemoës, Martin; Jensen, Kaj Frank;

    2011-01-01

    CTP synthase catalyzes the last committed step in de novo pyrimidine-nucleotide biosynthesis. Active CTP synthase is a tetrameric enzyme composed of a dimer of dimers. The tetramer is favoured in the presence of the substrate nucleotides ATP and UTP; when saturated with nucleotide, the tetramer c...

  16. Expression, crystallization and structure elucidation of γ-terpinene synthase from Thymus vulgaris.

    Science.gov (United States)

    Rudolph, Kristin; Parthier, Christoph; Egerer-Sieber, Claudia; Geiger, Daniel; Muller, Yves A; Kreis, Wolfgang; Müller-Uri, Frieder

    2016-01-01

    The biosynthesis of γ-terpinene, a precursor of the phenolic isomers thymol and carvacrol found in the essential oil from Thymus sp., is attributed to the activitiy of γ-terpinene synthase (TPS). Purified γ-terpinene synthase from T. vulgaris (TvTPS), the Thymus species that is the most widely spread and of the greatest economical importance, is able to catalyze the enzymatic conversion of geranyl diphosphate (GPP) to γ-terpinene. The crystal structure of recombinantly expressed and purified TvTPS is reported at 1.65 Å resolution, confirming the dimeric structure of the enzyme. The putative active site of TvTPS is deduced from its pronounced structural similarity to enzymes from other species of the Lamiaceae family involved in terpenoid biosynthesis: to (+)-bornyl diphosphate synthase and 1,8-cineole synthase from Salvia sp. and to (4S)-limonene synthase from Mentha spicata.

  17. Molecular cloning, functional expression and characterization of (E)-beta farnesene synthase from Citrus junos.

    Science.gov (United States)

    Maruyama, T; Ito, M; Honda, G

    2001-10-01

    We cloned the gene of the acyclic sesquiterpene synthase, (E)-beta-farnesene synthase (CJFS) from Yuzu (Citrus junos, Rutaceae). The function of CJFS was elucidated by the preparation of recombinant protein and subsequent enzyme assay. CJFS consisted of 1867 nucleotides including 1680 bp of coding sequence encoding a protein of 560 amino acids with a molecular weight of 62 kDa. The deduced amino acid sequence possessed characteristic amino acid residues, such as the DDxxD motif, which are highly conserved among terpene synthases. This is the first report of the cloning of a terpene synthase from a Rutaceous plant. A possible reaction mechanism for terpene biosynthesis is also discussed on the basis of sequence comparison of CJFS with known sesquiterpene synthase genes.

  18. Protons, the thylakoid membrane, and the chloroplast ATP synthase.

    Science.gov (United States)

    Junge, W

    1989-01-01

    According to the chemiosmotic theory, proton pumps and ATP synthases are coupled by lateral proton flow through aqueous phases. Three long-standing challenges to this concept, all of which have been loosely subsumed under 'localized coupling' in the literature, were examined in the light of experiments carried out with thylakoids: (1) Nearest neighbor interaction between pumps and ATP synthases. Considering the large distances between photosystem II and CFoCF1, in stacked thylakoids this is a priori absent. (2) Enhanced proton diffusion along the surface of the membrane. This could not be substantiated for the outer side of the thylakoid membrane. Even for the interface between pure lipid and water, two laboratories have reported the absence of enhanced diffusion. (3) Localized proton ducts in the membrane. Intramembrane domains that can transiently trap protons do exist in thylakoid membranes, but because of their limited storage capacity for protons, they probably do not matter for photophosphorylation under continuous light. Seemingly in favor of localized proton ducts is the failure of a supposedly permeant buffer to enhance the onset lag of photophosphorylation. However, it was found that failure of some buffers and the ability of others in this respect were correlated with their failure/ability to quench pH transients in the thylakoid lumen, as predicted by the chemiosmotic theory. It was shown that the chemiosmotic concept is a fair approximation, even for narrow aqueous phases, as in stacked thylakoids. These are approximately isopotential, and protons are taken in by the ATP synthase straight from the lumen. The molecular mechanism by which F0F1 ATPases couple proton flow to ATP synthesis is still unknown. The threefold structural symmetry of the headpiece that, probably, finds a corollary in the channel portion of these enzymes appeals to the common wisdom that structural symmetry causes functional symmetry. "Rotation catalysis" has been proposed. It is

  19. ATP synthase in slow- and fast-growing mycobacteria is active in ATP synthesis and blocked in ATP hydrolysis direction.

    NARCIS (Netherlands)

    Haagsma, A.C.; Driessen, N.N.; Hahn, M.M.; Lill, H.; Bald, D.

    2010-01-01

    ATP synthase is a validated drug target for the treatment of tuberculosis, and ATP synthase inhibitors are promising candidate drugs for the treatment of infections caused by other slow-growing mycobacteria, such as Mycobacterium leprae and Mycobacterium ulcerans. ATP synthase is an essential enzyme

  20. ATP synthase in slow- and fast-growing mycobacteria is active in ATP synthesis and blocked in ATP hydrolysis direction.

    NARCIS (Netherlands)

    Haagsma, A.C.; Driessen, N.N.; Hahn, M.M.; Lill, H.; Bald, D.

    2010-01-01

    ATP synthase is a validated drug target for the treatment of tuberculosis, and ATP synthase inhibitors are promising candidate drugs for the treatment of infections caused by other slow-growing mycobacteria, such as Mycobacterium leprae and Mycobacterium ulcerans. ATP synthase is an essential enzyme

  1. The essential cell division protein FtsN interacts with the murein (peptidoglycan) synthase PBP1B in Escherichia coli

    NARCIS (Netherlands)

    Müller, Patrick; Ewers, C.; Bertsche, U.; Anstett, M.; Kallis, T.; Breukink, E.J.; Fraipont, Claudine; Terrak, Mohammed; Nguyen-Distèche, Martine; Vollmer, W.

    2007-01-01

    Bacterial cell division requires the coordinated action of cell division proteins and murein (peptidoglycan) synthases. Interactions involving the essential cell division protein FtsN and murein synthases were studied by affinity chromatography with membrane fraction. The murein synthases PBP1A, PBP

  2. Biochemical and molecular responses underlying differential arsenic tolerance in rice (Oryza sativa L.).

    Science.gov (United States)

    Begum, Most Champa; Islam, Mohammad Saiful; Islam, Monirul; Amin, Ruhul; Parvez, Mohammad Sarwar; Kabir, Ahmad Humayan

    2016-07-01

    The arsenic (As) is a toxic element causing major health concern worldwide. Arsenate stress caused no significant reduction in growth parameters and shoot electrolyte leakage but showed increased root arsenate reductase activity along with relatively lower root As content and shoot translocation rate in As-tolerant BRRI 33 than in As-sensitive BRRI 51. It indicates that As inhibition and tolerance mechanisms are driven by root responses. Interestingly, As stress showed consistent decrease in phosphate content and expression of phosphate transporters (OsPT8, OsPT4, OsPHO1;2) under both high and low phosphate conditions in roots of BRRI 33, suggesting that limiting phosphate transport mainly mediated by OsPHO1;2 directs less As accumulation in BRRI 33. Further, BRRI 33 showed simultaneous increase in OsPCS1 (phytochelatin synthase) expression and phytochelatins (PCs) content in roots under As exposure supporting the hypothesis that root As sequestration acts as 'firewall system' in limiting As translocation in shoots. Furthermore, increased CAT, POD, SOD, GR, along with elevated glutathione, methionine, cysteine and proline suggests that strong antioxidant defense plays integral part to As tolerance in BRRI 33. Again, BRRI 33 self-grafts and plants having BRRI 33 rootstock combined with BRRI 51 scion had no adverse effect on morphological parameters but showed reduced As translocation rate, increased root arsenate reductase activity, shoot PC synthesis and root OsPHO1;2 expression due to As stress. It confirms that signal driving As tolerance mechanisms is generated in the roots. These findings can be implemented for As detoxification and As-free transgenic rice production for health safety.

  3. Hyperactivity: glycogen synthase kinase-3 as a therapeutic target.

    Science.gov (United States)

    Mines, Marjelo A

    2013-05-15

    The diagnosis of hyperactivity-associated disorders has increased within the past few years. The prevalence of hyperactivity-associated disorders is indicative of the need to more fully understand the underlying causes and to develop improved therapeutic interventions. There is increasing evidence that glycogen synthase kinase-3 (GSK3) mediates locomotor hyperactivity in a number of animal models, and therefore may be a potential target for therapeutic intervention in hyperactivity-associated behaviors. In this review, we discuss 1) the effect of manipulations of GSK3 in the absence of drugs and disorders on locomotor activity, 2) the role of GSK3 in drug-induced hyperactivity in rodents, and 3) regulation of locomotor activity by GSK3 in transgenic mouse models related to specific disorders. These studies link GSK3 regulation and activity to hyperactivity-associated behaviors and disease pathologies. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. Alterations in Nitric Oxide Synthase in the Aged CNS

    Directory of Open Access Journals (Sweden)

    Junyang Jung

    2012-01-01

    Full Text Available Aging is associated with neuronal loss, gross weight reduction of the brain, and glial proliferation in the cortex, all of which lead to functional changes in the brain. It is known that oxidative stress is a critical factor in the pathogenesis of aging; additionally, growing evidence suggests that excessive nitric oxide (NO production contributes to the aging process. However, it is still unclear how NO plays a role in the aging process. This paper describes age-related changes in the activity of NADPH-diaphorase (NADPH-d, a marker for neurons containing nitric oxide synthase (NOS, in many CNS regions. Understanding these changes may provide a novel perspective in identifying the aging mechanism.

  5. Recurrent encephalopathy: NAGS (N-acetylglutamate synthase) deficiency in adults.

    Science.gov (United States)

    Cartagena, A; Prasad, A N; Rupar, C A; Strong, M; Tuchman, M; Ah Mew, N; Prasad, C

    2013-01-01

    N-acetyl-glutamate synthase (NAGS) deficiency is a rare autosomal recessive urea cycle disorder (UCD) that uncommonly presents in adulthood. Adult presentations of UCDs include; confusional episodes, neuropsychiatric symptoms and encephalopathy. To date, there have been no detailed neurological descriptions of an adult onset presentation of NAGS deficiency. In this review we examine the clinical presentation and management of UCDs with an emphasis on NAGS deficiency. An illustrative case is provided. Plasma ammonia levels should be measured in all adult patients with unexplained encephalopathy, as treatment can be potentially life-saving. Availability of N-carbamylglutamate (NCG; carglumic acid) has made protein restriction largely unnecessary in treatment regimens currently employed. Genetic counselling remains an essential component of management of NAGS.

  6. Amino acids conferring herbicide resistance in tobacco acetohydroxyacid synthase.

    Science.gov (United States)

    Le, Dung Tien; Choi, Jung-Do; Tran, Lam-Son Phan

    2010-01-01

    Acetohydroxyacid synthase (AHAS) (EC 4.1.3.18) is a target of commercially available herbicides such as sulfonylurea, imidazolinone, and triazolopyrimidine. In plants and microorganisms, AHAS catalyzes the first common reaction in the biosynthesis pathways leading to leucine, isoleucine and valine. Intensive studies using different approaches - including site-directed mutagenesis, molecular modeling and structural analysis - on plant AHAS-s have contributed to the understanding of the herbicide-AHAS interaction. Knowledge of the critical roles of amino acid residues of plant AHAS in conferring herbicide resistance will enable the creation of new herbicide-tolerant AHAS which could be used to develop herbicide-resistant transgenic plants. Moreover, such information will also elucidate design strategies for more efficient herbicides that could also kill weeds resistant to previously used AHAS-inhibiting herbicides. In this review, we summarize the results of intensive searches for amino acid residues and their substitutions that confer herbicide resistance in tobacco AHAS.

  7. Dihydropteroate synthase gene mutations in Pneumocystis and sulfa resistance

    DEFF Research Database (Denmark)

    Huang, Laurence; Crothers, Kristina; Atzori, Chiara

    2004-01-01

    Pneumocystis pneumonia (PCP) remains a major cause of illness and death in HIV-infected persons. Sulfa drugs, trimethoprim-sulfamethoxazole (TMP-SMX) and dapsone are mainstays of PCP treatment and prophylaxis. While prophylaxis has reduced the incidence of PCP, its use has raised concerns about...... in the dihydropteroate synthase (DHPS) gene. Similar mutations have been observed in P. jirovecii. Studies have consistently demonstrated a significant association between the use of sulfa drugs for PCP prophylaxis and DHPS gene mutations. Whether these mutations confer resistance to TMP-SMX or dapsone plus trimethoprim...... for PCP treatment remains unclear. We review studies of DHPS mutations in P. jirovecii and summarize the evidence for resistance to sulfamethoxazole and dapsone....

  8. A new motif for inhibitors of geranylgeranyl diphosphate synthase.

    Science.gov (United States)

    Foust, Benjamin J; Allen, Cheryl; Holstein, Sarah A; Wiemer, David F

    2016-08-15

    The enzyme geranylgeranyl diphosphate synthase (GGDPS) is believed to receive the substrate farnesyl diphosphate through one lipophilic channel and release the product geranylgeranyl diphosphate through another. Bisphosphonates with two isoprenoid chains positioned on the α-carbon have proven to be effective inhibitors of this enzyme. Now a new motif has been prepared with one isoprenoid chain on the α-carbon, a second included as a phosphonate ester, and the potential for a third at the α-carbon. The pivaloyloxymethyl prodrugs of several compounds based on this motif have been prepared and the resulting compounds have been tested for their ability to disrupt protein geranylgeranylation and induce cytotoxicity in myeloma cells. The initial biological studies reveal activity consistent with GGDPS inhibition, and demonstrate a structure-function relationship which is dependent on the nature of the alkyl group at the α-carbon.

  9. Catalysis and Sulfa Drug Resistance in Dihydropteroate Synthase

    Energy Technology Data Exchange (ETDEWEB)

    Yun, Mi-Kyung; Wu, Yinan; Li, Zhenmei; Zhao, Ying; Waddell, M. Brett; Ferreira, Antonio M.; Lee, Richard E.; Bashford, Donald; White, Stephen W. (SJCH)

    2013-04-08

    The sulfonamide antibiotics inhibit dihydropteroate synthase (DHPS), a key enzyme in the folate pathway of bacteria and primitive eukaryotes. However, resistance mutations have severely compromised the usefulness of these drugs. We report structural, computational, and mutagenesis studies on the catalytic and resistance mechanisms of DHPS. By performing the enzyme-catalyzed reaction in crystalline DHPS, we have structurally characterized key intermediates along the reaction pathway. Results support an S{sub N}1 reaction mechanism via formation of a novel cationic pterin intermediate. We also show that two conserved loops generate a substructure during catalysis that creates a specific binding pocket for p-aminobenzoic acid, one of the two DHPS substrates. This substructure, together with the pterin-binding pocket, explains the roles of the conserved active-site residues and reveals how sulfonamide resistance arises.

  10. Identification of sucrose synthase as an actin-binding protein

    Science.gov (United States)

    Winter, H.; Huber, J. L.; Huber, S. C.; Davies, E. (Principal Investigator)

    1998-01-01

    Several lines of evidence indicate that sucrose synthase (SuSy) binds both G- and F-actin: (i) presence of SuSy in the Triton X-100-insoluble fraction of microsomal membranes (i.e. crude cytoskeleton fraction); (ii) co-immunoprecipitation of actin with anti-SuSy monoclonal antibodies; (iii) association of SuSy with in situ phalloidin-stabilized F-actin filaments; and (iv) direct binding to F-actin, polymerized in vitro. Aldolase, well known to interact with F-actin, interfered with binding of SuSy, suggesting that a common or overlapping binding site may be involved. We postulate that some of the soluble SuSy in the cytosol may be associated with the actin cytoskeleton in vivo.

  11. Plant diterpene synthases: exploring modularity and metabolic diversity for bioengineering.

    Science.gov (United States)

    Zerbe, Philipp; Bohlmann, Jörg

    2015-07-01

    Plants produce thousands of diterpenoid natural products; some of which are of significant industrial value as biobased pharmaceuticals (taxol), fragrances (sclareol), food additives (steviosides), and commodity chemicals (diterpene resin acids). In nature, diterpene synthase (diTPS) enzymes are essential for generating diverse diterpene hydrocarbon scaffolds. While some diTPSs also form oxygenated compounds, more commonly, oxygenation is achieved by cytochrome P450-dependent mono-oxygenases. Recent genome-, transcriptome-, and metabolome-guided gene discovery and enzyme characterization identified novel diTPS functions that form the core of complex modular pathway systems. Insights into diterpene metabolism may translate into the development of new bioengineered microbial and plant-based production systems.

  12. New insight into the catalytic properties of rice sucrose synthase.

    Science.gov (United States)

    Huang, Yu-Chiao; Hsiang, Erh-Chieh; Yang, Chien-Chih; Wang, Ai-Yu

    2016-01-01

    Sucrose synthase (SuS), which catalyzes the reversible conversion of sucrose and uridine diphosphate (UDP) into fructose and UDP-glucose, is a key enzyme in sucrose metabolism in higher plants. SuS belongs to family 4 of the glycosyltransferases (GT4) and contains an E-X7-E motif that is conserved in members of GT4 and two other GT families. To gain insight into the roles of this motif in rice sucrose synthase 3 (RSuS3), the two conserved glutamate residues (E678 and E686) in this motif and a phenylalanine residue (F680) that resides between the two glutamate residues were changed by site-directed mutagenesis. All mutant proteins maintained their tetrameric conformation. The mutants E686D and F680Y retained partial enzymatic activity and the mutants E678D, E678Q, F680S, and E686Q were inactive. Substrate binding assays indicated that UDP and fructose, respectively, were the leading substrates in the sucrose degradation and synthesis reactions of RSuS3. Mutations on E678, F680, and E686 affected the binding of fructose, but not of UDP. The results indicated that E678, F680, and E686 in the E-X7-E motif of RSuS3 are essential for the activity of the enzyme and the sequential binding of substrates. The sequential binding of the substrates implied that the reaction catalyzed by RSuS can be controlled by the availability of fructose and UDP, depending on the metabolic status of a tissue.

  13. Metallothionein prevents cardiac pathological changes in diabetes by modulating nitration and inactivation of cardiac ATP synthase.

    Science.gov (United States)

    Cong, Weitao; Zhao, Ting; Zhu, Zhongxin; Huang, Binbin; Ma, Weide; Wang, Yuehui; Tan, Yi; Chakrabarti, Subrata; Li, Xiaokun; Jin, Litai; Cai, Lu

    2014-04-01

    Mitochondrial ATP production is the main energy source for the cell. Diabetes reduces the efficient generation of ATP, possibly due to the inactivation of ATP synthase. However, the exact mechanism by which diabetes induces inactivation of ATP synthase remains unknown, as well as whether such inactivation has a role in the development of pathological abnormalities of the diabetic heart. To address these issues, we used cardiac metallothionein-transgenic (MT-TG) and wild-type (WT) mice with streptozotocin-induced diabetes, since we have demonstrated previously that diabetes-induced cardiac damage and remodeling were found in WT diabetic mice, but not in MT-TG diabetic mice. Immunohistochemical and biochemical assays were used to compare pathological and biochemical changes of the heart between MT-TG and WT diabetic mice, and a proteomic assay to evaluate ATP synthase expression and tyrosine nitration, with its activity. LC/MS analysis revealed that diabetes increased tyrosine nitration of the ATP synthase α subunit at Tyr(271), Tyr(311), and Tyr(476), and the β subunit at Tyr(269) and Tyr(508), and also significantly reduced ATP synthase activity by ~32%. These changes were not observed in MT-TG diabetic mice. Furthermore, parallel experiments with induced expression of cardiac MT by zinc supplementation in diabetic mice produced similar effects. These results suggest that MT can preserve ATP synthase activity in streptozotocin-induced diabetes, probably through the inhibition of ATP synthase nitration. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Evidence that nitric oxide synthase is involved in progesterone-induced acrosomal exocytosis in mouse spermatozoa.

    Science.gov (United States)

    Herrero, M B; Viggiano, J M; Pérez Martínez, S; de Gimeno, M F

    1997-01-01

    In a recent work, we detected nitric oxide synthase (NO synthase) in the acrosome and tail of mouse and human spermatozoa by an immunofluorescence technique. Also, NO-synthase inhibitors added during sperm capacitation in vitro reduced the percentage of oocytes fertilized in vitro, suggesting a role for NO synthase in sperm function. Therefore, in the present study the effect of three NO-synthase inhibitors, NG-nitro-L-arginine methyl ester (L-NAME), NG-nitro-D-arginine methyl ester (D-NAME) and L-NG-nitro-arginine (NO2-arg), and of a nitric oxide donor, spermine-NONOate, on the progesterone-induced acrosome reaction of mouse sperm was examined. NO-synthase inhibitors were added at 0, 60 or 90 min during capacitation; at 120 min, mouse epididymal spermatozoa were exposed to 15 microM progesterone for another 15 min. In another set of experiments, different concentrations of spermine-NONOate were added to capacitated spermatozoa for 15 min; in these experiments, progesterone was not included. NO2-arg and L-NAME blocked progesterone-induced exocytosis regardless of the time at which these inhibitors were added. Moreover, D-NAME did not inhibit exocytosis. In contrast, spermine-NONOate stimulated the acrosomal exocytosis in vitro directly. These results provide evidence that mouse sperm NO synthase participates in the progesterone-induced acrosome reaction in vitro and that nitric oxide induces this event.

  15. Cloning and Characterization of Cheilanthifoline and Stylopine Synthase Genes from Chelidonium majus.

    Science.gov (United States)

    Yahyazadeh, Mahdi; Ratmoyo, Purwanto; Bittner, Florian; Sato, Fumihiko; Selmar, Dirk

    2017-06-19

    The most prominent alkaloid of Chelidonium majus is dihydrocoptisine, revealing the characteristical benzophenanthridine skeleton. Up to now, any information about the enzymes responsible for its biosynthesis in C. majus and the related genes respectively, is lacking. Based on sequence similarities to the corresponding methylenedioxy bridge-forming cytochrome P450-enzymes involved in the isoquinoline alkaloid biosynthesis in Eschscholzia californica, genes for a cheilanthifoline synthase and a stylopine synthase from C. majus were isolated, sequenced and heterologously expressed in yeast.The activity of the heterologously expressed cytochrome P450-enzymes was determined in situ as well as on the basis of microsomal fractions. It was shown that the cheilanthifoline synthase (c8931) converts scoulerine into cheilanthifoline, the latter subsequently being converted to stylopine by the action of a stylopine synthase (c1128). Based on the well-known instability of stylopine, it can be assumed that in vivo - under the acidic conditions in the vacuole - this alkaloid is converted to dihydrocoptisine, which accumulates in C. majus leaves. Both methylenedioxy bridge-forming cytochrome P450-enzymes from C. majus are characterized by their high substrate specificity. Apart from their genuine substrates, i.e. scoulerine and cheilanthifoline, cheilanthifoline synthase and stylopine synthase do not accept other substrates tested; the only alternative substrate identified was scoulerine, which is converted by stylopine synthase to yield minor amounts of nandinine.Quantitative RT-PCR revealed that the expression of cheilanthifoline synthase and stylopine synthase genes is very similar in both, in roots and leaves from C. majus, although the alkaloid accumulation pattern in these organs are quite different. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  16. Production and characterisation of monoclonal antibodies to phytoene synthase of Lycopersicon esculentum.

    Science.gov (United States)

    Fraser, P D; Misawa, N; Sandmann, G; Johnson, J; Schuch, W; Bramley, P M

    1998-10-01

    Monoclonal antibodies have been prepared against the tomato (Lycopersicon esculentum Mill.) fruit ripening-enhanced phytoene synthase (PSY1). The antigen was prepared as a beta-galactosidase fusion protein by cloning a 1.13 kb fragment of Psy1 cDNA into pUR291, followed by transformation of E. coli. The fusion protein, induced by IPTG, was purified by preparative SDS-PAGE and used to elicit an immune response. The cell lines were screened for cross-reactivity against beta-galactosidase-phytoene synthase fusion protein in E. coli extracts using western blotting and ELISA detection procedures. Positive clones were further screened for their ability to cross-react with the mature phytoene synthase protein on western blots as well as their ability to inhibit enzyme activity. Eleven monoclonal lines were obtained. Nine of these, all of the IgM isotype, exhibited strong responses to phytoene synthase of ripe tomato fruit on western blots, but did not inhibit enzyme activity effectively. The other two lines (IgG/la 2 isotypes) inhibited phytoene synthase activity in ripe tomato stroma, but produced a poor response to the protein on western blots. The monoclonals identified a ripe fruit phytoene synthase of 38 kDa, exclusively located in the chromoplast. In contrast, antibodies were unable to detect microbial phytoene synthases, nor phytoene synthase of maize leaf, tomato chloroplast or mango fruit extracts, either on western blots or from inhibition of phytoene synthase activity. However, they did cross-react with a 44 kDa protein from carrot leaf stroma and with three different proteins (44, 41, and 37 kDa) in carrot root. Cross-reactivity was also found with a 37 kDa protein from pumpkin fruit stroma.

  17. The role of 1-deoxy-d-xylulose-5-phosphate synthase and phytoene synthase gene family in citrus carotenoid accumulation.

    Science.gov (United States)

    Peng, Gang; Wang, Chunyan; Song, Song; Fu, Xiumin; Azam, Muhammad; Grierson, Don; Xu, Changjie

    2013-10-01

    Three 1-deoxy-D-xylulose-5-phosphate synthases (DXS) and three phytoene synthases (PSY) were identified in citrus, from Affymetrix GeneChip Citrus Genome Array, GenBank and public orange genome databases. Tissue-specific expression analysis of these genes was carried out on fruit peel and flesh, flower and leaf of Satsuma mandarin (Citrus unshiu Marc.) in order to determine their roles in carotenoid accumulation in different tissues. Expression of CitDXS1 and CitPSY1 was highest in all test tissues, while that of CitDXS2 and CitPSY2 was lower, and that of CitDXS3 and CitPSY3 undetectable. The transcript profiles of CitDXS1 and CitPSY1 paralleled carotenoid accumulation in flesh of Satsuma mandarin and orange (Citrus sinensis Osbeck) during fruit development, and CitPSY1 expression was also associated with carotenoid accumulation in peel, while the CitDXS1 transcript level was only weakly correlated with carotenoid accumulation in peel. Similar results were obtained following correlation analysis between expression of CitDXS1 and CitPSY1 and carotenoid accumulation in peel and flesh of 16 citrus cultivars. These findings identify CitPSY1 and CitDXS1 as the main gene members controlling carotenoid biosynthesis in citrus fruit. Furthermore, chromoplasts were extracted from flesh tissue of these citrus, and chromoplasts of different shape (spindle or globular), different size, and color depth were observed in different cultivars, indicating chromoplast abundance, number per gram tissue, size and color depth were closely correlated with carotenoid content in most cultivars. The relationship between carotenoid biosynthesis and chromoplast development was discussed. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  18. Expression and activity of inducible nitric oxide synthase and endothelial nitric oxide synthase correlate with ethanol-induced liver injury

    Institute of Scientific and Technical Information of China (English)

    Guang-Jin Yuan; Xiao-Rong Zhou; Zuo-Jiong Gong; Pin Zhang; Xiao-Mei Sun; Shi-Hua Zheng

    2006-01-01

    AIM: To study the expression and activity of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in rats with ethanol-induced liver injury and their relation with liver damage, activation of nuclear factor-KB (NF-кB) and tumor necrosis factor-α (TNF-α)expression in the liver.METHODS: Female Sprague-Dawley rats were given fish oil (0.5 mL) along with ethanol or isocaloric dextrose daily via gastrogavage for 4 or 6 wk. Liver injury was assessed using serum alanine aminotransferase (ALT)activity and pathological analysis. Liver malondialdehyde (MDA), nitric oxide contents, iNOS and eNOS activity were determined. NF-KB p65, iNOS, eNOS and TNF-αprotein or mRNA expression in the liver were detected by immunohistochemistry or reverse transcriptase-polymerase chain reaction (RT-PCR).RESULTS: Chronic ethanol gavage for 4 wk caused steatosis, inflammation and necrosis in the liver, and elevated serum ALT activity. Prolonged ethanol administration (6 wk) enhanced the liver damage. These responses were accompanied with increased lipid peroxidation, NO contents, iNOS activity and reduced eNOS activity. NF-кB p65, iNOS and TNF-α protein or mRNA expression were markedly induced after chronic ethanol gavage, whereas eNOS mRNA expression remained unchanged. The enhanced iNOS activity and expression were positively correlated with the liver damage, especially the necro-inflammation, activation of NF-кB, and TNF-α mRNA expression.CONCLUSION: iNOS expression and activity are induced in the liver after chronic ethanol exposure in rats, which are correlated with the liver damage, especially the necro-inflammation, activation of NF-KB and TNF-αexpression. eNOS activity is reduced, but its mRNA expression is not affected.

  19. Surface exposed amino acid differences between mesophilic and thermophilic phosphoribosyl diphosphate synthase

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; McGuire, James N

    2004-01-01

    The amino acid sequence of 5-phospho-alpha-D-ribosyl 1-diphosphate synthase from the thermophile Bacillus caldolyticus is 81% identical to the amino acid sequence of 5-phospho-alpha-D-ribosyl 1-diphosphate synthase from the mesophile Bacillus subtilis. Nevertheless the enzyme from the two organisms...... competitive with respect to ATP. A predicted structure of the B. caldolyticus enzyme based on homology modelling with the structure of B. subtilis 5-phospho-alpha-D-ribosyl 1-diphosphate synthase shows 92% of the amino acid differences to be on solvent exposed surfaces in the hexameric structure....

  20. Microsatellite instability and the association with plasma homocysteine and thymidylate synthase in colorectal cancer

    DEFF Research Database (Denmark)

    Jensen, Lars Henrik; Lindebjerg, Jan; Crüger, Dorthe G.;

    2008-01-01

    The possible associations between microsatellite instability, homocysteine and thymidylate synthase were investigated in tumors and plasma from 130 patients with colorectal cancer. Other analyses included thymidylate synthase and 5,10-methylene-tetrahydrofolate reductase gene polymorphisms......, carcinoembryonic antigen, vitamin B12, and folate. Microsatellite instability of tumors was associated with higher levels of plasma homocysteine (p = 0.008) and higher protein expression of thymidylate synthase (p ... factors. CEA was not associated with neither homocysteine nor microsatellite instability. The data suggests that there is a more pronounced methyl unit deficiency in microsatellite instable tumors....

  1. Crystal structure of TruD, a novel pseudouridine synthase with a new protein fold.

    Science.gov (United States)

    Kaya, Yusuf; Del Campo, Mark; Ofengand, James; Malhotra, Arun

    2004-04-30

    TruD, a recently discovered novel pseudouridine synthase in Escherichia coli, is responsible for modifying uridine13 in tRNA(Glu) to pseudouridine. It has little sequence homology with the other 10 pseudouridine synthases in E. coli which themselves have been grouped into four related protein families. Crystal structure determination of TruD revealed a two domain structure consisting of a catalytic domain that differs in sequence but is structurally very similar to the catalytic domain of other pseudouridine synthases and a second large domain (149 amino acids, 43% of total) with a novel alpha/beta fold that up to now has not been found in any other protein.

  2. ESR-spektroskopische Untersuchungen der F0F1-ATP-Synthase aus Escherichia coli

    OpenAIRE

    Motz, Christian

    1999-01-01

    Die FoF1-ATP-Synthase katalysiert die Synthese von ATP aus ADP und Pi bei der oxidativen bzw. Photophosphorylierung. Der ATP-Synthase-Komplex läßt sich in zwei funktionelle Einheiten unterteilen: Fo ist ein integraler Membranproteinkomplex, der den Protonenkanal bildet. F1 hingegen ist ein wasserlöslicher Proteinkomplex, der die Nukleotidbindungsstellen trägt. Die ATP-Synthase aus Escherichia coli hat die Zusammensetzung alpha3beta3gamma delta epsilon für die F1 und ab2c9-12 für den Fo-Teil. ...

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

    Directory of Open Access Journals (Sweden)

    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.

  4. Solanapyrone synthase, a possible Diels-Alderase and iterative type I polyketide synthase encoded in a biosynthetic gene cluster from Alternaria solani.

    Science.gov (United States)

    Kasahara, Ken; Miyamoto, Takanori; Fujimoto, Takashi; Oguri, Hiroki; Tokiwano, Tetsuo; Oikawa, Hideaki; Ebizuka, Yutaka; Fujii, Isao

    2010-06-14

    The solanapyrone biosynthetic gene cluster was cloned from Alternaria solani. It consists of six genes-sol1-6-coding for a polyketide synthase, an O-methyltransferase, a dehydrogenase, a transcription factor, a flavin-dependent oxidase, and cytochrome P450. The prosolanapyrone synthase (PSS) encoded by sol1 was expressed in Aspergillus oryzae and its product was identified as desmethylprosolanapyrone I (8). Although PSS is closely related to the PKSs/Diels-Alderases LovB and MlcA of lovastatin and compactin biosynthesis, it did not catalyze cycloaddition. Sol5, encoding a flavin-dependent oxidase (solanapyrone synthase, SPS), was expressed in Pichia pastoris and purified. The purified recombinant SPS showed activity for the formation of (-)-solanapyrone A (1) from achiral prosolanapyrone II (2), establishing that this single enzyme catalyzes both the oxidation and the subsequent cycloaddition reaction, possibly as a Diels-Alder enzyme.

  5. Eukaryotic beta-alanine synthases are functionally related but have a high degree of structural diversity

    DEFF Research Database (Denmark)

    Gojkovic, Zoran; Sandrini, Michael; Piskur, Jure

    2001-01-01

    beta -Alanine synthase (EC 3.5.1.6), which catalyzes the final step of pyrimidine catabolism, has only been characterized in mammals. A Saccharomyces kluyveri pyd3 mutant that is unable to grow on N-carbamy-beta -alanine as the sole nitrogen source and exhibits diminished beta -alanine synthase...... no pyrimidine catabolic pathway, it enabled growth on N-carbamyl- beta -alanine as the sole nitrogen source. The D. discoideum and D. melanogaster PYD3 gene products are similar to mammalian beta -alanine synthases. In contrast, the S. kluyveri protein is quite different from these and more similar to bacterial...... N- carbamyl amidohydrolases. All three beta -alanine synthases are to some degree related to various aspartate transcarbamylases, which catalyze the second step of the de novo pyrimidine biosynthetic pathway. PYD3 expression in yeast seems to be inducible by dihydrouracil and N-carbamyl-beta...

  6. Lack of inhibition of endothelial nitric oxide synthase in the isolated rat aorta by doxorubicin.

    NARCIS (Netherlands)

    Hartog, den GJ; Boots, AW; Haenen, GR; Vijgh, van der W.J.F.

    2003-01-01

    Besides inducing cardiotoxicity, doxorubicin also affects the vasculature. Recent observations in cultured endothelial cells indicated that the endothelial form of nitric oxide synthase might be inhibited by doxorubicin thereby seriously interfering with vascular function. We have investigated the

  7. Prostaglandin-endoperoxide H synthase-2 expression and activity increases with term labor in human chorion

    National Research Council Canada - National Science Library

    Mijovic, J E; Zakar, T; Nairn, T K; Olson, D M

    1997-01-01

    We investigated the changes in prostaglandin-endoperoxide H synthase (PGHS) specific activity and the levels and distribution of PGHS-1 and PGHS-2 mRNA in chorion collected at term before the onset of labor (CS...

  8. The Structure of Sucrose Synthase-1 from Arabidopsis thaliana and Its Functional Implications

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Yi; Anderson, Spencer; Zhang, Yanfeng; Garavito, R. Michael (MSU); (NWU)

    2014-10-02

    Sucrose transport is the central system for the allocation of carbon resources in vascular plants. During growth and development, plants control carbon distribution by coordinating sites of sucrose synthesis and cleavage in different plant organs and different cellular locations. Sucrose synthase, which reversibly catalyzes sucrose synthesis and cleavage, provides a direct and reversible means to regulate sucrose flux. Depending on the metabolic environment, sucrose synthase alters its cellular location to participate in cellulose, callose, and starch biosynthesis through its interactions with membranes, organelles, and cytoskeletal actin. The x-ray crystal structure of sucrose synthase isoform 1 from Arabidopsis thaliana (AtSus1) has been determined as a complex with UDP-glucose and as a complex with UDP and fructose, at 2.8- and 2.85-{angstrom} resolutions, respectively. The AtSus1 structure provides insights into sucrose catalysis and cleavage, as well as the regulation of sucrose synthase and its interactions with cellular targets.

  9. Identification of amino acid networks governing catalysis in the closed complex of class I terpene synthases.

    Science.gov (United States)

    Schrepfer, Patrick; Buettner, Alexander; Goerner, Christian; Hertel, Michael; van Rijn, Jeaphianne; Wallrapp, Frank; Eisenreich, Wolfgang; Sieber, Volker; Kourist, Robert; Brück, Thomas

    2016-02-23

    Class I terpene synthases generate the structural core of bioactive terpenoids. Deciphering structure-function relationships in the reactive closed complex and targeted engineering is hampered by highly dynamic carbocation rearrangements during catalysis. Available crystal structures, however, represent the open, catalytically inactive form or harbor nonproductive substrate analogs. Here, we present a catalytically relevant, closed conformation of taxadiene synthase (TXS), the model class I terpene synthase, which simulates the initial catalytic time point. In silico modeling of subsequent catalytic steps allowed unprecedented insights into the dynamic reaction cascades and promiscuity mechanisms of class I terpene synthases. This generally applicable methodology enables the active-site localization of carbocations and demonstrates the presence of an active-site base motif and its dominating role during catalysis. It additionally allowed in silico-designed targeted protein engineering that unlocked the path to alternate monocyclic and bicyclic synthons representing the basis of a myriad of bioactive terpenoids.

  10. SCREENING OF 6-PYRUVOYL-TETRAHYDROPTERIN SYNTHASE ACTIVITY DEFICIENCY AMONG HYPERP HENYLALANINEMIC PATIENTS

    Directory of Open Access Journals (Sweden)

    DURDI QUJEQ

    1999-10-01

    Full Text Available A deficiency of the phenylalanine hydroxylase activity or its cofactor tetrahydrobiopterin may"nlead to hyperphenylalamnemia and as a result, loss of IQ, poor school performance, and"nbehavior problems occurs. Deficiency in 6-pyruvoyl-tetrahydropterin synthase activity is the"nmajor cause of tetrahydrobiopterin deficient phenylketonuria. In this study, blood specimens"nfrom 165 healthy volunteers and 127 children with phenylketonuria were used to determine"nthe 6-pyruvoyl-tetrahydropterin synthase activity. It was found that the activity of 6-"npyruvoyl- tetrahydropterin synthase was decreased in comparison with control [23.46 +/-"n2.94, (mean +/- SD, mmol/ ml/h, n=I27 vs. 127.63 +/- 4.52, n=165, p<0.05]. Results of"nthis study indicate that examination of 6-pyruvoyl-tetrahydropterin synthase activity is helpful"nand may lead to the diagnosis cause of hyperphenylalaninemia.

  11. Dihydroxyacetone synthase is localized in the peroxisomal matrix of methanol-grown Hansenula polymorpha

    NARCIS (Netherlands)

    Douma, Anneke C.; Veenhuis, Marten; de Koning, Willem; Evers, Melchior; Harder, Willem

    1985-01-01

    The subcellular localization of dihydroxyacetone synthase (DHAS) in the methylotrophic yeast Hansenula polymorpha was studied by various biochemical and immunocytochemical methods. After cell fractionation involving differential and sucrose gradient centrifugation of protoplast homogenates prepared

  12. EPSP合成酶的纯化与制备%Purification and Preparation of EPSP Synthase

    Institute of Scientific and Technical Information of China (English)

    向文胜; 王相晶; 覃兆海; 任天瑞; 张雅莉; 张文吉; 苏少泉

    2000-01-01

    The rapid purification(less than 1.5 h) of EPSP synthase from bean seedling by S ephadex G-50 and Mono-Q chromtography was reported. Specific activity of E PSP synthase obtained by the method was 175.2 nmol.min-1.mg-1.Conc entrated enzyme solution after adjusting to 50% glycerol(V/V) and 1mg.mL -1BSA, was stored at -20℃. EPSP synthase activity was stable at least f or 150 days.The activity of EPSP synthase was inhibited approximately 50% by 6.3 μmol.L-1 glyphosate. It showed that the purified EPSP synth ase as herbicide screening model is possible. This purified method has been used to study enzyme mechanism of the glyphosate resistant bean.

  13. Macro Simulations for PCs in the Classroom.

    Science.gov (United States)

    Case, Karl E.; Fair, Ray C.

    1985-01-01

    The macroeconomic model in Ray C. Fair's textbook, "Specification, Estimation, and Analysis of Macroeconometric Models" (Cambridge: Harvard University Press, 1984), has been programed to run on a personal computer. The model consists of 128 equations. The model's potential for use as a teaching tool is discussed. (RM)

  14. Multichannel CGI system based on PCs

    Science.gov (United States)

    Wang, Runjie; Tian, Jingquan; Ni, Zhengguo; Chen, Lei

    2005-02-01

    Computer Generation Image (CGI) system is main part of the flight simulator. Usually, the Image Generation Workstation to be used in CGI system is very expensive. This paper presents a low cost CGI system based on PC and we use it to develop a flight simulator, which has a 3 channels display system with wide field-of-view (horizontal 240°× vertical 60°). First, the paper discusses the architectural principles of CGI system and provides a CGI system based on PC. Second, a type of 3 channels display system is described. The display system is designed in folded flat screen using rear project curtain. Adjacent screen shapes a 100° corner and each channel provides 80°×60° field-of-view. Furthermore, some key technologies, such as image connection, channels synchronization and channels communication, are discussed in detail. At the end of paper, a flight simulator with 3 channels visual scene is described and it is successfully implemented in training pilot. PC-Based CGI system also can be applied to a lot of fields including multimedia commerce, medical media databases visualization, CAD/CAM, scientific computing visualization, and entertainment.

  15. PCs all-in-one for dummies

    CERN Document Server

    Chambers, Mark L

    2013-01-01

    A perfect companion for your PC! Whether you use your PC for work or play, there's a lot to learn and a lot of territory to discover, so take along a good guide. Serving up nine meaty minibooks, this All-in-One guide covers essential PC topics from soup through nuts, including the latest on PC hardware, Windows 8, the Internet, all the tools in Office 2013, digital media, troubleshooting and maintenance, upgrading your PC, home networking, and PC gaming. You'll get to know your PC inside and out and find yourself turning to this terrific resource again and again. This new

  16. Tablet PCs, Academic Results and Educational Inequalities

    Science.gov (United States)

    Ferrer, Ferran; Belvis, Esther; Pamies, Jordi

    2011-01-01

    This article is the result of a study carried out in 2008 and 2009 by a team from the Autonomous University of Barcelona in order to evaluate the implementation of the Digital Whiteboard Program in public schools in the region of Aragon (Spain). The following pages present some of the results obtained during the study. More specifically, this…

  17. PCs and networking for oceanographic research vessels

    Digital Repository Service at National Institute of Oceanography (India)

    Desai, R.G.P.; Desa, E.; Vithayathil, G.

    on IBM PC compatibles. The computers are located in different laboratories and are dedicated to data collection from one or more instruments. They are integratEd. by a local area network for real time sharing and integration of data. The special...

  18. Transcriptional Modulation of Squalene Synthase Genes in Barley Treated with PGPR

    OpenAIRE

    Anam eYousaf; Abdul eQadir; Tehmina eAnjum; Aqeel eAhmad

    2015-01-01

    Phytosterol contents and food quality of plant produce is directly associated with transcription of gene Squalene Synthase (SS). In current study, barley plants were treated with different rhizobacterial strains under semi controlled (27±3°C) greenhouse conditions in order to modulate expression of SS gene. Plant samples were analysed through semi-quantitative PCR to evaluate effect of rhizobacterial application on transcriptional status of squalene synthase. Results revealed that among four ...

  19. Seasonal influence on gene expression of monoterpene synthases in Salvia officinalis (Lamiaceae).

    Science.gov (United States)

    Grausgruber-Gröger, Sabine; Schmiderer, Corinna; Steinborn, Ralf; Novak, Johannes

    2012-03-01

    Garden sage (Salvia officinalis L., Lamiaceae) is one of the most important medicinal and aromatic plants and possesses antioxidant, antimicrobial, spasmolytic, astringent, antihidrotic and specific sensorial properties. The essential oil of the plant, formed mainly in very young leaves, is in part responsible for these activities. It is mainly composed of the monoterpenes 1,8-cineole, α- and β-thujone and camphor synthesized by the 1,8-cineole synthase, the (+)-sabinene synthase and the (+)-bornyl diphosphate synthase, respectively, and is produced and stored in epidermal glands. In this study, the seasonal influence on the formation of the main monoterpenes in young, still expanding leaves of field-grown sage plants was studied in two cultivars at the level of mRNA expression, analyzed by qRT-PCR, and at the level of end-products, analyzed by gas chromatography. All monoterpene synthases and monoterpenes were significantly influenced by cultivar and season. 1,8-Cineole synthase and its end product 1,8-cineole remained constant until August and then decreased slightly. The thujones increased steadily during the vegetative period. The transcript level of their corresponding terpene synthase, however, showed its maximum in the middle of the vegetative period and declined afterwards. Camphor remained constant until August and then declined, exactly correlated with the mRNA level of the corresponding terpene synthase. In summary, terpene synthase mRNA expression and respective end product levels were concordant in the case of 1,8-cineole (r=0.51 and 0.67 for the two cultivars, respectively; p<0.05) and camphor (r=0.75 and 0.82; p<0.05) indicating basically transcriptional control, but discordant for α-/β-thujone (r=-0.05 and 0.42; p=0.87 and 0.13, respectively).

  20. Synthèse bibliographique: la divinyl éther synthase de plantes

    OpenAIRE

    Fauconnier M.L.; Hoyaux P.; Delcarte J.; Marlier M.; du Jardin P.

    2001-01-01

    Divinyl ether synthase in plants: a review. Divinyl ether synthase, an enzyme of the lipoxygenase pathway transforms, in potato tubers, 9-hydroperoxides of fatty acids into colneleic and colnelenic acid, two divinyl ethers of fatty acids. The enzyme has been described in a limited number of quite different plants. The enzyme has also been detected in tomato roots, garlic bulbs, tobacco plants and in marine algae. The enzyme is bound to membranes and is located in the microsomal fraction. The ...