WorldWideScience

Sample records for hyda hydrogenase ldh

  1. Spectroelectrochemical characterization of the active site of the [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii.

    Science.gov (United States)

    Silakov, Alexey; Kamp, Christina; Reijerse, Eduard; Happe, Thomas; Lubitz, Wolfgang

    2009-08-25

    Hydrogenases catalyze the reversible oxidation of molecular hydrogen. The active site of the [FeFe] hydrogenases (H-cluster) contains a catalytically active binuclear subcluster ([2Fe](H)) connected to a "cubane" [4Fe4S](H) subcluster. Here we present an IR spectroelectrochemical study of the [FeFe] hydrogenase HydA1 isolated from the green alga Chlamydomonas reinhardtii. The enzyme shows IR bands similar to those observed for bacterial [FeFe] hydrogenases. They are assigned to the stretching vibrations of the CN(-) and CO ligands on both irons of the [2Fe](H) subcluster. By following changes in frequencies of the IR bands during electrochemical titrations, two one-electron redox processes of the active enzyme could be distinguished. The reduction of the oxidized state (H(ox)) occurred at a midpoint potential of -400 mV vs NHE (H(ox)/H(red) transition) and relates to a change of the formal oxidation state of the binuclear subcluster. A subsequent reduction (H(red)/H(sred) transition) was determined to have a midpoint potential of -460 mV vs NHE. On the basis of the IR spectra, it is suggested that the oxidation state of the binuclear subcluster does not change in this transition. Tentatively, a reduction of the [4Fe4S](H) cluster has been proposed. In contrast to the bacterial [FeFe] hydrogenases, where the bridging CO ligand becomes terminal when going from H(ox) to H(red), in HydA1 the bridging CO is present in both the H(ox) and H(red) state. The removal of the bridging CO moiety has been observed in the H(red) to H(sred) transition. The significance of this result for the hydrogen conversion mechanism of this class of enzymes is discussed.

  2. Profiling the hydA gene and hydA gene transcript levels of Clostridium butyricum during continuous, mixed-culture hydrogen fermentation

    Energy Technology Data Exchange (ETDEWEB)

    Tolvanen, Katariina E.S.; Koskinen, Perttu E.P.; Santala, Ville P.; Karp, Matti T. [Department of Chemistry and Bioengineering, Tampere University of Technology, P.O. Box 541, FI-33101 Tampere (Finland); Raussi, Hanna-Mari; Ylikoski, Alice I.; Hemmilae, Ilkka A. [PerkinElmer Life Sciences, Wallac Oy, P.O. Box 10, FI-20101 Turku (Finland)

    2008-10-15

    The purpose of the research was to investigate the hydrogenase (hydA) gene and the corresponding transcript levels of Clostridium butyricum during continuous hydrogen fermentation. A quantitative real-time PCR (qrt-PCR) method was developed to specifically target the C. butyricum hydrogenase gene and mRNA in samples collected from a continuous, mixed-culture bioreactor over a period of 30 days (operation days 114-144). The detection limit of the qrt-PCR was 3.9 x 10{sup 2}hydA copies and the linear range 3.9 x 10{sup 2}-3.9 x 10{sup 7}hydA copies. The results showed that after a re-inoculation of the bioreactor on day 120 the hydA copy numbers started to rise and stabilized after day 127. The number of hydA transcript continued to rise until day 142. The results demonstrate that this method is suitable for detecting the hydA gene and gene transcript levels of C. butyricum from bioreactor samples. The expression level of hydA gene changed during continuous operation and can, therefore, be a useful target for process performance monitoring. (author)

  3. Relationship among growth parameters for Clostridium butyricum, hydA gene expression, and biohydrogen production in a sucrose-supplemented batch reactor.

    Science.gov (United States)

    Wang, Mei-Yun; Olson, Betty H; Chang, Jo-Shu

    2008-03-01

    This study was undertaken to identify the relationship between the performance of dark H2 fermentation and expression of the key functional gene (i.e., hydrogenase gene) involved in the bioH2 production process. Clostridium butyricum CGS5 isolated from anaerobic sewage sludge was used as the model strain for this study. Copy number of the hydrogenase gene (hydA) and mRNA transcripts (cDNA hydA) (after amplification) and the total DNA and RNA (before amplification) were measured over the course of the growth of strain CGS5. Cell concentration was also determined by optical density and converted to dry weight. After amplification, the hydA gene increased 1,500-fold during late exponential growth phase after normalization to the copy number at time 0, and cDNA from mRNA transcripts of hydA also increased 500-fold after normalization. mRNA transcripts of hydA lagged behind the increase of total DNA and RNA, and increases in hydA more closely mimicked those of total DNA. Increases in both of these parameters corresponded with hydrogen production. Transcripts of 16s ribosomal RNA reached a maximum value earlier (38 h) than did those of hydA (47 h). All molecular characteristics matched those for sucrose utilization, growth, and hydrogen production. These experiments indicated that transcription as measured by cDNA can be related to hydrogen production and possesses the potential to be used as tool for process control.

  4. Optimized Expression and Purification for High-Activity Preparations of Algal [FeFe]-Hydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Yacoby, I.; Tegler, L. T.; Pochekailov, S.; Zhang, S.; King, P. W.

    2012-04-01

    Recombinant expression and purification of metallo-enzymes, including hydrogenases, at high-yields is challenging due to complex, and enzyme specific, post-translational maturation processes. Low fidelities of maturation result in preparations containing a significant fraction of inactive, apo-protein that are not suitable for biophysical or crystallographic studies. We describe the construction, overexpression and high-yield purification of a fusion protein consisting of the algal [2Fe2S]-ferredoxin PetF (Fd) and [FeFe]-hydrogenase HydA1. The maturation of Fd-HydA1 was optimized through improvements in culture conditions and media components used for expression. We also demonstrated that fusion of Fd to the N-terminus of HydA1, in comparison to the C-terminus, led to increased expression levels that were 4-fold higher. Together, these improvements led to enhanced HydA1 activity and improved yield after purification. The strong binding-affinity of Fd for DEAE allowed for two-step purification by ion exchange and StrepTactin affinity chromatography. In addition, the incorporation of a TEV protease site in the Fd-HydA1 linker allowed for the proteolytic removal of Fd after DEAE step, and purification of HydA1 alone by StrepTactin. In combination, this process resulted in HydA1 purification yields of 5 mg L{sup -1} of culture from E. coli with specific activities of 1000 U (U = 1 {micro}mol hydrogen evolved mg{sup -1} min{sup -1}). The [FeFe]-hydrogenases are highly efficient enzymes and their catalytic sites provide model structures for synthetic efforts to develop robust hydrogen activation catalysts. In order to characterize their structure-function properties in greater detail, and to use hydrogenases for biotechnological applications, reliable methods for rapid, high-yield expression and purification are required.

  5. Optimized over-expression of [FeFe] hydrogenases with high specific activity in Clostridium acetobutylicum

    Energy Technology Data Exchange (ETDEWEB)

    von Abendroth, Gregory; Stripp, Sven; Happe, Thomas [Ruhr-Universitaet Bochum, Lehrstuhl fuer Biochemie der Pflanzen, AG Photobiotechnologie, 44780 Bochum (Germany); Silakov, Alexey [Max-Planck-Institut fuer Bioanorganische Chemie, 45470 Muelheim an der Ruhr (Germany); Croux, Christian; Soucaille, Philippe; Girbal, Laurence [UMR5504, UMR792 Ingenierie des Systemes Biologiques et des Procedes, CNRS, INRA, INSA, 31400 Toulouse (France)

    2008-11-15

    It was previously shown that Clostridium acetobutylicum is capable to over-express various [FeFe] hydrogenases although the protein yield was low. In this study we report on doubling the yield of the clostridial hydrogenase by replacing the native gene hydA1{sub Ca} with a recombinant one via homologous recombination. The purified protein HydA1{sub Ca} shows an unexpected high specific activity (up to 2257 {mu}mol H{sub 2} min{sup -1} mg{sup -1}) for hydrogen evolution. Furthermore, the highly active green algal hydrogenase HydA1{sub Cr} from Chlamydomonas reinhardtii was heterologously expressed in C. acetobutylicum, and purified with increased yield (1 mg protein per liter of cells) and high activity (625 {mu}mol H{sub 2} min{sup -1} mg{sup -1}). EPR studies demonstrate intact H-clusters for homologously and heterologously expressed [FeFe] hydrogenases in the CO-inhibited oxidized redox state, and prove the high efficiency of the C. acetobutylicum expression system. (author)

  6. [FeFe]-Hydrogenase-Catalyzed H2 Production in a Photoelectrochemical Biofuel Cell

    Energy Technology Data Exchange (ETDEWEB)

    Hambourger, M.; Gervaldo, M.; Svedruzic, D.; King, P. W.; Gust, D.; Ghirardi, M.; Moore, A. L.; Moore, T. A.

    2008-01-01

    The Clostridium acetobutylicum [FeFe]-hydrogenase HydA has been investigated as a hydrogen production catalyst in a photoelectrochemical biofuel cell. Hydrogenase was adsorbed to pyrolytic graphite edge and carbon felt electrodes. Cyclic voltammograms of the immobilized hydrogenase films reveal cathodic proton reduction and anodic hydrogen oxidation, with a catalytic bias toward hydrogen evolution. When corrected for the electrochemically active surface area, the cathodic current densities are similar for both carbon electrodes, and 40% of those obtained with a platinum electrode. The high surface area carbon felt/hydrogenase electrode was subsequently used as the cathode in a photoelectrochemical biofuel cell. Under illumination, this device is able to oxidize a biofuel substrate and reduce protons to hydrogen. Similar photocurrents and hydrogen production rates were observed in the photoelectrochemical biofuel cell using either hydrogenase or platinum cathodes.

  7. H2-Producing Bacterial Community during Rice Straw Decomposition in Paddy Field Soil: Estimation by an Analysis of [FeFe]-Hydrogenase Gene Transcripts.

    Science.gov (United States)

    Baba, Ryuko; Asakawa, Susumu; Watanabe, Takeshi

    2016-09-29

    The transcription patterns of [FeFe]-hydrogenase genes (hydA), which encode the enzymes responsible for H2 production, were investigated during rice straw decomposition in paddy soil using molecular biological techniques. Paddy soil amended with and without rice straw was incubated under anoxic conditions. RNA was extracted from the soil, and three clone libraries of hydA were constructed using RNAs obtained from samples in the initial phase of rice straw decomposition (day 1 with rice straw), methanogenic phase of rice straw decomposition (day 14 with rice straw), and under a non-amended condition (day 14 without rice straw). hydA genes related to Proteobacteria, Firmicutes, Bacteroidetes, Chloroflexi, and Thermotogae were mainly transcribed in paddy soil samples; however, their proportions markedly differed among the libraries. Deltaproteobacteria-related hydA genes were predominantly transcribed on day 1 with rice straw, while various types of hydA genes related to several phyla were transcribed on day 14 with rice straw. Although the diversity of transcribed hydA was significantly higher in the library on day 14 with rice straw than the other two libraries, the composition of hydA transcripts in the library was similar to that in the library on day 14 without rice straw. These results indicate that the composition of active H2 producers and/or H2 metabolic patterns dynamically change during rice straw decomposition in paddy soil.

  8. [FeFe]-Hydrogenase Abundance and Diversity along a Vertical Redox Gradient in Great Salt Lake, USA

    Directory of Open Access Journals (Sweden)

    Eric S. Boyd

    2014-11-01

    Full Text Available The use of [FeFe]-hydrogenase enzymes for the biotechnological production of H2 or other reduced products has been limited by their sensitivity to oxygen (O2. Here, we apply a PCR-directed approach to determine the distribution, abundance, and diversity of hydA gene fragments along co-varying salinity and O2 gradients in a vertical water column of Great Salt Lake (GSL, UT. The distribution of hydA was constrained to water column transects that had high salt and relatively low O2 concentrations. Recovered HydA deduced amino acid sequences were enriched in hydrophilic amino acids relative to HydA from less saline environments. In addition, they harbored interesting variations in the amino acid environment of the complex H-cluster metalloenzyme active site and putative gas transfer channels that may be important for both H2 transfer and O2 susceptibility. A phylogenetic framework was created to infer the accessory cluster composition and quaternary structure of recovered HydA protein sequences based on phylogenetic relationships and the gene contexts of known complete HydA sequences. Numerous recovered HydA are predicted to harbor multiple N- and C-terminal accessory iron-sulfur cluster binding domains and are likely to exist as multisubunit complexes. This study indicates an important role for [FeFe]-hydrogenases in the functioning of the GSL ecosystem and provides new target genes and variants for use in identifying O2 tolerant enzymes for biotechnological applications.

  9. Molecular characterization and homologous overexpression of [FeFe]-hydrogenase in Clostridium tyrobutyricum JM1

    Energy Technology Data Exchange (ETDEWEB)

    Jo, Ji Hye [Biosciences Center, National Renewable Energy Laboratory, 1617 Cole Blvd, Golden, CO 80401-3393 (United States); Jeon, Che Ok [Department of Life Science, Chung-Ang University, Seoul 156-756 (Korea); Lee, Seung Yoon [K-water Research Institute, Korea Water Resources Corporation, 462-1, Jeonmin-dong, Yuseong-gu, Daejon 305-703 (Korea); Lee, Dae Sung [Department of Environmental Engineering, Kyungpook National University, 1370 Sankyuk-dong, Buk-gu, Daegu 702-701 (Korea); Park, Jong Moon [Department of Chemical Engineering, Pohang University of Science and Technology, San 31, Hyoja-dong, Nam-gu, Pohang, Gyeongbuk 790-784 (Korea)

    2010-02-15

    The H{sub 2}-evoving [FeFe]-hydrogenase in Clostridium tyrobutyricum JM1 was isolated to elucidate molecular characterization and modular structure of the hydrogenase. Then, homologous overexpression of the hydrogenase gene was for the first time performed to enhance hydrogen production. The hydA open reading frame (ORF) was 1734-bp, encodes 577 amino acids with a predicted molecular mass of 63,970 Da, and presents 80% and 75% identity at the amino acid level with the [FeFe]-hydrogenase genes of Clostridium kluyveri DSM 555 and Clostridium acetobutylicum ATCC 824, respectively. One histidine residue and 19 cysteine residues, known to fasten one [2Fe-2S] cluster, three [4Fe-4S] clusters and one H-cluster, were conserved in hydA of C. tyrobutyricum. A 2327-bp DNA region containing the ORF and the putative promoter region was amplified and subcloned into a pJIR418 shuttle vector. The gene transfer of the recombinant plasmid into C. tyrobutyricum JM1 was performed by a modified electrotransformation method. Homologous overexpression of the [FeFe]-hydrogenase gene resulted in a 1.7-fold and 1.5-fold increase in hydrogenase activity and hydrogen yield concomitant with the shift of metabolic pathway. (author)

  10. Shewanella oneidensis: a new and efficient System for Expression and Maturation of heterologous [Fe-Fe] Hydrogenase from Chlamydomonas reinhardtii

    Directory of Open Access Journals (Sweden)

    Sybirna Kateryna

    2008-09-01

    Full Text Available Abstract Background The eukaryotic green alga, Chlamydomonas reinhardtii, produces H2 under anaerobic conditions, in a reaction catalysed by a [Fe-Fe] hydrogenase HydA1. For further biochemical and biophysical studies a suitable expression system of this enzyme should be found to overcome its weak expression in the host organism. Two heterologous expression systems used up to now have several advantages. However they are not free from some drawbacks. In this work we use bacterium Shewanella oneidensis as a new and efficient system for expression and maturation of HydA1 from Chlamydomonas reinhardtii. Results Based on codon usage bias and hydrogenase maturation ability, the bacterium S. oneidensis, which possesses putative [Fe-Fe] and [Ni-Fe] hydrogenase operons, was selected as the best potential host for C. reinhardtii [Fe-Fe] hydrogenase expression. Hydrogen formation by S. oneidensis strain AS52 (ΔhydAΔhyaB transformed with a plasmid bearing CrHydA1 and grown in the presence of six different substrates for anaerobic respiration was determined. A significant increase in hydrogen evolution was observed for cells grown in the presence of trimethylamine oxide, dimethylsulfoxide and disodium thiosulfate, showing that the system of S. oneidensis is efficient for heterologous expression of algal [Fe-Fe] hydrogenase. Conclusion In the present work a new efficient system for heterologous expression and maturation of C. reinhardtii hydrogenase has been developed. HydA1 of C. reinhardtii was purified and shown to contain 6 Fe atoms/molecule of protein, as expected. Using DMSO, TMAO or thiosulfate as substrates for anaerobic respiration during the cell growth, 0.4 – 0.5 mg l-1(OD600 = 1 of catalytically active HydA1 was obtained with hydrogen evolution rate of ~700 μmol H2 mg-1 min-1.

  11. Expression of a clostridial [FeFe]-hydrogenase in Chlamydomonas reinhardtii prolongs photo-production of hydrogen from water splitting

    Energy Technology Data Exchange (ETDEWEB)

    Noone, Seth; Ratcliff, Kathleen; Davis, ReAnna; Subramanian, Venkataramanan; Meuser, Jonathan; Posewitz, Matthew C.; King, Paul W.; Ghirardi, Maria L.

    2017-03-01

    The high oxygen (O2) sensitivity of green algal [FeFe]-hydrogenases is a significant limitation for the sustained production of hydrogen gas (H2) from photosynthetic water splitting. To address this limitation we replaced the native [FeFe]-hydrogenases with a more O2-tolerant clostridial [FeFe]-hydrogenase CaI in Chlamydomonas reinhardtii strain D66..delta..HYD (hydA1-hydA2-) that contains insertionally inactivated [FeFe]-hydrogenases genes. Expression and translocation of CaI in D66..delta..HYD led to the recovery of H2 photoproduction at ~ 20% of the rates of the wild-type parent strain D66. We show for the first time that a bacterial [FeFe]-hydrogenase can be expressed, localized and matured to a catalytically active form that couples to photosynthetic electron transport in the green alga C. reinhardtii. The lower rates of O2 inactivation of CaI led to more sustained H2 photoproduction when cultures were challenged with O2 or kept under prolonged illumination at solar intensities. These results provide new insights into the requisites for attaining photobiological H2 production from water splitting using a more O2-tolerant hydrogenase.

  12. Heterologous expression of an algal hydrogenase in a heterocystous cyanobacterium

    Energy Technology Data Exchange (ETDEWEB)

    Thorsten Heidorn; Peter Lindblad [Dept. of Physiological Botany, Uppsala University, Villavogen 6, SE-752 36 Uppsala, (Sweden)

    2006-07-01

    For the expression of an active algal [FeFe] hydrogenase in the heterocystous cyanobacterium Nostoc punctiforme A TCC 29133 the Chlamydomonas reinhardtii hydrogenase gene hydA1 and the accessory genes hydEF and hydG are to be introduced into the cyanobacteria cells. The genes were amplified by PCR from EST clones, cloned into the cloning vector pBluescript SK+ and sequenced. An expression vector for multi-cistronic cloning, based on pSCR202, was constructed and for a functional test GFP was inserted as a reporter gene. The GFP construct was transformed into Nostoc punctiforme A TCC 29133 by electroporation and expression of GFP was visualized by fluorescence microscopy. (authors)

  13. H₂-dependent azoreduction by Shewanella oneidensis MR-1: involvement of secreted flavins and both [Ni-Fe] and [Fe-Fe] hydrogenases.

    Science.gov (United States)

    Le Laz, Sébastien; Kpebe, Arlette; Lorquin, Jean; Brugna, Myriam; Rousset, Marc

    2014-03-01

    In this paper, the hydrogen (H2)-dependent discoloration of azo dye amaranth by Shewanella oneidensis MR-1 was investigated. Experiments with hydrogenase-deficient strains demonstrated that periplasmic [Ni-Fe] hydrogenase (HyaB) and periplasmic [Fe-Fe] hydrogenase (HydA) are both respiratory hydrogenases of dissimilatory azoreduction in S. oneidensis MR-1. These findings suggest that HyaB and HydA can function as uptake hydrogenases that couple the oxidation of H2 to the reduction of amaranth to sustain cellular growth. This constitutes to our knowledge the first report of the involvement of [Fe-Fe] hydrogenase in a bacterial azoreduction process. Assays with respiratory inhibitors indicated that a menaquinone pool and different cytochromes were involved in the azoreduction process. High-performance liquid chromatography analysis revealed that flavin mononucleotide and riboflavin were secreted in culture supernatant by S. oneidensis MR-1 under H2-dependent conditions with concentration of 1.4 and 2.4 μmol g protein(-1), respectively. These endogenous flavins were shown to significantly accelerate the reduction of amaranth at micromolar concentrations acting as electron shuttles between the cell surface and the extracellular azo dye. This work may facilitate a better understanding of the mechanisms of azoreduction by S. oneidensis MR-1 and may have practical applications for microbiological treatments of dye-polluted industrial effluents.

  14. Development and application of a functional CE-SSCP fingerprinting method based on [Fe-Fe]-hydrogenase genes for monitoring hydrogen-producing Clostridium in mixed cultures

    Energy Technology Data Exchange (ETDEWEB)

    Quemeneur, Marianne; Hamelin, Jerome; Latrille, Eric; Steyer, Jean-Philippe; Trably, Eric [INRA, UR050, Laboratoire de Biotechnologie de l' Environnement, avenue des Etangs, Narbonne F-11100 (France)

    2010-12-15

    A Capillary Electrophoresis Single Strand Conformation Polymorphism (CE-SSCP) method based on functional [Fe-Fe]-hydrogenase genes was developed for monitoring the hydrogen (H{sub 2})-producing clostridial population in mixed-culture bioprocesses. New non-degenerated primers were designed and then validated on their specific PCR detection of a broad range of clostridial hydA genes. The hydA-based CE-SSCP method gave a specific and discriminating profile for each of the Clostridium strains tested. This method was validated using H{sub 2}-producing mixed cultures incubated at temperatures ranging from 25 C to 45 C. The hydA CE-SSCP profiles clearly differed between temperatures tested. Hence, they varied according to variations of the H{sub 2} production performances. The HydA sequences amplified with the new primer set indicated that diverse Clostridium strains impacted the H{sub 2} production yields. The highest performances were related to the dominance of Clostridium sporogenes-like hydA sequences. This CE-SSCP tool offers highly reliable and throughput analysis of the functional diversity and structure of the hydA genes for better understanding of the H{sub 2}-producing clostridial population dynamics in H{sub 2} dark fermentation bioreactors. (author)

  15. Tyrosine, cysteine, and S-adenosyl methionine stimulate in vitro [FeFe] hydrogenase activation.

    Directory of Open Access Journals (Sweden)

    Jon M Kuchenreuther

    Full Text Available BACKGROUND: [FeFe] hydrogenases are metalloenzymes involved in the anaerobic metabolism of H(2. These proteins are distinguished by an active site cofactor known as the H-cluster. This unique [6Fe-6S] complex contains multiple non-protein moieties and requires several maturation enzymes for its assembly. The pathways and biochemical precursors for H-cluster biosynthesis have yet to be elucidated. PRINCIPAL FINDINGS: We report an in vitro maturation system in which, for the first time, chemical additives enhance [FeFe] hydrogenase activation, thus signifying in situ H-cluster biosynthesis. The maturation system is comprised of purified hydrogenase apoprotein; a dialyzed Escherichia coli cell lysate containing heterologous HydE, HydF, and HydG maturases; and exogenous small molecules. Following anaerobic incubation of the Chlamydomonas reinhardtii HydA1 apohydrogenase with S-adenosyl methionine (SAM, cysteine, tyrosine, iron, sulfide, and the non-purified maturases, hydrogenase activity increased 5-fold relative to incubations without the exogenous substrates. No conditions were identified in which addition of guanosine triphosphate (GTP improved hydrogenase maturation. SIGNIFICANCE: The in vitro system allows for direct investigation of [FeFe] hydrogenase activation. This work also provides a foundation for studying the biosynthetic mechanisms of H-cluster biosynthesis using solely purified enzymes and chemical additives.

  16. Microoxic Niches within the Thylakoid Stroma of Air-Grown Chlamydomonas reinhardtii Protect [FeFe]-Hydrogenase and Support Hydrogen Production under Fully Aerobic Environment1[OPEN

    Science.gov (United States)

    Liran, Oded; Milrad, Yuval; Eilenberg, Haviva; Weiner, Iddo

    2016-01-01

    Photosynthetic hydrogen production in the microalga Chlamydomonas reinhardtii is catalyzed by two [FeFe]-hydrogenase isoforms, HydA1 and HydA2, both irreversibly inactivated upon a few seconds exposure to atmospheric oxygen. Until recently, it was thought that hydrogenase is not active in air-grown microalgal cells. In contrast, we show that the entire pool of cellular [FeFe]-hydrogenase remains active in air-grown cells due to efficient scavenging of oxygen. Using membrane inlet mass spectrometry, 18O2 isotope, and various inhibitors, we were able to dissect the various oxygen uptake mechanisms. We found that both chlororespiration, catalyzed by plastid terminal oxidase, and Mehler reactions, catalyzed by photosystem I and Flavodiiron proteins, significantly contribute to oxygen uptake rate. This rate is considerably enhanced with increasing light, thus forming local anaerobic niches at the proximity of the stromal face of the thylakoid membrane. Furthermore, we found that in transition to high light, the hydrogen production rate is significantly enhanced for a short duration (100 s), thus indicating that [FeFe]-hydrogenase functions as an immediate sink for surplus electrons in aerobic as well as in anaerobic environments. In summary, we show that an anaerobic locality in the chloroplast preserves [FeFe]-hydrogenase activity and supports continuous hydrogen production in air-grown microalgal cells. PMID:27443604

  17. Heterologous expression of an algal hydrogenase in a hetero-cystous cyanobacterium

    Energy Technology Data Exchange (ETDEWEB)

    Thorsten Heidorn; Peter Lindblad [Dept. of Physiological Botany, Uppsala University, V illavagen 6, SE-752 36 Uppsala, (Sweden)

    2006-07-01

    For the expression of an active algal [FeFe] hydrogenase in the hetero-cystous cyanobacterium Nostoc punctiforme A TCC 29133 the Chlamydomonas reinhardtii hydrogenase gene hydA1 and the accessory genes hydEF and hydG are to be introduced into the cyano-bacterial cells. The genes were amplified by PCR from EST clones, cloned into the cloning vector pBluescript SK+ and sequenced. An expression vector for multi-cistronic cloning, based on pSCR202, was constructed and for a functional test GFP was inserted as a reporter gene. The GFP construct was transformed into Nostoc punctiforme A TCC 29133 by electroporation and expression of GFP was visualized by fluorescence microscopy. (authors)

  18. A cell-free microtiter plate screen for improved [FeFe] hydrogenases.

    Directory of Open Access Journals (Sweden)

    James A Stapleton

    Full Text Available BACKGROUND: [FeFe] hydrogenase enzymes catalyze the production and dissociation of H(2, a potential renewable fuel. Attempts to exploit these catalysts in engineered systems have been hindered by the biotechnologically inconvenient properties of the natural enzymes, including their extreme oxygen sensitivity. Directed evolution has been used to improve the characteristics of a range of natural catalysts, but has been largely unsuccessful for [FeFe] hydrogenases because of a lack of convenient screening platforms. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe an in vitro screening technology for oxygen-tolerant and highly active [FeFe] hydrogenases. Despite the complexity of the protocol, we demonstrate a level of reproducibility that allows moderately improved mutants to be isolated. We have used the platform to identify a mutant of the Chlamydomonas reinhardtii [FeFe] hydrogenase HydA1 with a specific activity approximately 4 times that of the wild-type enzyme. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate the feasibility of using the screen presented here for large-scale efforts to identify improved biocatalysts for energy applications. The system is based on our ability to activate these complex enzymes in E. coli cell extracts, which allows unhindered access to the protein maturation and assay environment.

  19. Metagenomic and PCR-Based Diversity Surveys of [FeFe]-Hydrogenases Combined with Isolation of Alkaliphilic Hydrogen-Producing Bacteria from the Serpentinite-Hosted Prony Hydrothermal Field, New Caledonia

    Science.gov (United States)

    Mei, Nan; Postec, Anne; Monnin, Christophe; Pelletier, Bernard; Payri, Claude E.; Ménez, Bénédicte; Frouin, Eléonore; Ollivier, Bernard; Erauso, Gaël; Quéméneur, Marianne

    2016-01-01

    High amounts of hydrogen are emitted in the serpentinite-hosted hydrothermal field of the Prony Bay (PHF, New Caledonia), where high-pH (~11), low-temperature (< 40°C), and low-salinity fluids are discharged in both intertidal and shallow submarine environments. In this study, we investigated the diversity and distribution of potentially hydrogen-producing bacteria in Prony hyperalkaline springs by using metagenomic analyses and different PCR-amplified DNA sequencing methods. The retrieved sequences of hydA genes, encoding the catalytic subunit of [FeFe]-hydrogenases and, used as a molecular marker of hydrogen-producing bacteria, were mainly related to those of Firmicutes and clustered into two distinct groups depending on sampling locations. Intertidal samples were dominated by new hydA sequences related to uncultured Firmicutes retrieved from paddy soils, while submarine samples were dominated by diverse hydA sequences affiliated with anaerobic and/or thermophilic submarine Firmicutes pertaining to the orders Thermoanaerobacterales or Clostridiales. The novelty and diversity of these [FeFe]-hydrogenases may reflect the unique environmental conditions prevailing in the PHF (i.e., high-pH, low-salt, mesothermic fluids). In addition, novel alkaliphilic hydrogen-producing Firmicutes (Clostridiales and Bacillales) were successfully isolated from both intertidal and submarine PHF chimney samples. Both molecular and cultivation-based data demonstrated the ability of Firmicutes originating from serpentinite-hosted environments to produce hydrogen by fermentation, potentially contributing to the molecular hydrogen balance in situ. PMID:27625634

  20. [FeFe]-hydrogenase maturation: insights into the role HydE plays in dithiomethylamine biosynthesis.

    Science.gov (United States)

    Betz, Jeremiah N; Boswell, Nicholas W; Fugate, Corey J; Holliday, Gemma L; Akiva, Eyal; Scott, Anna G; Babbitt, Patricia C; Peters, John W; Shepard, Eric M; Broderick, Joan B

    2015-03-10

    HydE and HydG are radical S-adenosyl-l-methionine enzymes required for the maturation of [FeFe]-hydrogenase (HydA) and produce the nonprotein organic ligands characteristic of its unique catalytic cluster. The catalytic cluster of HydA (the H-cluster) is a typical [4Fe-4S] cubane bridged to a 2Fe-subcluster that contains two carbon monoxides, three cyanides, and a bridging dithiomethylamine as ligands. While recent studies have shed light on the nature of diatomic ligand biosynthesis by HydG, little information exists on the function of HydE. Herein, we present biochemical, spectroscopic, bioinformatic, and molecular modeling data that together map the active site and provide significant insight into the role of HydE in H-cluster biosynthesis. Electron paramagnetic resonance and UV-visible spectroscopic studies demonstrate that reconstituted HydE binds two [4Fe-4S] clusters and copurifies with S-adenosyl-l-methionine. Incorporation of deuterium from D2O into 5'-deoxyadenosine, the cleavage product of S-adenosyl-l-methionine, coupled with molecular docking experiments suggests that the HydE substrate contains a thiol functional group. This information, along with HydE sequence similarity and genome context networks, has allowed us to redefine the presumed mechanism for HydE away from BioB-like sulfur insertion chemistry; these data collectively suggest that the source of the sulfur atoms in the dithiomethylamine bridge of the H-cluster is likely derived from HydE's thiol containing substrate.

  1. Properties of hydrogenase from Megasphaera elsdenii

    NARCIS (Netherlands)

    Dijk, van C.

    1980-01-01

    This thesis is concerned with the purification and properties of hydrogenase from the obligate anaerobic rumen bacterium Megasphaera elsdenii. In chapter 1 the motives underlying this thesis, the physiological role of hydrogenase in some heterotrophs, including

  2. Fundamental Studies of Recombinant Hydrogenases

    Energy Technology Data Exchange (ETDEWEB)

    Adams, Michael W

    2014-01-25

    This research addressed the long term goals of understanding the assembly and organization of hydrogenase enzymes, of reducing them in size and complexity, of determining structure/function relationships, including energy conservation via charge separation across membranes, and in screening for novel H2 catalysts. A key overall goal of the proposed research was to define and characterize minimal hydrogenases that are produced in high yields and are oxygen-resistant. Remarkably, in spite of decades of research carried out on hydrogenases, it is not possible to readily manipulate or design the enzyme using molecular biology approaches since a recombinant form produced in a suitable host is not available. Such resources are essential if we are to understand what constitutes a “minimal” hydrogenase and design such catalysts with certain properties, such as resistance to oxygen, extreme stability and specificity for a given electron donor. The model system for our studies is Pyrococcus furiosus, a hyperthermophile that grows optimally at 100°C, which contains three different nickel-iron [NiFe-] containing hydrogenases. Hydrogenases I and II are cytoplasmic while the other, MBH, is an integral membrane protein that functions to both evolve H2 and pump protons. Three important breakthroughs were made during the funding period with P. furiosus soluble hydrogenase I (SHI). First, we produced an active recombinant form of SHI in E. coli by the co-expression of sixteen genes using anaerobically-induced promoters. Second, we genetically-engineered P. furiosus to overexpress SHI by an order of magnitude compared to the wild type strain. Third, we generated the first ‘minimal’ form of SHI, one that contained two rather than four subunits. This dimeric form was stable and active, and directly interacted with a pyruvate-oxidizing enzyme with any intermediate electron carrier. The research resulted in five peer-reviewed publications.

  3. Lactate dehydrogenase (LDH activity in retinoblastoma

    Directory of Open Access Journals (Sweden)

    Maurya OPS

    1987-01-01

    Full Text Available LDH estimation in aqueous and serum was carried out in 10 cases of retinoblastoma and 10 control cases (normal. LDH estimation was done by calorimetric method devised by King-Wooten (1964 (1. LDH levels were quite high in serum and aqueous in retinoblastoma, as compared to control cases LDH aqueous/serum ratio was significantly higher in retinoblastoma as compared to control

  4. Hydrogenases from the Hyperthermophilic Archaeon Pyrococcus furiosus

    NARCIS (Netherlands)

    van Haaster, D.J.

    2007-01-01

    Hydrogenase is an electron-transfer protein and catalyses the simplest chemical redox reaction, the reversible two-electron oxidation of molecular hydrogen in aerobic and anaerobic microorganisms. A kinetic study of the hydrogen oxidation reaction by Fe-hydrogenase from Desulfovibrio vulgaris

  5. Properties of hydrogenase from Megasphaera elsdenii

    NARCIS (Netherlands)

    Dijk, van C.

    1980-01-01

    This thesis is concerned with the purification and properties of hydrogenase from the obligate anaerobic rumen bacterium Megasphaera elsdenii. In chapter 1 the motives underlying this thesis, the physiological role of hydrogenase in some heterotrophs, including Megasphaera elsdenii, as well as a com

  6. Hydrogenases from the Hyperthermophilic Archaeon Pyrococcus furiosus

    NARCIS (Netherlands)

    van Haaster, D.J.

    2007-01-01

    Hydrogenase is an electron-transfer protein and catalyses the simplest chemical redox reaction, the reversible two-electron oxidation of molecular hydrogen in aerobic and anaerobic microorganisms. A kinetic study of the hydrogen oxidation reaction by Fe-hydrogenase from Desulfovibrio vulgaris (Hilde

  7. Total LDH and LDH isoenzyme distribution in the serum of normal children

    NARCIS (Netherlands)

    Heiden, C.V.D.; Desplanque, J.; Stoop, J.W.; Wadman, S.K.

    Total LDH activity and LDH isoenzyme distribution were determined in sera of in normal children from 4 to 13 years old and compared to a control group of adult sera. It was found that in children the level of total LDH activity and the isoenzyme distribution did not differ significantly from that in

  8. Total LDH and LDH isoenzyme distribution in the serum of normal children

    NARCIS (Netherlands)

    Heiden, C.V.D.; Desplanque, J.; Stoop, J.W.; Wadman, S.K.

    1968-01-01

    Total LDH activity and LDH isoenzyme distribution were determined in sera of in normal children from 4 to 13 years old and compared to a control group of adult sera. It was found that in children the level of total LDH activity and the isoenzyme distribution did not differ significantly from that in

  9. Hydrogenase polypeptide and methods of use

    Energy Technology Data Exchange (ETDEWEB)

    Adams, Michael W.W.; Hopkins, Robert C.; Jenney, JR, Francis E.; Sun, Junsong

    2016-02-02

    Provided herein are polypeptides having hydrogenase activity. The polypeptide may be multimeric, and may have hydrogenase activity of at least 0.05 micromoles H.sub.2 produced min.sup.-1 mg protein.sup.-1. Also provided herein are polynucleotides encoding the polypeptides, genetically modified microbes that include polynucleotides encoding one or more subunits of the multimeric polypeptide, and methods for making and using the polypeptides.

  10. Cobaloxime-based artificial hydrogenases.

    Science.gov (United States)

    Bacchi, Marine; Berggren, Gustav; Niklas, Jens; Veinberg, Elias; Mara, Michael W; Shelby, Megan L; Poluektov, Oleg G; Chen, Lin X; Tiede, David M; Cavazza, Christine; Field, Martin J; Fontecave, Marc; Artero, Vincent

    2014-08-01

    Cobaloximes are popular H2 evolution molecular catalysts but have so far mainly been studied in nonaqueous conditions. We show here that they are also valuable for the design of artificial hydrogenases for application in neutral aqueous solutions and report on the preparation of two well-defined biohybrid species via the binding of two cobaloxime moieties, {Co(dmgH)2} and {Co(dmgBF2)2} (dmgH2 = dimethylglyoxime), to apo Sperm-whale myoglobin (SwMb). All spectroscopic data confirm that the cobaloxime moieties are inserted within the binding pocket of the SwMb protein and are coordinated to a histidine residue in the axial position of the cobalt complex, resulting in thermodynamically stable complexes. Quantum chemical/molecular mechanical docking calculations indicated a coordination preference for His93 over the other histidine residue (His64) present in the vicinity. Interestingly, the redox activity of the cobalt centers is retained in both biohybrids, which provides them with the catalytic activity for H2 evolution in near-neutral aqueous conditions.

  11. Magnetisme i LDH og produktene etter dekomposisjon

    OpenAIRE

    Glesne, Erik

    2015-01-01

    Oppgaven omfatter studier av strukturelle og magnetiske egenskaper til materialer med lagdelt struktur i klassen lagdelte dobbelthydroksider (LDH). LDHer er lagdelte leirematerialer der det sitter vann og ladningsbalanserende anioner mellom lagene. Mange ulike kombinasjoner kationer med ulik ladning kan danne lagene, og LDHer har derfor en rekke interessante potensielle bruksområder. Dette omfatter blant annet multifunksjonelle nanokompositter med både polymerer og andre lagdelte strukturer i...

  12. Significance of CSF-LDH in various types of meningitis

    OpenAIRE

    Vekaria, Parul N; Jasani, Jasmin H; Dhruva, Gauravi; Kotadia, Tarun

    2015-01-01

    The cerebrospinal fluid concentration of Lactate dehydrogenase (LDH) was studied in patients with pyogenic and tubercular meningitis. Significant increase in LDH level (P<0.001) were observed in the test group when compared to the control group. LDH may  useful in differentiating viral from other meningitis. It may act as corroborative evidence of meningitis.

  13. The surprising diversity of clostridial hydrogenases: a comparative genomic perspective

    OpenAIRE

    Calusinska, Magdalena; Happe, Thomas; Joris, Bernard; Wilmotte, Annick

    2010-01-01

    Among the large variety of micro-organisms capable of fermentative hydrogen production, strict anaerobes such as members of the genus Clostridium are the most widely studied. They can produce hydrogen by a reversible reduction of protons accumulated during fermentation to dihydrogen, a reaction which is catalysed by hydrogenases. Sequenced genomes provide completely new insights into the diversity of clostridial hydrogenases. Building on previous reports, we found that [FeFe] hydrogenases are...

  14. Polypropylene/Layered Double Hydroxide (LDH) Nanocomposites: Influence of LDH Particle Size on the Crystallization Behavior of Polypropylene.

    Science.gov (United States)

    Nagendra, Baku; Mohan, Kiran; Gowd, E Bhoje

    2015-06-17

    Highly dispersed isotactic polypropylene (iPP) nanocomposites were prepared by incorporating two different sized Mg-Al LDH nanoparticles with different loadings from 1 to 10 wt % using a modified solvent mixing method. Larger sized LDH nanoparticles (∼3-4 μm) were prepared from the gel form of Mg-Al LDH, and the smaller sized nanoparticles (∼50-200 nm) were prepared by sonication of as-synthesized LDH particles. Such obtained LDH nanoparticles were carefully characterized using wide-angle X-ray diffraction (WAXD), transmission electron microscopy, and scanning electron microscopy. WAXD and atomic force microscopy results indicate that the LDH nanoparticles were highly dispersed in the iPP matrix. The influence of LDH nanoparticles size and concentration on the thermal stability, spherulitic morphology, melting behavior, isothermal crystallization kinetics, and lamellar structure of iPP were investigated. Incorporation of low loadings of sonicated LDH particles (e.g., 1-2.5 wt %) show substantial effect on thermal stability, spherulite size, crystallinity, and crystallization half-time and lamellar morphology of iPP compared to the pure iPP and that of nanocomposites with larger LDH particles with same loadings. The better nucleation ability of iPP in the presence of sonicated LDH can be attributed to the high surface area of LDH nanoparticles along with its better dispersibility within the polymer matrix. The incorporation of LDH nanoparticles does not change the crystallization growth mechanism and crystal structure of iPP.

  15. The surprising diversity of clostridial hydrogenases: a comparative genomic perspective.

    Science.gov (United States)

    Calusinska, Magdalena; Happe, Thomas; Joris, Bernard; Wilmotte, Annick

    2010-06-01

    Among the large variety of micro-organisms capable of fermentative hydrogen production, strict anaerobes such as members of the genus Clostridium are the most widely studied. They can produce hydrogen by a reversible reduction of protons accumulated during fermentation to dihydrogen, a reaction which is catalysed by hydrogenases. Sequenced genomes provide completely new insights into the diversity of clostridial hydrogenases. Building on previous reports, we found that [FeFe] hydrogenases are not a homogeneous group of enzymes, but exist in multiple forms with different modular structures and are especially abundant in members of the genus Clostridium. This unusual diversity seems to support the central role of hydrogenases in cell metabolism. In particular, the presence of multiple putative operons encoding multisubunit [FeFe] hydrogenases highlights the fact that hydrogen metabolism is very complex in this genus. In contrast with [FeFe] hydrogenases, their [NiFe] hydrogenase counterparts, widely represented in other bacteria and archaea, are found in only a few clostridial species. Surprisingly, a heteromultimeric Ech hydrogenase, known to be an energy-converting [NiFe] hydrogenase and previously described only in methanogenic archaea and some sulfur-reducing bacteria, was found to be encoded by the genomes of four cellulolytic strains: Clostridum cellulolyticum, Clostridum papyrosolvens, Clostridum thermocellum and Clostridum phytofermentans.

  16. Lactate dehydrogenase (LDH isoenzymes patterns in ocular tumours

    Directory of Open Access Journals (Sweden)

    Singh Rajendra

    1991-01-01

    Full Text Available Estimation of lactate dehydrogenase (LDH isoenzymes in the serum and aqueous humor was carried out in 15 cases of benign ocular tumour, 15 cases of malignant tumor and 15 normal cases. Cases of both sexes aged between 1 year and 75 years were included. LDH, isoenzymes specially LDH4 and LDH5 are higher and LDH1 and LDH2 lower in sera of patients with malignant tumor specially retinoblastoma as compared to benign tumor cases and control cases. LDH isoenzymes in aqueous humor are significantly higher and show a characteristic pattern in retinoblastoma cases, the concentration was presumably too low in the control, malignant tumor other than retinoblastoma and benign tumor cases as its fractionation was not possible.

  17. Purification and characterization of the [NiFe]-hydrogenase of Shewanella oneidensis MR-1.

    Science.gov (United States)

    Shi, Liang; Belchik, Sara M; Plymale, Andrew E; Heald, Steve; Dohnalkova, Alice C; Sybirna, Kateryna; Bottin, Hervé; Squier, Thomas C; Zachara, John M; Fredrickson, James K

    2011-08-15

    Shewanella oneidensis MR-1 possesses a periplasmic [NiFe]-hydrogenase (MR-1 [NiFe]-H(2)ase) that has been implicated in H(2) production and oxidation as well as technetium [Tc(VII)] reduction. To characterize the roles of MR-1 [NiFe]-H(2)ase in these proposed reactions, the genes encoding both subunits of MR-1 [NiFe]-H(2)ase were cloned and then expressed in an MR-1 mutant without hyaB and hydA genes. Expression of recombinant MR-1 [NiFe]-H(2)ase in trans restored the mutant's ability to produce H(2) at 37% of that for the wild type. Following purification, MR-1 [NiFe]-H(2)ase coupled H(2) oxidation to reduction of Tc(VII)O(4)(-) and methyl viologen. Change of the buffers used affected MR-1 [NiFe]-H(2)ase-mediated reduction of Tc(VII)O(4)(-) but not methyl viologen. Under the conditions tested, all Tc(VII)O(4)(-) used was reduced in Tris buffer, while in HEPES buffer, only 20% of Tc(VII)O(4)(-) was reduced. The reduced products were soluble in Tris buffer but insoluble in HEPES buffer. Transmission electron microscopy analysis revealed that Tc precipitates reduced in HEPES buffer were aggregates of crystallites with diameters of ∼5 nm. Measurements with X-ray absorption near-edge spectroscopy revealed that the reduction products were a mixture of Tc(IV) and Tc(V) in Tris buffer but only Tc(IV) in HEPES buffer. Measurements with extended X-ray adsorption fine structure showed that while the Tc bonding environment in Tris buffer could not be determined, the Tc(IV) product in HEPES buffer was very similar to Tc(IV)O(2)·nH(2)O, which was also the product of Tc(VII)O(4)(-) reduction by MR-1 cells. These results shows for the first time that MR-1 [NiFe]-H(2)ase catalyzes Tc(VII)O(4)(-) reduction directly by coupling to H(2) oxidation.

  18. Insights into the Synthesis of Layered Double Hydroxide (LDH) Nanoparticles: Part 2. Formation Mechanisms of LDH

    OpenAIRE

    Sun, Xiaodi; Dey, Sandwip K.

    2015-01-01

    This study demonstrates the effect of (co)intercalated anion compositions on nanostructure evolution to understand the formation mechanisms of layered double hydroxide (LDH) nanoparticles following coprecipitation and hydrothermal treatments (HT). Initially, the room temperature coprecipitation resulted in amorphous primary nanoparticles that agglomerated at the edges due to low surface charge densities. The reversibility of such agglomeration was determined by the crystalline quality upon HT...

  19. Replacing Electron Transport Cofactors with Hydrogenases

    KAUST Repository

    Laamarti, Rkia

    2016-12-01

    Enzymes have found applications in a broad range of industrial production processes. While high catalytic activity, selectivity and mild reaction conditions are attractive advantages of the biocatalysts, particularly costs arising from required cofactors pose a sever limitation. While cofactor-recycling systems are available, their use implies constraints for process set-up and conditions, which are a particular problem e.g. for solid-gas-phase reactions. Several oxidoreductases are able to directly exchange electrons with electrodes. Hence, the co-immobilization of both, an electron-utilizing and an electron-generating oxidoreductase on conductive nanoparticles should facilitate the direct electron flow from an enzymatic oxidation to a reduction reaction circumventing redox-cofactors requirements. In such a set-up, hydrogenases could generate and provide electrons directly form gaseous hydrogen. This thesis describes the co-immobilization of the oxygen tolerant hydrogenases from C. eutropha or C. metallidurans and cytochrome P450BM3 as test system. Conductive material in the form of carbon nanotubes (CNT) serves as a suitable support. A combination of the hydrogenase and the catalytic domain of P450BM3 immobilized on carbon nanotubes were tested for the oxidation of lauric acid in the presence of hydrogen instead of an electron-transport cofactor. The GC-MS analysis reveals the conversion of 4% of lauric acid (LA) into three products, which correspond to the hydroxylated lauric acid in three different positions with a total turnover (TON) of 34. The product distribution is similar to that obtained when using the wildtype P450BM3 with the nicotinamide adenine dinucleotide phosphate (NADPH) cofactor. Such electronic coupling couldn’t be achieved for the conversion of other substrates such as propane and cyclohexane, probably due to the high uncoupling rate within the heme-domain of cytochrome P450BM3 when unnatural substrates are introduced.

  20. Tumor LDH-A expression and serum LDH status are two metabolic predictors for triple negative breast cancer brain metastasis.

    Science.gov (United States)

    Dong, Tieying; Liu, Zhaoliang; Xuan, Qijia; Wang, Zhuozhong; Ma, Wenjie; Zhang, Qingyuan

    2017-07-20

    There are limited therapeutic methods for triple negative breast cancer in the clinic, which is easy to progress into the brain to form metastatic lesions and evolve into the terminal stage. Because both the primary cancer and the brain metastasis have high glycolysis, we hypothesize that lactate dehydrogenase (LDH), which catalyzes the final step of glycolysis, may be a predictor, as well as a treatment target, for breast cancer brain metastasis. Therefore, the expression of LDH-A was detected on 119 triple negative breast cancer tissues with immunohistochemistry, and the serum LDH levels were also measured. Our results showed that the LDH-A expression inside the tumor was significantly higher than the matched normal tissues. Tumor LDH-A expression, serum LDH status, and the slope of serum LDH status were closely associated with triple negative breast cancer brain metastasis and brain metastasis free survival. This study indicates that tumor LDH and serum LDH status are two predictors for triple negative breast cancer brain metastasis.

  1. Structure and function of [NiFe] hydrogenases.

    Science.gov (United States)

    Ogata, Hideaki; Lubitz, Wolfgang; Higuchi, Yoshiki

    2016-11-01

    Hydrogenases catalyze the reversible conversion of molecular hydrogen to protons and electrons via a heterolytic splitting mechanism. The active sites of [NiFe] hydrogenases comprise a dinuclear Ni-Fe center carrying CO and CN(-) ligands. The catalytic activity of the standard (O2-sensitive) [NiFe] hydrogenases vanishes under aerobic conditions. The O2-tolerant [NiFe] hydrogenases can sustain H2 oxidation activity under atmospheric conditions. These hydrogenases have very similar active site structures that change the ligand sphere during the activation/catalytic process. An important structural difference between these hydrogenases has been found for the proximal iron-sulphur cluster located in the vicinity of the active site. This unprecedented [4Fe-3S]-6Cys cluster can supply two electrons, which lead to rapid recovery of the O2 inactivation, to the [NiFe] active site. © The Authors 2016. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  2. Opportunities for improving pLDH-based malaria diagnostic tests

    Directory of Open Access Journals (Sweden)

    Choi Young

    2011-08-01

    Full Text Available Abstract Background Monoclonal antibodies to Plasmodium lactate dehydrogenase (pLDH have been previously used to format immunochromatographic tests for the diagnosis of malaria. Using pLDH as an antigen has several advantages as a sensitive measure of the presence of parasites within patient blood samples. However, variable results in terms of specificity and sensitivity among different commercially available diagnostic kits have been reported and it has not been clear from these studies whether the performance of an individual test is due simply to how it is engineered or whether it is due to the biochemical nature of the pLDH-antibody reaction itself. Methods A series of systematic studies to determine how various pLDH monoclonal antibodies work in combination was undertaken. Different combinations of anti-pLDH monoclonal antibodies were used in a rapid-test immunochromatographic assay format to determine parameters of sensitivity and specificity with regard to individual Plasmodium species. Results Dramatic differences were found in both species specificity and overall sensitivity depending on which antibody is used on the immunochromatographic strip and which is used on the colorimetric colloidal-gold used for visual detection. Discussion The results demonstrate the feasibility of different test formats for the detection and speciation of malarial infections. In addition, the data will enable the development of a universal rapid test algorithm that may potentially provide a cost-effective strategy to diagnose and manage patients in a wide range of clinical settings. Conclusion These data emphasize that using different anti-pLDH antibody combinations offers a tractable way to optimize immunochromatographic pLDH tests.

  3. Oxygen-tolerant hydrogenases in hydrogen-based technologies.

    Science.gov (United States)

    Friedrich, Bärbel; Fritsch, Johannes; Lenz, Oliver

    2011-06-01

    To develop a viable H2 technology, production of H2 has to be significantly enlarged by using renewable resources. One option of generating H2 is the photosynthetic conversion of sunlight and water directly to H2 and O2. Photosystems and hydrogenases are currently being exploited for the design of efficient H2-producing systems that require highly active and O2-tolerant biocatalysts. This communication focuses on two challenging features: hydrogenases that produce H2 in the presence of O2, and direct electron transfer between photosystem I (PS I) and hydrogenase. The latter is accomplished by connecting both modules through a protein fusion or a synthetic molecular wire. These are first steps toward a photosynthetic microbial cell or a semi-synthetic system that may be employed in future H2-based technologies.

  4. Maturation of [FeFe]-hydrogenases: Structures and mechanisms

    Energy Technology Data Exchange (ETDEWEB)

    Nicolet, Yvain; Fontecilla-Camps, Juan C. [Laboratoire de Cristallographie et Cristallogenese des Proteines, Institut de Biologie Structurale J.P. Ebel, Universite Joseph Fourier, Grenoble, CNRS, UMR 5075, CEA, DSV/IBS, 41 rue J. Horowitz F-38027 Grenoble cedex 1 (France); Fontecave, Marc [Laboratoire de Chimie et Biologie des Metaux; Universite Joseph Fourier, Grenoble, CNRS, UMR 5249, CEA, DSV/iRTSV, 17 rue des Martyrs F-38054 Grenoble cedex 9 (France); College de France, 11 place Marcelin-Berthelot 75231 Paris cedex 05 (France)

    2010-10-15

    Maturation of [FeFe]-hydrogenases, consisting in the synthesis and assembly of a di-iron center with a dithiolate bridging ligand as well as CO and CN ligands, depends on the concerted action of three metalloproteins, HydE, HydF and HydG. HydE and HydG are ''Radical-SAM'' enzymes involved in the synthesis of the ligands. HydF is proposed to function as a scaffold protein in which the di-iron center is assembled before being transferred to the hydrogenase. Here we review the current knowledge regarding the structure of the three maturases and the mechanisms of synthesis and assembly of the di-iron center of [FeFe]-hydrogenases. (author)

  5. Recombinant antibodies for specific detection of clostridial [Fe-Fe] hydrogenases

    Science.gov (United States)

    Mangayil, Rahul; Karp, Matti; Lamminmäki, Urpo; Santala, Ville

    2016-01-01

    Biological hydrogen production is based on activity of specific enzymes called hydrogenases. Hydrogenases are oxygen sensitive metalloenzymes containing Ni and/or Fe atoms at the active site, catalyzing reversible reduction of protons. Generally, [Fe-Fe] hydrogenases prefer proton reduction to molecular hydrogen, a potential energy carrier molecule that can be produced by bioprocesses in sustainable manner. Thus, monitoring tools have been developed to study the relationship between [Fe-Fe] hydrogenases and biohydrogen production in bioreactors at DNA and RNA levels. In the present study, novel molecular tools are introduced for quantitative monitoring of clostridial [Fe-Fe] hydrogenases at the protein level. Aerobic and anaerobic biopanning (for inactive and active [Fe-Fe] hydrogenase, respectively) of phage displayed single-chain variable fragment (scFv) antibody libraries aided in isolating nine potential scFvs. The enriched antibodies demonstrated high specificity towards Clostridium spp. [Fe-Fe] hydrogenases allowing detection from pure and mixed cultures. Additionally, the antibodies showed different binding characteristics towards hydrogenase catalytic states, providing a possible means for functional detection of clostridial [Fe-Fe] hydrogenases. From hydrogenase-antibody interaction studies we observed that though antibody binding reduced the enzyme catalytic activity, it facilitated to retain hydrogen evolution from oxygen exposed hydrogenases. PMID:27786270

  6. Recombinant antibodies for specific detection of clostridial [Fe-Fe] hydrogenases.

    Science.gov (United States)

    Mangayil, Rahul; Karp, Matti; Lamminmäki, Urpo; Santala, Ville

    2016-10-27

    Biological hydrogen production is based on activity of specific enzymes called hydrogenases. Hydrogenases are oxygen sensitive metalloenzymes containing Ni and/or Fe atoms at the active site, catalyzing reversible reduction of protons. Generally, [Fe-Fe] hydrogenases prefer proton reduction to molecular hydrogen, a potential energy carrier molecule that can be produced by bioprocesses in sustainable manner. Thus, monitoring tools have been developed to study the relationship between [Fe-Fe] hydrogenases and biohydrogen production in bioreactors at DNA and RNA levels. In the present study, novel molecular tools are introduced for quantitative monitoring of clostridial [Fe-Fe] hydrogenases at the protein level. Aerobic and anaerobic biopanning (for inactive and active [Fe-Fe] hydrogenase, respectively) of phage displayed single-chain variable fragment (scFv) antibody libraries aided in isolating nine potential scFvs. The enriched antibodies demonstrated high specificity towards Clostridium spp. [Fe-Fe] hydrogenases allowing detection from pure and mixed cultures. Additionally, the antibodies showed different binding characteristics towards hydrogenase catalytic states, providing a possible means for functional detection of clostridial [Fe-Fe] hydrogenases. From hydrogenase-antibody interaction studies we observed that though antibody binding reduced the enzyme catalytic activity, it facilitated to retain hydrogen evolution from oxygen exposed hydrogenases.

  7. Insights into the synthesis of layered double hydroxide (LDH) nanoparticles: Part 2. Formation mechanisms of LDH.

    Science.gov (United States)

    Sun, Xiaodi; Dey, Sandwip K

    2015-11-15

    This study demonstrates the effect of (co)intercalated anion compositions on nanostructure evolution to understand the formation mechanisms of layered double hydroxide (LDH) nanoparticles following coprecipitation and hydrothermal treatments (HT). Initially, the room temperature coprecipitation resulted in amorphous primary nanoparticles that agglomerated at the edges due to low surface charge densities. The reversibility of such agglomeration was determined by the crystalline quality upon HT and consequent surface charge density, which in turn were strongly influenced by the composition of the intercalated anions. Upon crystallization, the agglomerated Zn2Al(OH)6(NO3)0.3(CO3)0.35⋅xH2O primary nanoparticles re-dispersed, but the Zn2Al(OH)6(NO3)⋅xH2O nanoparticles with much lower stability and higher disorder (especially at the edges) exhibited irreversible agglomeration, and transformed into secondary nanoparticles via aggregational growth. Additionally, the stability studies on Zn2Al(OH)6(NO3)y(CO3)0.5(1-y)⋅xH2O nanoparticles (y=0-1) showed that the size difference between the cointercalated anions caused phase separation when 0.9⩾y⩾0.6, leading to bimodal size distributions. Moreover, the coarsening rates were controlled through the cointercalated anion compositions. By gradually varying the ratio of cointercalated NO3(-) to CO3(2-), monodispersed Zn2Al(OH)6(NO3)y(CO3)0.5(1-y)⋅xH2O (0.5⩾y⩾0) nanoparticles with systematic variation in the particle size of ∼200-400nm were obtained after HT at 85°C for 12h.

  8. The behaviour of LDH-3 in patients with malignant diseases during therapy with cytostatic drugs and prednisone, studied by LDH-isoenzyme electrophoresis on cellulose acetate

    NARCIS (Netherlands)

    Gennip, A.H. van; Tabak-van Gorcum, J.A.; Taminiau, J.A.J.M.; Wadman, S.K.

    1975-01-01

    A modified method used for the quantitative estimation of LDH-isoenzymes in serum after electrophoresis on cellulose acetate is described. Total LDH-activity and isoenzyme distribution in serum samples of capillary blood are compared to those in samples collected by venipuncture. Total LDH-values

  9. Structural and functional models for [NiFe] hydrogenase

    NARCIS (Netherlands)

    Angamuthu, Raja

    2009-01-01

    The main goal of the research presented in this thesis is the synthesis of suitable structural and functional models for the enzyme [NiFe] hydrogenase, which can reduce protons into dihydrogen. A brief survey of the roles of all the known nickel containing enzymes in biological systems with a focus

  10. Process and genes for expression and overexpression of active [FeFe] hydrogenases

    Science.gov (United States)

    Seibert, Michael; King, Paul W; Ghirardi, Maria Lucia; Posewitz, Matthew C; Smolinski, Sharon L

    2014-09-16

    A process for expression of active [FeFe]-hydrogenase in a host organism that does not contain either the structural gene(s) for [FeFe]-hydrogenases and/or homologues for the maturation genes HydE, HydF and HyG, comprising: cloning the structural hydrogenase gene(s) and/or the maturation genes HydE, HydF and HydG from an organisms that contains these genes into expression plasmids; transferring the plasmids into an organism that lacks a native [FeFe]-hydrogenase or that has a disrupted [FeFe]-hydrogenase and culturing it aerobically; and inducing anaerobiosis to provide [FeFe] hydrogenase biosynthesis and H?2#191 production.

  11. Process and genes for expression and overexpression of active [FeFe] hydrogenases

    Energy Technology Data Exchange (ETDEWEB)

    Seibert, Michael; King, Paul W; Ghirardi, Maria Lucia; Posewitz, Matthew C; Smolinski, Sharon L

    2014-09-16

    A process for expression of active [FeFe]-hydrogenase in a host organism that does not contain either the structural gene(s) for [FeFe]-hydrogenases and/or homologues for the maturation genes HydE, HydF and HyG, comprising: cloning the structural hydrogenase gene(s) and/or the maturation genes HydE, HydF and HydG from an organisms that contains these genes into expression plasmids; transferring the plasmids into an organism that lacks a native [FeFe]-hydrogenase or that has a disrupted [FeFe]-hydrogenase and culturing it aerobically; and inducing anaerobiosis to provide [FeFe] hydrogenase biosynthesis and H?2#191 production.

  12. Serum LDH in chronic cough: a potential marker of airway inflammation.

    Science.gov (United States)

    Faruqi, Shoaib; Wilmot, Rachel; Wright, Caroline; Morice, Alyn Hugh

    2012-04-01

    Lactate dehydrogenase (LDH) is found in almost all tissues of the body and five different isoenzymes are known (LDH-1 to LDH-5). LDH can be elevated in many pathological conditions. We have observed serum LDH to be increased in patients with chronic cough. We wanted to confirm this finding, study the reproducibility and determine the origin of the LDH. Patients prospectively seen at the Hull Cough Clinic had total and specific LDH isoenzyme levels in serum determined. A subgroup of patients also had a serum creatine phosphokinase (CK) measured. Patients completed cough symptom scores and the Hull Airway Reflux Questionnaire (HARQ). Spirometry was performed. Eighty-three patients were included. Forty-two percent had LDH values above the reference range and 78% had LDH values in the fourth quartile of the reference range or above. This increase in LDH was predominantly because of a rise in isoenzymes 4 and 5. The increase in LDH was found to be reproducible at 8 weeks. Ten percent had CK values above the normal range. There was no correlation observed between LDH values and the cough scores, HARQ scores or lung function. Serum LDH levels are elevated in a substantial proportion of patients with chronic cough. This rise is likely to be due to airway inflammation known to be associated with chronic cough. © 2011 Blackwell Publishing Ltd.

  13. Hydrogenase activity in aged, nonviable Desulfovibrio vulgaris cultures and its significance in anaerobic biocorrosion.

    Science.gov (United States)

    Chatelus, C; Carrier, P; Saignes, P; Libert, M F; Berlier, Y; Lespinat, P A; Fauque, G; Legall, J

    1987-07-01

    Batch cultures of Desulfovibrio vulgaris stored at 32 degrees C for 10 months have been found to retain 50% of the hydrogenase activity of a 1-day culture. The hydrogenase found in old cultures needs reducing conditions for its activation. Viable cell counts are negative after 6 months, showing that the hydrogenase activity does not depend on the presence of viable cells. These observations are of importance in the understanding of anaerobic biocorrosion of metals caused by depolarization phenomena.

  14. Oxygen-resistant hydrogenases and methods for designing and making same

    Science.gov (United States)

    King, Paul; Ghirardi, Maria Lucia; Seibert, Michael

    2014-03-04

    The invention provides oxygen-resistant iron-hydrogenases ([Fe]-hydrogenases) for use in the production of H.sub.2. Methods used in the design and engineering of the oxygen-resistant [Fe]-hydrogenases are disclosed, as are the methods of transforming and culturing appropriate host cells with the oxygen-resistant [Fe]-hydrogenases. Finally, the invention provides methods for utilizing the transformed, oxygen insensitive, host cells in the bulk production of H.sub.2 in a light catalyzed reaction having water as the reactant.

  15. The [FeFe] hydrogenase of Nyctotherus ovalis has a chimeric origin

    Directory of Open Access Journals (Sweden)

    Jouany Jean-Pierre

    2007-11-01

    Full Text Available Abstract Background The hydrogenosomes of the anaerobic ciliate Nyctotherus ovalis show how mitochondria can evolve into hydrogenosomes because they possess a mitochondrial genome and parts of an electron-transport chain on the one hand, and a hydrogenase on the other hand. The hydrogenase permits direct reoxidation of NADH because it consists of a [FeFe] hydrogenase module that is fused to two modules, which are homologous to the 24 kDa and the 51 kDa subunits of a mitochondrial complex I. Results The [FeFe] hydrogenase belongs to a clade of hydrogenases that are different from well-known eukaryotic hydrogenases. The 24 kDa and the 51 kDa modules are most closely related to homologous modules that function in bacterial [NiFe] hydrogenases. Paralogous, mitochondrial 24 kDa and 51 kDa modules function in the mitochondrial complex I in N. ovalis. The different hydrogenase modules have been fused to form a polyprotein that is targeted into the hydrogenosome. Conclusion The hydrogenase and their associated modules have most likely been acquired by independent lateral gene transfer from different sources. This scenario for a concerted lateral gene transfer is in agreement with the evolution of the hydrogenosome from a genuine ciliate mitochondrion by evolutionary tinkering.

  16. Isotopic fractionation associated with [NiFe]- and [FeFe]-hydrogenases

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Hui; Gandhi, Hasand; Cornish, Adam J.; Moran, James J.; Kreuzer, Helen W.; Ostrom, Nathaniel; Hegg, Eric L.

    2016-01-30

    Hydrogenases catalyze the reversible formation of H2 from electrons and protons with high efficiency. Understanding the relationships between H2 production, H2 uptake, and H2-H2O exchange can provide insight into the metabolism of microbial communities in which H2 is an essential component in energy cycling. In this manuscript, we used stable H isotopes (1H and 2H) to probe the isotope effects associated with three [FeFe]-hydrogenases and three [NiFe]-hydrogenases. All six hydrogenases displayed fractionation factors for H2 formation that were significantly less than 1, producing H2 that was severely depleted in 2H relative to the substrate, water. Consistent with differences in their active site structure, the fractionation factors for each class appear to cluster, with the three [NiFe]-hydrogenases (α = 0.27-0.40) generally having smaller values than the three [FeFe]-hydrogenases (α = 0.41-0.55). We also obtained isotopic fractionation factors associated with H2 uptake and H2-H2O exchange under conditions similar to those utilized for H2 production, providing us with a more complete picture of the three reactions catalyzed by hydrogenases. The fractionation factors determined in our studies can be used as signatures for different hydrogenases to probe their activity under different growth conditions and to ascertain which hydrogenases are most responsible for H2 production and/or uptake in complex microbial communities.

  17. Production and Application of a Soluble Hydrogenase from Pyrococcus furiosus

    Directory of Open Access Journals (Sweden)

    Chang-Hao Wu

    2015-01-01

    Full Text Available Hydrogen gas is a potential renewable alternative energy carrier that could be used in the future to help supplement humanity’s growing energy needs. Unfortunately, current industrial methods for hydrogen production are expensive or environmentally unfriendly. In recent years research has focused on biological mechanisms for hydrogen production and specifically on hydrogenases, the enzyme responsible for catalyzing the reduction of protons to generate hydrogen. In particular, a better understanding of this enzyme might allow us to generate hydrogen that does not use expensive metals, such as platinum, as catalysts. The soluble hydrogenase I (SHI from the hyperthermophile Pyrococcus furiosus, a member of the euryarchaeota, has been studied extensively and used in various biotechnological applications. This review summarizes the strategies used in engineering and characterizing three different forms of SHI and the properties of the recombinant enzymes. SHI has also been used in in vitro systems for hydrogen production and NADPH generation and these systems are also discussed.

  18. [FeFe]-hydrogenase models assembled into vesicular structures.

    Science.gov (United States)

    Menzel, Kristin; Apfel, Ulf-Peter; Wolter, Nonio; Rüger, Ronny; Alpermann, Theodor; Steiniger, Frank; Gabel, Detlef; Förster, Stephan; Weigand, Wolfgang; Fahr, Alfred

    2014-03-01

    Compartmentalization is a major prerequisite for the origin of life on earth according to Wächtershäuser "Iron-Sulfur-World". The hypothesis is mainly based on an autocatalytic inorganic energy reproducing redox system consisting of iron and sulfur as requirement for the subsequent synthesis of complex organic structures. Here, we modified [FeFe]-hydrogenase models by means of covalent coupling to either oleic acid or the amphiphilic block copolymer polybutadiene-polyethyleneoxide (PB-PEO) and incorporated those into the membranes of vesicles composed of phospholipids (liposomes) or the unmodified amphiphilic polymer (polymersomes). We employed a [2Fe-2S] cluster as a hydrogenase model, since these structures are known to be suitable catalysts for the generation of H2 in the presence of weak acids. Successful incorporation was confirmed by spectrophotometric iron quantification and the vesicles formed were characterized by size determination (photon correlation spectroscopy (PCS)), and zeta potential as well as by cryo-transmission electron microscopy (Cryo-TEM). The modified models could be incorporated into liposomes or polymersomes up to molar proportions of 3.15% and 28%, respectively. Due to the immobilization in vesicular bilayers the [FeFe]-hydrogenase models can even exhibit catalytic action under the particular conditions of the intravesicular microenvironment. Our results suggest that the vesicular systems described may be applied as a nanoreactor for the reduction of encapsulated substances by generating hydrogen and thus as a minimal cell model.

  19. Wiring of Photosystem II to Hydrogenase for Photoelectrochemical Water Splitting.

    Science.gov (United States)

    Mersch, Dirk; Lee, Chong-Yong; Zhang, Jenny Zhenqi; Brinkert, Katharina; Fontecilla-Camps, Juan C; Rutherford, A William; Reisner, Erwin

    2015-07-08

    In natural photosynthesis, light is used for the production of chemical energy carriers to fuel biological activity. The re-engineering of natural photosynthetic pathways can provide inspiration for sustainable fuel production and insights for understanding the process itself. Here, we employ a semiartificial approach to study photobiological water splitting via a pathway unavailable to nature: the direct coupling of the water oxidation enzyme, photosystem II, to the H2 evolving enzyme, hydrogenase. Essential to this approach is the integration of the isolated enzymes into the artificial circuit of a photoelectrochemical cell. We therefore developed a tailor-made hierarchically structured indium-tin oxide electrode that gives rise to the excellent integration of both photosystem II and hydrogenase for performing the anodic and cathodic half-reactions, respectively. When connected together with the aid of an applied bias, the semiartificial cell demonstrated quantitative electron flow from photosystem II to the hydrogenase with the production of H2 and O2 being in the expected two-to-one ratio and a light-to-hydrogen conversion efficiency of 5.4% under low-intensity red-light irradiation. We thereby demonstrate efficient light-driven water splitting using a pathway inaccessible to biology and report on a widely applicable in vitro platform for the controlled coupling of enzymatic redox processes to meaningfully study photocatalytic reactions.

  20. Nickel availability to pea (Pisum sativum L.) plants limits hydrogenase activity of Rhizobium leguminosarum bv. viciae bacteroids by affecting the processing of the hydrogenase structural subunits.

    Science.gov (United States)

    Brito, B; Palacios, J M; Hidalgo, E; Imperial, J; Ruiz-Argüeso, T

    1994-01-01

    Rhizobium leguminosarum bv. viciae UPM791 induces the synthesis of an [NiFe] hydrogenase in pea (Pisum sativum L.) bacteroids which oxidizes the H2 generated by the nitrogenase complex inside the root nodules. The synthesis of this hydrogenase requires the genes for the small and large hydrogenase subunits (hupS and hupL, respectively) and 15 accessory genes clustered in a complex locus in the symbiotic plasmid. We show here that the bacteroid hydrogenase activity is limited by the availability of nickel to pea plants. Addition of Ni2+ to plant nutrient solutions (up to 10 mg/liter) resulted in sharp increases (up to 15-fold) in hydrogenase activity. This effect was not detected when other divalent cations (Zn2+, Co2+, Fe2+, and Mn2+) were added at the same concentrations. Determinations of the steady-state levels of hupSL-specific mRNA indicated that this increase in hydrogenase activity was not due to stimulation of transcription of structural genes. Immunoblot analysis with antibodies raised against the large and small subunits of the hydrogenase enzyme demonstrated that in the low-nickel situation, both subunits are mainly present in slow-migrating, unprocessed forms. Supplementation of the plant nutrient solution with increasing nickel concentrations caused the conversion of the slow-migrating forms of both subunits into fast-moving, mature forms. This nickel-dependent maturation process of the hydrogenase subunits is mediated by accessory gene products, since bacteroids from H2 uptake-deficient mutants carrying Tn5 insertions in hupG and hupK and in hypB and hypE accumulated the immature forms of both hydrogenase subunits even in the presence of high nickel levels. Images PMID:8071205

  1. Structural Characterization of the Novel and Thermal Stable Hydrogenases from the Purple Sulfur Bacteria Thiocapsa Roseopersicina and Lamprobacter Modestohalophilus

    Science.gov (United States)

    2011-08-01

    a molecules of hydrogenase from T. roseopersicina form a ring-shaped hexameric complex with D3 symmetry. It is assumed that the formation of such...Optimization of purification of the hydrogenase from L. modestohalophilus 8 3.4. Optimization of the purification of hydrogenase complex ...activity were pooled and concentrated by ultrafiltration . Methods of preparation of crystal and study of 3D structure of hydrogenase from T

  2. Enhanced Hydrogen Production by Co-cultures of Hydrogenase and Nitrogenase in Escherichia coli.

    Science.gov (United States)

    Lee, Hyun Jeong; Sekhon, Simranjeet Singh; Kim, Young Su; Park, Ju-Yong; Kim, Yang-Hoon; Min, Jiho

    2016-03-01

    Rhodobacter sphaeroides is a bacterium that can produce hydrogen by interaction with hydrogenase and nitrogenase. We report a hydrogen production system using co-cultivation of hydrogenase in liquid medium and immobilized nitrogenase in Escherichia coli. The recombinant plasmid has been constructed to analyze the effect of hydrogen production on the expression of hupSL hydrogenase and nifHDK nitrogenase isolated from R. sphaeroides. All recombinant E. coli strains were cultured anaerobically, and cells for nitrogenase were immobilized in agar gel, whereas cells for hydrogenase were supplemented on the nitrogenase agar gel. The hupSL hydrogenase has been observed to enhance hydrogen production and hydrogenase activity under co-culture with nifHDK nitrogenase. The maximum hydrogen production has been obtained at an agar gel concentration and a cell concentration for co-culture of 2 % and 6.4 × 10(8) CFU. Thus, co-culture of hupSL hydrogenase and nifHDK nitrogenase provides a promising route for enhancing the hydrogen production and hydrogenase activity.

  3. The [FeFe] hydrogenase of Nyctotherus ovalis has a chimeric origin

    NARCIS (Netherlands)

    Boxma, B.; Ricard, G.; Hoek, van A.H.A.M.; Severing, E.; Moon-van der Staay, S.Y.; Staay, van der G.W.M.; Alen, T.A.; Graaf, de R.M.; Cremers, G.; Kwantes, M.; McEwan, N.R.; Newbold, C.J.; Jouany, J.P.; Michalowski, T.; Pristas, P.; Huynen, M.A.; Hackstein, J.H.P.

    2007-01-01

    Background The hydrogenosomes of the anaerobic ciliate Nyctotherus ovalis show how mitochondria can evolve into hydrogenosomes because they possess a mitochondrial genome and parts of an electron-transport chain on the one hand, and a hydrogenase on the other hand. The hydrogenase permits direct

  4. The [FeFe] hydrogenase of Nyctotherus ovalis has a chimeric origin

    NARCIS (Netherlands)

    Boxma, B.; Ricard, G.; Hoek, A.H.A.M. van; Severing, E.; Moon-van der Staay, S.Y.; Staay, G.W.M. van der; Alen, T.A. van; Graaf, R.M. de; Cremers, G.; Kwantes, M.; McEwan, N.R.; Newbold, C.J.; Jouany, J.P.; Michalowski, T.; Pristas, P.; Huynen, M.A.; Hackstein, J.H.P.

    2007-01-01

    BACKGROUND: The hydrogenosomes of the anaerobic ciliate Nyctotherus ovalis show how mitochondria can evolve into hydrogenosomes because they possess a mitochondrial genome and parts of an electron-transport chain on the one hand, and a hydrogenase on the other hand. The hydrogenase permits direct

  5. [NiFe]-hydrogenases: spectroscopic and electrochemical definition of reactions and intermediates.

    NARCIS (Netherlands)

    F.A. Armstrong; S.P.J. Albracht

    2005-01-01

    Production and usage of di-hydrogen, H2, in micro-organisms is catalysed by highly active, 'ancient' metalloenzymes known as hydrogenases. Based on the number and identity of metal atoms in their active sites, hydrogenases fall into three main classes, [NiFe]-, [FeFe]- and [Fe]-. All contain the unu

  6. Practical applications of hydrogenase I from Pyrococcus furiosus for NADPH generation and regeneration

    NARCIS (Netherlands)

    Mertens, R.; Greiner, L.; Ban, van den E.C.D.; Haaker, H.B.C.M.; Liese, A.

    2003-01-01

    The soluble hydrogenase I (H-2:NADP(+) oxidoreductase, EC 1.18.99.1) from the marine hyperthermophilic strain of the archaeon Pyrococcus furiosus was partially purified by anion-exchange chromatography. This P furiosus hydrogenase I preparation (PF H(2)ase I) has been used as biocatalyst in the enzy

  7. Enhancement of photoheterotrophic biohydrogen production at elevated temperatures by the expression of a thermophilic clostridial hydrogenase.

    Science.gov (United States)

    Lo, Shou-Chen; Shih, Shau-Hua; Chang, Jui-Jen; Wang, Chun-Ying; Huang, Chieh-Chen

    2012-08-01

    The working temperature of a photobioreactor under sunlight can be elevated above the optimal growth temperature of a microorganism. To improve the biohydrogen productivity of photosynthetic bacteria at higher temperatures, a [FeFe]-hydrogenase gene from the thermophile Clostridium thermocellum was expressed in the mesophile Rhodopseudomonas palustris CGA009 (strain CGA-CThydA) using a log-phase expression promoter P( pckA ) to drive the expression of heterogeneous hydrogenase gene. In contrast, a mesophilic Clostridium acetobutylicum [FeFe]-hydrogenase gene was also constructed and expressed in R. palustris (strain CGA-CAhydA). Both transgenic strains were tested for cell growth, in vivo hydrogen production rate, and in vitro hydrogenase activity at elevated temperatures. Although both CGA-CThydA and CGA-CAhydA strains demonstrated enhanced growth over the vector control at temperatures above 38 °C, CGA-CThydA produced more hydrogen than the other strains. The in vitro hydrogenase activity assay, measured at 40 °C, confirmed that the activity of the CGA-CThydA hydrogenase was higher than the CGA-CAhydA hydrogenase. These results showed that the expression of a thermophilic [FeFe]-hydrogenase in R. palustris increased the growth rate and biohydrogen production at elevated temperatures. This transgenic strategy can be applied to a broad range of purple photosynthetic bacteria used to produce biohydrogen under sunlight.

  8. A hydrogenosomal [Fe]-hydrogenase from the anaerobic chytrid Neocallimastix sp. L2

    NARCIS (Netherlands)

    Voncken, Frank G.J.; Boxma, Brigitte; Hoek, Angela H.A.M. van; Akhmanova, Anna S.; Vogels, Godfried D.; Huynen, Martijn; Veenhuis, Marten; Hackstein, Johannes H.P.

    2002-01-01

    The presence of a [Fe]-hydrogenase in the hydrogenosomes of the anaerobic chytridiomycete fungus Neocallimastix sp. L2 has been demonstrated by immunocytochemistry, subcellular fractionation, Western-blotting and measurements of hydrogenase activity in the presence of various concentrations of carbo

  9. A hydrogenosomal [Fe]-hydrogenase from the anaerobic chytrid Neocallimastix sp L2

    NARCIS (Netherlands)

    Voncken, Frank G.J.; Boxma, Brigitte; Hoek, Angela H.A.M. van; Akhmanova, Anna S.; Vogels, Godfried D.; Huynen, Martijn; Veenhuis, Marten; Hackstein, Johannes H.P.

    2002-01-01

    The presence of a [Fe]-hydrogenase in the hydrogenosomes of the anaerobic chytridiomycete fungus Neocallimastix sp. L2 has been demonstrated by immunocytochemistry, subcellular fractionation, Western-blotting, and measurements of hydrogenase activity in the presence of various concentrations of carb

  10. New hypotheses for hydrogenase implication in the corrosion of mild steel

    Energy Technology Data Exchange (ETDEWEB)

    Mehanna, Maha; Basseguy, Regine; Delia, Marie-Line [Laboratoire de Genie Chimique (LGC) CNRS-INPT, 5 rue Paulin Talabot BP 1301, 31106 Toulouse (France); Girbal, Laurence; Demuez, Marie [Laboratoire d' Ingenierie des Systemes Biologiques et des Procedes (LISBP) CNRS-INSA, 135 Avenue de Rangueil, 31077 Toulouse (France); Bergel, Alain [Laboratoire de Genie Chimique (LGC) CNRS-INPT, 5 rue Paulin Talabot BP 1301, 31106 Toulouse (France)], E-mail: Alain.Bergel@ensiacet.fr

    2008-12-01

    The influence of [Fe]-hydrogenase from Clostridium acetobutylicum was studied on the anaerobic corrosion of mild steel. Two short-circuited mild steel electrodes were exposed to the same solution and hydrogenase was retained on the surface of only one electrode thanks to a dialysis membrane. The galvanic current and the electrode potential were measured as a function of time in order to monitor the difference in electrochemical behaviour induced by the presence of hydrogenase. A sharp potential decrease of around 500 mV was controlled by the deoxygenating phase. When hydrogenase was introduced after complete deoxygenation, significant heterogeneous corrosion was observed under the vivianite deposit on the electrode in contact with hydrogenase, while the other electrode only showed the vivianite deposit, which was analysed by MEB and EDX. The effect of hydrogenase was then confirmed by monitoring the free potential of single coupons exposed or not to the enzyme in a classical cell after complete deoxygenating. In both phosphate and Tris-HCl buffers, the presence of hydrogenase increased the free potential around 60 mV and induced marked general corrosion. It was concluded that [Fe]-hydrogenase acts in the absence of any final electron acceptor by catalysing direct proton reduction on the mild steel surface.

  11. Amplification of LDH gene from Indian strains of Plasmodium vivax

    Directory of Open Access Journals (Sweden)

    Ritu Berwal, N. Gopalan, Kshitij Chandel, Shri Prakash ,K. Sekhar

    2006-09-01

    Full Text Available Background & objectives: Plasmodium vivax is geographically widespread and responsible for >50% of malaria cases in India. Increased drug resistance of the parasite highlights the immediaterequirement of early and accurate diagnosis as well as new therapeutics. In view of this, the presentstudy was undertaken to amplify P. vivax (Indian strains lactate dehydrogenase gene (PvLDHwhich has been identified as a good target for antimalarials as well as diagnostics.Methods: P. vivax infected clinical blood samples were collected from southern part of India andwere tested with established diagnostic parameters (ICT, Giemsa staining. Total DNA was extractedfrom blood samples and subjected to PCR using two sets of primers, one for the amplification of fullPvLDH gene (951bp and the other for a partial PvLDH gene fragment (422bp, covering a variableantigenic region (140aa as compared to other plasmodial species.Results & conclusion: PCRs for both the full and partial gene targets were optimised and found to beconsistent when tested on several P. vivax positive clinical samples. In addition, full gene PCR wasfound to specifically detect only P. vivax DNA and could be used as a specific molecular diagnostictool. These amplified products can be cloned and expressed as a recombinant protein that might beuseful for the development and screening of antimalarials as well as for diagnostic purposes.

  12. Production of biohydrogen by recombinant expression of [NiFe]-hydrogenase 1 in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Kim Jaoon YH

    2010-07-01

    Full Text Available Abstract Background Hydrogenases catalyze reversible reaction between hydrogen (H2 and proton. Inactivation of hydrogenase by exposure to oxygen is a critical limitation in biohydrogen production since strict anaerobic conditions are required. While [FeFe]-hydrogenases are irreversibly inactivated by oxygen, it was known that [NiFe]-hydrogenases are generally more tolerant to oxygen. The physiological function of [NiFe]-hydrogenase 1 is still ambiguous. We herein investigated the H2 production potential of [NiFe]-hydrogenase 1 of Escherichia coli in vivo and in vitro. The hyaA and hyaB genes corresponding to the small and large subunits of [NiFe]-hydrogenase 1 core enzyme, respectively, were expressed in BL21, an E. coli strain without H2 producing ability. Results Recombinant BL21 expressing [NiFe]-hydrogenase 1 actively produced H2 (12.5 mL H2/(h·L in 400 mL glucose minimal medium under micro-aerobic condition, whereas the wild type BL21 did not produce H2 even when formate was added as substrate for formate hydrogenlyase (FHL pathway. The majority of recombinant protein was produced as an insoluble form, with translocation of a small fraction to the membrane. However, the membrane fraction displayed high activity (~65% of total cell fraction, based on unit protein mass. Supplement of nickel and iron to media showed these metals contribute essentially to the function of [NiFe]-hydrogenase 1 as components of catalytic site. In addition, purified E. coli [NiFe]-hydrogenase 1 using his6-tag displayed oxygen-tolerant activity of ~12 nmol H2/(min·mg protein under a normal aeration environment, compared to [FeFe]-hydrogenase, which remains inactive under this condition. Conclusions This is the first report on physiological function of E. coli [NiFe]-hydrogenase 1 for H2 production. We found that [NiFe]-hydrogenase 1 has H2 production ability even under the existence of oxygen. This oxygen-tolerant property is a significant advantage because it is

  13. [FeFe]-hydrogenase gene quantification and melting curve analysis from hydrogen-fermenting bioreactor samples

    Energy Technology Data Exchange (ETDEWEB)

    Tolvanen, Katariina E.S.; Santala, Ville P.; Karp, Matti T. [Department of Chemistry and Bioengineering, Tampere University of Technology, P.O. Box 541, FI-33101 Tampere (Finland)

    2010-04-15

    In this study, quantitative PCR (qPCR) was used to quantify [FeFe]-hydrogenases and subsequently melting curves were analyzed from hydrogen-fermenting, mixed-culture bioreactor samples. Denaturing gradient gel electrophoresis (DGGE) analysis was also performed to the reactor samples revealing a clostridial dominance in the reactor. Primers targeting [FeFe]-hydrogenases were designed based on known clostridial [FeFe]-hydrogenase gene sequences and tested with several clostridial strains. The results show that amplification efficiencies of four different clostridia are highly similar and melting curves of the clostridial strains were within 1 C of each other. We compared the melting curves to the hydrogen percentage and observed a correlation between the results. The closer the melting curves were to those of clostridia, the better the hydrogen production. Based on these results, the primers and melting curve analysis of [FeFe]-hydrogenase amplicons can be used for analysing hydrogenase genes from bioreactor samples. (author)

  14. Presence and expression of hydrogenase specific C-terminal endopeptidases in cyanobacteria

    Directory of Open Access Journals (Sweden)

    Lindblad Peter

    2003-05-01

    Full Text Available Abstract Background Hydrogenases catalyze the simplest of all chemical reactions: the reduction of protons to molecular hydrogen or vice versa. Cyanobacteria can express an uptake, a bidirectional or both NiFe-hydrogenases. Maturation of those depends on accessory proteins encoded by hyp-genes. The last maturation step involves the cleavage of a ca. 30 amino acid long peptide from the large subunit by a C-terminal endopeptidase. Until know, nothing is known about the maturation of cyanobacterial NiFe-hydrogenases. The availability of three complete cyanobacterial genome sequences from strains with either only the uptake (Nostoc punctiforme ATCC 29133/PCC 73102, only the bidirectional (Synechocystis PCC 6803 or both NiFe-hydrogenases (Anabaena PCC 7120 prompted us to mine these genomes for hydrogenase maturation related genes. In this communication we focus on the presence and the expression of the NiFe-hydrogenases and the corresponding C-terminal endopeptidases, in the three strains mentioned above. Results We identified genes encoding putative cyanobacterial hydrogenase specific C-terminal endopeptidases in all analyzed cyanobacterial genomes. The genes are not part of any known hydrogenase related gene cluster. The derived amino acid sequences show only low similarity (28–41% to the well-analyzed hydrogenase specific C-terminal endopeptidase HybD from Escherichia coli, the crystal structure of which is known. However, computational secondary and tertiary structure modeling revealed the presence of conserved structural patterns around the highly conserved active site. Gene expression analysis shows that the endopeptidase encoding genes are expressed under both nitrogen-fixing and non-nitrogen-fixing conditions. Conclusion Anabaena PCC 7120 possesses two NiFe-hydrogenases and two hydrogenase specific C-terminal endopeptidases but only one set of hyp-genes. Thus, in contrast to the Hyp-proteins, the C-terminal endopeptidases are the only known

  15. In vitro hydrogen production by glucose dehydrogenase and hydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Woodward, J.; Mattingly, S.M. [Oak Ridge National Lab., TN (United States); Danson, M. [Univ. of Bath (United Kingdom)] [and others

    1996-07-01

    A new in vitro enzymatic pathway for the generation of molecular hydrogen from glucose has been demonstrated. The reaction is based on the oxidation of glucose by Thermoplasma acidophilum glucose dehydrogenase with the concomitant oxidation of NADPH by Pyrococcus furiosus hydrogenase. Stoichiometric yields of hydrogen were produced from glucose with the continuous recycling of cofactor. This simple system may provide a method for the biological production of hydrogen from renewable sources. In addition, the other product of this reaction, gluconic acid, is a high-value chemical commodity. 23 refs., 5 figs.

  16. In vitro hydrogen production by glucose dehydrogenase and hydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Woodward, J. [Oak Ridge National Lab., TN (United States)

    1996-10-01

    A new in vitro enzymatic pathway for the generation of molecular hydrogen from glucose has been demonstrated. The reaction is based upon the oxidation of glucose by Thermoplasma acidophilum glucose dehydrogenase with the concomitant oxidation of NADPH by Pyrococcus furiosus hydrogenase. Stoichiometric yields of hydrogen were produced from glucose with continuous cofactor recycle. This simple system may provide a method for the biological production of hydrogen from renewable sources. In addition, the other product of this reaction, gluconic acid, is a high-value commodity chemical.

  17. The effect of extracellular alkalinization on lactate metabolism of breast cancer stem cells: Overview of LDH-A, LDH-B, MCT1 and MCT4 gene expression

    Science.gov (United States)

    Neolaka, G. M. G.; Yustisia, I.; Sadikin, M.; Wanandi, S. I.

    2017-08-01

    Changes in the metabolic status of cancer cells are presumed to be correlated with the adjustment of these cells to extracellular changes. Cell glycolysis increases the production of intracellular lactate catalyzed by the lactate dehydrogenases, both LDH-A and LDH-B. An increase in intracellular lactate can affect extracellular pH balance through monocarboxylate transporters, particularly MCT1 and MCT4. This study aimed to analyze the effects of extracellular alkalinization on the lactate metabolism of human breast cancer stem cells (BCSCs). In this study, human primary BCSCs (CD24-/CD44+ cells) were treated with 100 mM sodium bicarbonate for 0.5, 24, and 48 h in DMEM F12/HEPES. After incubation, extracellular pH was measured and cells were harvested to extract the total RNA and protein. The expression of LDH-A, LDH-B, MCT1, and MCT4 mRNA genes were analyzed using qRT-PCR method. Our study shows that administration of sodium bicarbonate in the BCSC culture medium could increase extracellular pH. To balance the increase of extracellular pH, BCSCs regulated the expression of LDH-A, LDH-B, MCT1, and MCT4 genes. As the extracellular pH increases, the expression of LDH-A that converts pyruvate to lactate increased along with the increase of MCT 4 and MCT 1 expression, which act as lactate transporters. As the incubation time increases, the pH decreases, leading to the suppression of LDH-A and increase of LDH-B expression that converts lactate into pyruvate. Therefore, we suggest that the extracellular alkalinization by sodium bicarbonate in BCSCs affected the genes that regulate lactate metabolism.

  18. Epilepsy treatment. Targeting LDH enzymes with a stiripentol analog to treat epilepsy.

    Science.gov (United States)

    Sada, Nagisa; Lee, Suni; Katsu, Takashi; Otsuki, Takemi; Inoue, Tsuyoshi

    2015-03-20

    Neuronal excitation is regulated by energy metabolism, and drug-resistant epilepsy can be suppressed by special diets. Here, we report that seizures and epileptiform activity are reduced by inhibition of the metabolic pathway via lactate dehydrogenase (LDH), a component of the astrocyte-neuron lactate shuttle. Inhibition of the enzyme LDH hyperpolarized neurons, which was reversed by the downstream metabolite pyruvate. LDH inhibition also suppressed seizures in vivo in a mouse model of epilepsy. We further found that stiripentol, a clinically used antiepileptic drug, is an LDH inhibitor. By modifying its chemical structure, we identified a previously unknown LDH inhibitor, which potently suppressed seizures in vivo. We conclude that LDH inhibitors are a promising new group of antiepileptic drugs.

  19. A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism

    Directory of Open Access Journals (Sweden)

    Grandl Gerald

    2011-05-01

    Full Text Available Abstract Background FeFe-hydrogenases are the most active class of H2-producing enzymes known in nature and may have important applications in clean H2 energy production. Many potential uses are currently complicated by a crucial weakness: the active sites of all known FeFe-hydrogenases are irreversibly inactivated by O2. Results We have developed a synthetic metabolic pathway in E. coli that links FeFe-hydrogenase activity to the production of the essential amino acid cysteine. Our design includes a complementary host strain whose endogenous redox pool is insulated from the synthetic metabolic pathway. Host viability on a selective medium requires hydrogenase expression, and moderate O2 levels eliminate growth. This pathway forms the basis for a genetic selection for O2 tolerance. Genetically selected hydrogenases did not show improved stability in O2 and in many cases had lost H2 production activity. The isolated mutations cluster significantly on charged surface residues, suggesting the evolution of binding surfaces that may accelerate hydrogenase electron transfer. Conclusions Rational design can optimize a fully heterologous three-component pathway to provide an essential metabolic flux while remaining insulated from the endogenous redox pool. We have developed a number of convenient in vivo assays to aid in the engineering of synthetic H2 metabolism. Our results also indicate a H2-independent redox activity in three different FeFe-hydrogenases, with implications for the future directed evolution of H2-activating catalysts.

  20. Potential Applications of Hybrid Layered Double Hydroxide (LDH Particles in Pulp and Paper Production

    Directory of Open Access Journals (Sweden)

    Sophia von Haartman

    2014-03-01

    Full Text Available Functionalization of papermaking pulp fibers using inorganic particles was investigated as a novel approach. Different layered double hydroxide (LDH particles were used in peroxide bleaching of thermomechanical pulp (TMP and in oxygen bleaching of eucalyptus kraft pulp. LDH particles were also tested as binding sites for optical brightening agents (OBA that are commonly used in paper production. The surface chemistry of LDH-treated pulps was examined using X-ray photoelectron spectroscopy (XPS and apparent contact angle with water. Adsorbed LDH was not detected by XPS on the fiber surfaces after the bleaching trials, but it had a clear impact on the processes. LDH particles modified with terephthalate anions decreased the consumption of hydrogen peroxide and increased opacity by 3 units in TMP. Unmodified LDH particles enhanced the selectivity in oxygen delignification of kraft pulp, leading to 10% gain in ISO brightness and reduction of 2 units in Kappa number in comparison with conventional processes. Paper strength properties were unaffected in the presented system. After bleaching with LDH, the amount of anionic groups on pulp surfaces was increased. Also, the retention of OBA onto TMP fibers was improved with modified LDH particles. LDH proved to have great potential for current and prospective applications in pulp and paper manufacture.

  1. Turning cellulose waste into electricity: hydrogen conversion by a hydrogenase electrode.

    Directory of Open Access Journals (Sweden)

    Sergey M Abramov

    Full Text Available Hydrogen-producing thermophilic cellulolytic microorganisms were isolated from cow faeces. Rates of cellulose hydrolysis and hydrogen formation were 0.2 mM L(-1 h(-1 and 1 mM L(-1 h(-1, respectively. An enzymatic fuel cell (EFC with a hydrogenase anode was used to oxidise hydrogen produced in a microbial bioreactor. The hydrogenase electrode was exposed for 38 days (912 h to a thermophilic fermentation medium. The hydrogenase activity remaining after continuous operation under load was 73% of the initial value.

  2. Insights into the Synthesis of Layered Double Hydroxide (LDH) Nanoparticles: Part 1. Optimization and Controlled Synthesis of Chloride-Intercalated LDH

    OpenAIRE

    Sun, Xiaodi; Neuperger, Erica; Dey, Sandwip K.

    2015-01-01

    Layered double hydroxide (LDH) nanoparticles have excellent anion-intercalating property, and their potential as theranostic nanovectors is high. However, understanding of the control of the mean particle size (MPS) and achievement of monodispersed particle size distribution (PSD) remains elusive. Herein, with the aid of statistical design of experiments on a model system of Cl−-intercalated (Zn, Al)-LDH, controlled synthesis of single crystalline nanoparticles using the coprecipitation metho...

  3. Iron-dependent hydrogenases of Entamoeba histolytica and Giardia lamblia: activity of the recombinant entamoebic enzyme and evidence for lateral gene transfer.

    Science.gov (United States)

    Nixon, Julie E J; Field, Jessica; McArthur, Andrew G; Sogin, Mitchell L; Yarlett, Nigel; Loftus, Brendan J; Samuelson, John

    2003-02-01

    Entamoeba histolytica and Spironucleus barkhanus have genes that encode short iron-dependent hydrogenases (Fe-hydrogenases), even though these protists lack hydrogenosomes. To understand better the biochemistry of the protist Fe-hydrogenases, we prepared a recombinant E. histolytica short Fe-hydrogenase and measured its activity in vitro. A Giardia lamblia gene encoding a short Fe-hydrogenase was identified from shotgun genomic sequences, and RT-PCR showed that cultured entamoebas and giardias transcribe short Fe-hydrogenase mRNAs. A second E. histolytica gene, which encoded a long Fe-hydrogenase, was identified from shotgun genomic sequences. Phylogenetic analyses suggested that the short Fe-hydrogenase genes of entamoeba and diplomonads share a common ancestor, while the long Fe-hydrogenase gene of entamoeba appears to have been laterally transferred from a bacterium. These results are discussed in the context of competing ideas for the origins of genes encoding fermentation enzymes of these protists.

  4. Preliminary Study on Serum Lactate Dehydrogenase (LDH)-Prognostic Biomarker in Carcinoma Breast

    Science.gov (United States)

    Gandhe, Mahendra Bhauraoji; Gupta, Dilip; Reddy, M.V.R.

    2016-01-01

    Introduction Serum Lactate Dehydrogenase (LDH) is one of the biochemical markers for breast cancer. Serum LDH is enzyme required for anaerobic glycolysis. One of its isoenzyme is increased in breast cancer due to up-regulation in its gene. It leads to increase in serum LDH level in breast cancer patients. Serum LDH is economical, easily available and easy to estimate. Aim In the present study, we evaluated the LDH levels in circulation of newly diagnosed patients of breast cancer and tried to correlate it with different TNM staging of carcinoma breast before interventions and after adjuvant therapy of these patients. Materials and Methods This prospective study was done on 83 diagnosed patients of breast cancer was conducted among poor patients in rural area. This study was conducted in the Department of Surgery between October 2008 to October 2010, at MGIMS, Sevagram, Maharashtra, a rural medical college located in Central India. Out of total 83 participants, 10 participants were having adverse events following surgery and remaining 73 participants were without adverse events following surgery. The significant difference in serum LDH levels between two groups, with and without adverse surgical outcome was calculated by Mann-Whitney U test. Results Patients with higher clinical TNM staging were having higher serum LDH levels. The serum LDH levels at sixth months following surgery showed a trend of statistically significant difference between patients with and without adverse events. As increased serum LDH levels in breast cancer patients shows poor prognosis, surgical outcome or advanced metastases. Conclusion Serum LDH monitoring can be used as a prognostic biomarker in patients of breast cancer. For confirmation of this finding, we require further more studies on larger sample size and long-term follow-up in patients specifically with higher serum LDH levels. PMID:27134855

  5. Metallocene supported core@LDH catalysts for slurry phase ethylene polymerisation.

    Science.gov (United States)

    Buffet, Jean-Charles; Byles, Coral F H; Felton, Ryan; Chen, Chunping; O'Hare, Dermot

    2016-03-14

    We report the synthesis of solid catalysts based on a zirconocene supported on either silica@AMO-LDH or zeolite@AMO-LDH for the slurry phase polymerisation of ethylene. The hybrid catalysts demonstrate synergistic effects in which the polymerisation activity is up to three times higher than the zirconocene supported on analogous single phase silica or zeolite supports.

  6. Structural Evolvement of Heating Treatment of Mg/Al-LDH and Preparation of Mineral Mesoporous Materials

    Institute of Scientific and Technical Information of China (English)

    CHEN Tianhu; XU Huifang; WANG Yifeng; QING Chengsong; FAN Mingde; CHEN Gang

    2006-01-01

    Although hydrotalcite, or layered double hydroxides (LDHs), is not a common mineral, it is an important material that can be easily synthesized in laboratory. In this study, structural evolvement and BET surface area changes of heat treated Mg/Al-LDH is evaluated by XRD, TEM and N2-BET analyses. The results indicate that the magnesium-aluminum LDH with carbonate as interlayer anion,periclase-like oxides was formed at temperatures of 400-800℃. Meanwhile, 2-3 nanometer mesoporous were formed during decomposition of LDH. However, the heat treated samples still preserve the morphology of the original LDH plates. Periclase-like formed from LDH heat treatment may re-hydrolyze and recover the structure of LDH. However, crystallinity of the recovered LDH is lower than that of the original LDH. This heat treatment will result in formation of (Mg, Al)-oxide nano-crystals and nanopores among the nano-crystals. When heating temperature exceeds 1000, the periclase-like (Mg, Al)-oxide is transformed into a composite with periclase (MgO) and spinel phases.The periclase can be re-hydrolyzed and dissolved in HCl solution. After acid treatment, the sample with a high surface area is composed of spinel nano-crystals and nanopores among them. Our results will provide a new and economic way to synthesize mesoporous materials through pathways of phase transformation of precursor materials with different composition.

  7. A novel endo-hydrogenase activity recycles hydrogen produced by nitrogen fixation.

    Directory of Open Access Journals (Sweden)

    Gordon Ng

    Full Text Available BACKGROUND: Nitrogen (N(2 fixation also yields hydrogen (H(2 at 1:1 stoichiometric amounts. In aerobic diazotrophic (able to grow on N(2 as sole N-source bacteria, orthodox respiratory hupSL-encoded hydrogenase activity, associated with the cell membrane but facing the periplasm (exo-hydrogenase, has nevertheless been presumed responsible for recycling such endogenous hydrogen. METHODS AND FINDINGS: As shown here, for Azorhizobium caulinodans diazotrophic cultures open to the atmosphere, exo-hydrogenase activity is of no consequence to hydrogen recycling. In a bioinformatic analysis, a novel seven-gene A. caulinodans hyq cluster encoding an integral-membrane, group-4, Ni,Fe-hydrogenase with homology to respiratory complex I (NADH: quinone dehydrogenase was identified. By analogy, Hyq hydrogenase is also integral to the cell membrane, but its active site faces the cytoplasm (endo-hydrogenase. An A. caulinodans in-frame hyq operon deletion mutant, constructed by "crossover PCR", showed markedly decreased growth rates in diazotrophic cultures; normal growth was restored with added ammonium--as expected of an H(2-recycling mutant phenotype. Using A. caulinodans hyq merodiploid strains expressing beta-glucuronidase as promoter-reporter, the hyq operon proved strongly and specifically induced in diazotrophic culture; as well, hyq operon induction required the NIFA transcriptional activator. Therefore, the hyq operon is constituent of the nif regulon. CONCLUSIONS: Representative of aerobic N(2-fixing and H(2-recycling alpha-proteobacteria, A. caulinodans possesses two respiratory Ni,Fe-hydrogenases: HupSL exo-hydrogenase activity drives exogenous H(2 respiration, and Hyq endo-hydrogenase activity recycles endogenous H(2, specifically that produced by N(2 fixation. To benefit human civilization, H(2 has generated considerable interest as potential renewable energy source as its makings are ubiquitous and its combustion yields no greenhouse gases. As

  8. Electrocatalytic mechanism of reversible hydrogen cycling by enzymes and distinctions between the major classes of hydrogenases

    OpenAIRE

    Hexter, Suzannah V.; Grey, Felix; Happe, Thomas; Climent, Victor; Armstrong, Fraser A.

    2012-01-01

    The extraordinary ability of Fe- and Ni-containing enzymes to catalyze rapid and efficient H+/H2 interconversion—a property otherwise exclusive to platinum metals—has been investigated in a series of experiments combining variable-temperature protein film voltammetry with mathematical modeling. The results highlight important differences between the catalytic performance of [FeFe]-hydrogenases and [NiFe]-hydrogenases and justify a simple model for reversible catalytic electron flow in enzymes...

  9. A hydrogen-producing, hydrogenase-free mutant strain of Nostoc punctiforme ATCC 29133

    Energy Technology Data Exchange (ETDEWEB)

    Lindberg, P.; Lindblad, P. [Uppsala Univ. (Sweden). Dept. of Physiological Botany; Schuetz, K.; Happe, T. [Universitaet Bonn (Germany). Botanisches Inst.

    2002-12-01

    The hupL gene, encoding the uptake hydrogenase large subunit, in Nostoc sp. strain ATCC 29133, a strain lacking a bidirectional hydrogenase, was inactivated by insertional mutagenesis. Recombinant strains were isolated and analysed, and one hupL{sup -} strain, NHM5, was selected for further study. Cultures of NHM5 were grown under nitrogen-fixing conditions and H{sub 2} evolution under air was observed using an H{sub 2} electrode. (Author)

  10. Aquifex aeolicus membrane hydrogenase for hydrogen biooxidation: Role of lipids and physiological partners in enzyme stability and activity

    Energy Technology Data Exchange (ETDEWEB)

    Infossi, Pascale; Lojou, Elisabeth; Giudici-Orticoni, Marie-Therese [Unite de Bioenergetique et Ingenierie des Proteines, UPR 9036, Institut de Microbiologie de la Mediterranee - CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20 (France); Chauvin, Jean-Paul [Institut de Biologie du developpement de Marseille Luminy, UMR 6216, Parc Scientifique de Luminy, 163 Avenue de Luminy, BP 907, 13009 Marseille (France); Herbette, Gaetan [Spectropole FI 1739, Aix-Marseille Universite case 511, Faculte de St Jerome Avenue Escadrille Normandie Niemen, 13397 Marseille Cedex 20 (France); Brugna, Myriam [Unite de Bioenergetique et Ingenierie des Proteines, UPR 9036, Institut de Microbiologie de la Mediterranee - CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20 (France); Universite de Provence, 3 Place Victor Hugo, 13331 Marseille Cedex 03 (France)

    2010-10-15

    Hydrogenase I from the hyperthermophilic bacterium Aquifex aeolicus is a good candidate for biotechnological devices thanks to its ability to oxidize hydrogen at high temperature, even in the presence of oxygen and CO. In order to enhance the enzyme stability and the catalytic efficiency, we investigated the hydrogen oxidation process with hydrogenase I embedded in a physiological-like environment. Hydrogenase I partners in the metabolic chain, namely membrane quinone and cytochrome b, were purified and fully characterized. The complex hydrogenase I-cytochrome b was inserted into liposomes. Surface Plasmon Resonance revealed that quinone took part in the stabilization of the complex. By use of molecular modelization and electrochemistry analysis, enzyme stability has been demonstrated to be stronger and enzymatic efficiency to be five times higher when hydrogenase is embedded into the liposomes. This result raises the possibility of using hydrogenases as biocatalysts in fuel cells. (author)

  11. Studies of Hybrid Nano-Bio-System: Single-Walled Carbon Nanotubes and Hydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Svedruzic-Chang, D.; Blackburn, J. L.; McDonald, T. J.; Heben, M. J.; King, P. W.

    2008-01-01

    We have examined changes in single-walled carbon nanotubes (SWNT) optical signals upon addition of recombinant [FeFe] hydrogenases from Clostridium acetobutylicum or Chlamydomonas reinhardtii. We found evidence that novel and stable charge-transfer complexes are formed only under conditions of hydrogenase catalytic turnover. Formation of the complex sensitizes the nanotubes to the proton-to-hydrogen redox half-reaction. Thus, the experimental potential can be altered by changing the pH or molecular hydrogen concentration. In the presence of molecular hydrogen, hydrogenase mediates electron injection into the conduction band of semiconducting SWNT, which was observed as a quenching of the photoluminescence signals. Here, we will present recent Raman studies, which revealed that SWNTs in a complex with hydrogenase may undergo either oxidation or reduction, depending on the electronic structure of the SWNT and the oxidation state of the enzyme. In addition, we will describe our efforts to prepare stable, solubilized SWNT/hydrogenase complexes in the absence of detergent. This work shows that SWNT/hydrogenase complexes have potential applications as a component of an energy conversion device.

  12. Using directed evolution to improve hydrogen production in chimeric hydrogenases from Clostridia species.

    Science.gov (United States)

    Plummer, Scott M; Plummer, Mark A; Merkel, Patricia A; Hagen, Moira; Biddle, Jennifer F; Waidner, Lisa A

    2016-11-01

    Hydrogenases are enzymes that play a key role in controlling excess reducing equivalents in both photosynthetic and anaerobic organisms. This enzyme is viewed as potentially important for the industrial generation of hydrogen gas; however, insufficient hydrogen production has impeded its use in a commercial process. Here, we explore the potential to circumvent this problem by directly evolving the Fe-Fe hydrogenase genes from two species of Clostridia bacteria. In addition, a computational model based on these mutant sequences was developed and used as a predictive aid for the isolation of enzymes with even greater efficiency in hydrogen production. Two of the improved mutants have a logarithmic increase in hydrogen production in our in vitro assay. Furthermore, the model predicts hydrogenase sequences with hydrogen productions as high as 540-fold over the positive control. Taken together, these results demonstrate the potential of directed evolution to improve the native bacterial hydrogenases as a first step for improvement of hydrogenase activity, further in silico prediction, and finally, construction and demonstration of an improved algal hydrogenase in an in vivo assay of C. reinhardtii hydrogen production. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Integration of an [FeFe]-hydrogenase into the anaerobic metabolism of Escherichia coli

    Science.gov (United States)

    Kelly, Ciarán L.; Pinske, Constanze; Murphy, Bonnie J.; Parkin, Alison; Armstrong, Fraser; Palmer, Tracy; Sargent, Frank

    2015-01-01

    Biohydrogen is a potentially useful product of microbial energy metabolism. One approach to engineering biohydrogen production in bacteria is the production of non-native hydrogenase activity in a host cell, for example Escherichia coli. In some microbes, hydrogenase enzymes are linked directly to central metabolism via diaphorase enzymes that utilise NAD+/NADH cofactors. In this work, it was hypothesised that heterologous production of an NAD+/NADH-linked hydrogenase could connect hydrogen production in an E. coli host directly to its central metabolism. To test this, a synthetic operon was designed and characterised encoding an apparently NADH-dependent, hydrogen-evolving [FeFe]-hydrogenase from Caldanaerobacter subterranus. The synthetic operon was stably integrated into the E. coli chromosome and shown to produce an active hydrogenase, however no H2 production was observed. Subsequently, it was found that heterologous co-production of a pyruvate::ferredoxin oxidoreductase and ferredoxin from Thermotoga maritima was found to be essential to drive H2 production by this system. This work provides genetic evidence that the Ca.subterranus [FeFe]-hydrogenase could be operating in vivo as an electron-confurcating enzyme. PMID:26839796

  14. Integration of an [FeFe]-hydrogenase into the anaerobic metabolism of Escherichia coli

    Directory of Open Access Journals (Sweden)

    Ciarán L. Kelly

    2015-12-01

    Full Text Available Biohydrogen is a potentially useful product of microbial energy metabolism. One approach to engineering biohydrogen production in bacteria is the production of non-native hydrogenase activity in a host cell, for example Escherichia coli. In some microbes, hydrogenase enzymes are linked directly to central metabolism via diaphorase enzymes that utilise NAD+/NADH cofactors. In this work, it was hypothesised that heterologous production of an NAD+/NADH-linked hydrogenase could connect hydrogen production in an E. coli host directly to its central metabolism. To test this, a synthetic operon was designed and characterised encoding an apparently NADH-dependent, hydrogen-evolving [FeFe]-hydrogenase from Caldanaerobacter subterranus. The synthetic operon was stably integrated into the E. coli chromosome and shown to produce an active hydrogenase, however no H2 production was observed. Subsequently, it was found that heterologous co-production of a pyruvate::ferredoxin oxidoreductase and ferredoxin from Thermotoga maritima was found to be essential to drive H2 production by this system. This work provides genetic evidence that the Ca.subterranus [FeFe]-hydrogenase could be operating in vivo as an electron-confurcating enzyme.

  15. Decolorization and degradation of textile dyes with biosulfidogenic hydrogenases.

    Science.gov (United States)

    Mutambanengwe, C C Z; Togo, C A; Whiteley, C G

    2007-01-01

    Successful decolorization of azo dyes (Orange II, Amido Black 10, Reactive Black 5, and Reactive Red 120) and industrial textile dye influents and effluents with sulfate-reducing bacteria from within a biosulfidogenic reactor was achieved with decolorizations ranging from 96% to 49% over 144 h. Concomitant with the decrease in absorbance of the dye in the visible region (480-620 nm) was an increase in the absorbance at 280 nm, over 48 h, suggesting an increase in concentration of single aromatic amines. With an extended period of time there was a subsequent decrease in the absorbance at 280 nm indicating that the aromatic amines had been degraded. The anthraquinone dye, Reactive Blue 2, remained unchanged after 144 h of incubation in the biosulfidogenic reactor and was only rapidly decolored at 192 h, implying that certain factors are induced in the reactor to break down this non-azo dye. The fastest decolorization/degradation rates and highest hydrogenase enzyme production were observed with Orange II, while the slowest decolorization/degradation rate and least enzyme production were with Reactive Blue 2, suggesting that these processes are controlled, to a certain degree, by an enzymatic mechanism. With sulfate-reducing bacteria that had been cultured on a lactate medium, there was complete decolorization of both authentic dyes and industrial influents and effluents as monitored by the decrease of absorbance in the visible region (480-620 nm). There was, however, very little breakdown of the single aromatic compounds as the absorbance at 280 nm remained fairly significant. This supports the suggestion that, within the biosulfidogenic reactor, there are factors other than the identified hydrogenases that are responsible for degradation of the aromatic compounds.

  16. Synthesis and Characterization of PE-g-MA/MgAI-LDH Exfoliation Nanocomposite via Solution Intercalation

    Institute of Scientific and Technical Information of China (English)

    陈伟; 瞿保钧

    2003-01-01

    An organo-modified MgAl-layered double hydroxide (OMgAl-LDH) was mecessfully exfoliated in the xyleue solution of polyethylene-grafted-malelc anhydride (PE-g-MA) under refluxing condition. A PE-g-MA/MgAI-LDH exfoliaUon nanocomposite was formed after the precipitaUon of PE-g-MA from the dispersion system. The structure and thermal property of the PE-g-MA/MgAI-LDH exfoliaUon nanocomposite were characterizd by X-ray diffraction(XRD),transmission electron microscopy(TEM),and thermogravimetry analysis(TGA).The disappearance of d001 XRD peak of OMgAl-LDH at 2θ=3.2° suggests that the MgAl hydroxide sheets are exfoliated in the nanocomposite.The TEM image shows that the MgAl hydroxide sheets of less than 70nm in length or width are exfoliated and dispersed disorderly in PE-g-MA matrix.TGA profiles indicate that the PE-g-MA/MgAl-LDH nanocomposite with 5wt% OMgAl-LDH loading shows a faster charring process in temperature range from 210 to 390℃ and a greater thermal stability beyond 390℃ than PE-g-MA does.The decompostion temperature of the nanocomposite is 25℃ higher than that of PE-g-MA as measured at 50% weight loss.The PE-g-MA/MgAl-LDH nanocomposite is promising for application of flame-retardant polymeric materials.

  17. The prognostic value of serum LDH levels in patients with hepatocellualr carcinoma after hepatic resection.

    Science.gov (United States)

    Hu, Wen-Jie; Fu, Shun-Jun; Diao, Jing-Fang; Wan, Dai-Wei; Tan, Zhi-Jian; Zhi, Qiao-Ming; He, Jun-Ming

    2015-07-16

    Serum lactate dehydrogenase (LDH) is a prognostic marker in many tumor types. The present study is to determine the value of preoperative peripheral LDH in predicting the prognosis of patients with HCC after hepatectomy. Clinicopathological and follow-up data selected 323 HCC patients who underwent hepatectomy. These patients were categorized as high LDH(>240 U/I) and low LDH group (≤ 240 U/I). Significant differences in tumor size, capsulation, tumor number, vascular invasion and TNM stage were observed between these two groups (P 240 U/I group were poorer than those in the LDH ≤ 240 U/I group respectively. Multivariate analysis demonstrated that LDH > 240 U/I served as an independent prognostic indicator of worse disease-free survival [hazard ratio (HR) = 1.711; 95% confidence interval (CI), 1.275-2.297; P < 0.001] and overall survival (HR = 1.568; 95% CI, 1.144-2.149; P = 0.005). Stratification analysis showed that LDH exhibited a greater predictive value for DFS and OS in HCC patients with with AFP < 200 ng/ml. Our results suggested that serum LDH level might be a potential and reliable prognostic biomarker in HCC patients after liver resection.

  18. Preparation and enhanced properties of polyaniline/grafted intercalated ZnAl-LDH nanocomposites

    Science.gov (United States)

    Hu, Jinlong; Gan, Mengyu; Ma, Li; Zhang, Jun; Xie, Shuang; Xu, Fenfang; Shen, JiYue Zheng Xiaoyu; Yin, Hui

    2015-02-01

    The polymeric nanocomposites (PANI/AD-LDH) were prepared by in situ polymerization based on polyaniline (PANI) and decavanadate-intercalated and γ-aminopropyltriethoxysilane (APTS)-grafted ZnAl-layered double hydroxide (AD-LDH). FTIR and XRD studies confirm the grafting of APTS with decavanadate-intercalated LDH (D-LDH). The extent of grafting (wt%) has also been estimated on the basis of the residue left in nitrogen atmosphere at 800 °C in TGA. SEM and XPS studies show the partial exfoliation of grafted LDH in the PANI matrix and the interfacial interaction between PANI and grafted LDH, respectively. The grafted intercalated layered double hydroxide in reinforcing the properties of the PANI nanocomposites has also been investigated by open circuit potential (OCP), tafel polarization curves (TAF), electrochemical impendence spectroscopy (EIS), salt spray test and TGA-DTA. The experimental results indicate that the PANI/AD-LDH has a higher thermal stability and anticorrosion properties relative to the PANI.

  19. Effect of Hypoxia on Ldh-c Expression in Somatic Cells of Plateau Pika.

    Science.gov (United States)

    Wei, Dengbang; Wei, Linna; Li, Xiao; Wang, Yang; Wei, Lian

    2016-08-01

    Sperm specific lactate dehydrogenases (LDH-C₄) is a lactate dehydrogenase that catalyzes the conversion of pyruvate to lactate. In mammals, Ldh-c was originally thought to be expressed only in testes and spermatozoa. Plateau pika (Ochotona curzoniae), which belongs to the genus Ochotona of the Ochotonidea family, is a hypoxia-tolerant mammal living 3000-5000 m above sea level on the Qinghai-Tibet Plateau, an environment which is strongly hypoxic. Ldh-c is expressed not only in testes and sperm, but also in the somatic tissues of plateau pika. To reveal the effect of hypoxia on pika Ldh-c expression, we investigated the mRNA and protein level of Ldh-c as well as the biochemical index of anaerobic glycolysis in pika somatic tissues at the altitudes of 2200 m, 3200 m and 3900 m. Our results showed that mRNA and protein expression levels of Ldh-c in the tissues of pika's heart, liver, brain and skeletal muscle were increased significantly from 2200 m to 3200 m, but had no difference from 3200 m to 3900 m; the activities of LDH and the contents of lactate showed no difference from 2200 m to 3200 m, but were increased significantly from 3200 m to 3900 m. Hypoxia up-regulated and maintained the expression levels of Ldh-c in the pika somatic cells. Under the hypoxia condition, plateau pikas increased anaerobic glycolysis in somatic cells by LDH-C₄, and that may have reduced their dependence on oxygen and enhanced their adaptation to the hypoxic environment.

  20. [Role of layered double hydroxide (LDH) in the protection of herring testis DNA from heavy metals].

    Science.gov (United States)

    Tang, Yi-Ni; Wu, Ping-Xiao; Zhu, Neng-Wu

    2012-10-01

    The role of layered double hydroxide (LDH) in the protection of herring testis DNA from heavy metals Cd2+ and Pb2+ was studied by X-ray diffraction ( XRD) spectra, Fourier transform infrared (FTIR) spectra, Scanning Electron Microscopy (SEM), Cyclic Voltammetry and Ultraviolet Spectrometry. Size expansion of the basal spacing (003) from 0. 76 nm in LDH to 2. 30 nm was observed in the resulting DNA-LDH nanohybrids and it gave peaks corresponding to C=O (1 534 cm(-1) and 1488 cm(-1)) in skeleton and bases, C-O stretching vibration (1228 cm(-1)), and P-O symmetrical stretching vibration (1096 cm(-1)) in functional groups of DNA, indicating that DNA were intercalated into the LDH by the ion exchange. However, the displacement of NO3(-) was not fully complete (partial intercalation of DNA). The DNA outside LDH interlayers was absorbed on the surface of LDH. The cyclic voltammetric curves showed that DNA in the composites exhibited a very similar peaks, which corresponded to the two reduction current peaks (E(P) = - 1.2 mV and E(P) = -2.4 mV) of free DNA. Also there was no cathode sag emerging in cyclic voltammetric curves, suggesting that both Cd2+ and Pb2+ cannot insert into the groove of DNA to associate with base pairs or other groups when DNA was bound on LDH. The results showed that, on the one hand, both Cd2+ and Pb2+ were absorbed on the external surface of LDH for immobilization, on the other hand, the layer of LDH provided ideal space for DNA by the action of protecting DNA molecules from Cd2+ and Pb2+.

  1. Effect of Hypoxia on Ldh-c Expression in Somatic Cells of Plateau Pika

    OpenAIRE

    Wei, Dengbang; Wei, Linna; Li, Xiao; Wang, Yang; Wei, Lian

    2016-01-01

    Sperm specific lactate dehydrogenases (LDH-C4) is a lactate dehydrogenase that catalyzes the conversion of pyruvate to lactate. In mammals, Ldh-c was originally thought to be expressed only in testes and spermatozoa. Plateau pika (Ochotona curzoniae), which belongs to the genus Ochotona of the Ochotonidea family, is a hypoxia-tolerant mammal living 3000–5000 m above sea level on the Qinghai-Tibet Plateau, an environment which is strongly hypoxic. Ldh-c is expressed not only in testes and sper...

  2. Hydrogenase Activity of Mineral-Associated and Suspended Populations of Desulfovibrio desulfuricans Essex 6

    Energy Technology Data Exchange (ETDEWEB)

    C.L. Reardon; T.S. Magnuson; E.S. Boyd; W.D. Leavitt; D.W. Reed; G.G. Geesey

    2014-02-01

    The interactions between sulfate-reducing microorganisms and iron oxides influence a number of important redox-sensitive biogeochemical processes including the formation of iron sulfides. Enzymes, such as hydrogenase which catalyze the reversible oxidation of molecular hydrogen, are known to mediate electron transfer to metals and may contribute to the formation and speciation of ferrous sulfides formed at the cell–mineral interface. In the present study, we compared the whole cell hydrogenase activity of Desulfovibrio desulfuricans strain Essex 6 growing as biofilms on hematite (hematite-associated) or as suspended populations using different metabolic pathways. Hematite-associated cells exhibited significantly greater hydrogenase activity than suspended populations during sulfate respiration but not during pyruvate fermentation. The enhanced activity of the hematite-associated, sulfate-grown cells appears to be dependent on iron availability rather than a general response to surface attachment since the activity of glass-associated cells did not differ from that of suspended populations. Hydrogenase activity of pyruvate-fermenting cells was stimulated by addition of iron as soluble Fe(II)Cl2 and, in the absence of added iron, both sulfate-reducing and pyruvate-fermenting cells displayed similar rates of hydrogenase activity. These data suggest that iron exerts a stronger influence on whole cell hydrogenase activity than either metabolic pathway or mode of growth. The location of hydrogenase to the cell envelope and the enhanced activity at the hematite surface in sulfate-reducing cells may influence the redox conditions that control the species of iron sulfides on the mineral surface.

  3. HupW Protease Specifically Required for Processing of the Catalytic Subunit of the Uptake Hydrogenase in the Cyanobacterium Nostoc sp. Strain PCC 7120

    Science.gov (United States)

    Lindberg, Pia; Devine, Ellenor; Stensjö, Karin

    2012-01-01

    The maturation process of [NiFe] hydrogenases includes a proteolytic cleavage of the large subunit. We constructed a mutant of Nostoc strain PCC 7120 in which hupW, encoding a putative hydrogenase-specific protease, is inactivated. Our results indicate that the protein product of hupW selectively cleaves the uptake hydrogenase in this cyanobacterium. PMID:22020512

  4. Anoxia- and hypoxia-induced expression of LDH-A* in the Amazon Oscar, Astronotus crassipinis

    Directory of Open Access Journals (Sweden)

    Vera Maria Fonseca Almeida-Val

    2011-01-01

    Full Text Available Adaptation or acclimation to hypoxia occurs via the modulation of physiologically relevant genes, such as erythropoietin, transferrin, vascular endothelial growth factor, phosphofructokinase and lactate dehydrogenase A. In the present study, we have cloned, sequenced and examined the modulation of the LDH-A gene after an Amazonian fish species, Astronotus crassipinis (the Oscar, was exposed to hypoxia and anoxia. In earlier studies, we have discovered that adults of this species are extremely tolerant to hypoxia and anoxia, while the juveniles are less tolerant. Exposure of juveniles to acute hypoxia and anoxia resulted in increased LDH-A gene expression in skeletal and cardiac muscles. When exposed to graded hypoxia juveniles show decreased LDH-A expression. In adults, the levels of LDH-A mRNA did not increase in hypoxic or anoxic conditions. Our results demonstrate that, when given time for acclimation, fish at different life-stages are able to respond differently to survive hypoxic episodes.

  5. Investigation of the electrochemical and photoelectrochemical properties of Ni-Al LDH photocatalysts.

    Science.gov (United States)

    Iguchi, Shoji; Kikkawa, Soichi; Teramura, Kentaro; Hosokawa, Saburo; Tanaka, Tsunehiro

    2016-05-18

    Layered double hydroxide (LDH) photocatalysts, including Ni-Al LDH, are active for the photocatalytic conversion of CO2 in water under UV light irradiation. In this study, we found that a series of LDHs exhibited anodic photocurrent which is a characteristic feature corresponding to n-type materials. Also, we estimated the potentials of photogenerated electrons and holes for LDHs, which are responsible for the photocatalytic reactions, using electrochemical techniques. The flat band potential of the Ni-Al LDH photocatalyst was estimated to be -0.40 V vs. NHE (pH = 0), indicating that the potential of the photogenerated electron is sufficient to reduce CO2 to CO. Moreover, we revealed that the flat band potentials of M(2+)-M(3+) LDH are clearly influenced by the type of trivalent metal (M(3+)) components.

  6. New insights into [FeFe] hydrogenase activation and maturase function.

    Directory of Open Access Journals (Sweden)

    Jon M Kuchenreuther

    Full Text Available [FeFe] hydrogenases catalyze H(2 production using the H-cluster, an iron-sulfur cofactor that contains carbon monoxide (CO, cyanide (CN(-, and a dithiolate bridging ligand. The HydE, HydF, and HydG maturases assist in assembling the H-cluster and maturing hydrogenases into their catalytically active form. Characterization of these maturases and in vitro hydrogenase activation methods have helped elucidate steps in the H-cluster biosynthetic pathway such as the HydG-catalyzed generation of the CO and CN(- ligands from free tyrosine. We have refined our cell-free approach for H-cluster synthesis and hydrogenase maturation by using separately expressed and purified HydE, HydF, and HydG. In this report, we illustrate how substrates and protein constituents influence hydrogenase activation, and for the first time, we show that each maturase can function catalytically during the maturation process. With precise control over the biomolecular components, we also provide evidence for H-cluster synthesis in the absence of either HydE or HydF, and we further show that hydrogenase activation can occur without exogenous tyrosine. Given these findings, we suggest a new reaction sequence for the [FeFe] hydrogenase maturation pathway. In our model, HydG independently synthesizes an iron-based compound with CO and CN(- ligands that is a precursor to the H-cluster [2Fe](H subunit, and which we have termed HydG-co. We further propose that HydF is a transferase that stabilizes HydG-co and also shuttles the complete [2Fe](H subcluster to the hydrogenase, a translocation process that may be catalyzed by HydE. In summary, this report describes the first example of reconstructing the [FeFe] hydrogenase maturation pathway using purified maturases and subsequently utilizing this in vitro system to better understand the roles of HydE, HydF, and HydG.

  7. Molecular and kinetic properties of sperm specific LDH after radiation inactivation.

    Science.gov (United States)

    Gupta, G S; Kang, B P

    2000-03-01

    Radiation inactivation of sperm specific lactate dehydrogenase-C4 (LDH-C4) has been studied and compared with the somatic LDH in aqueous solution. D37 of C isozyme was 470 Gy and that of B isozyme was 520 Gy. Semi-log plots of log N/No versus dose suggested that the inactivation of two LDH isozymes in presence of normal saline follows a single hit kinetics. Target molecular weight calculated by radiation analysis was found as 1.52 x 10(5) gm/mole for LDH-C4 and 1.38 x 10(5) gm/mole for LDH-B4. SDS-PAGE of irradiated enzymes showed a band of 35 kDa but did not indicate the presence of any other extra band, when compared with sham-irradiated enzymes. Chemical kinetics of residual activity following irradiation at D37 showed decrease in Vmax with coenzymes and primary substrates. However, decrease in Km was seen with pyruvate as increasing substrate. Nevertheless, K did not change when NAD+ was the leading substrate for LDH-B4 or LDH-C4. A hyperchromicity in intrinsic fluorescence and a blue shift in lambdamax over sham-irradiated LDH-C4 revealed the exposure of buried tryptophan residues to the surface after radiation inactivation. Results suggest that inspite of presence of variant amino acids, the conformations of two isozymes are stabilized by similar forces which behave in a similar way for radiation inactivation in aqueous phase.

  8. Pleural LDH as a prognostic marker in adenocarcinoma lung with malignant pleural effusion

    OpenAIRE

    Verma, Akash; Phua, Chee Kiang; Sim, Wen Yuan; Algoso, Reyes Elmer; Tee, Kuan Sen; Lew, Sennen J. W.; Lim, Albert Y.H.; Goh, Soon Keng; Tai, Dessmon Y. H.; Kor, Ai Ching; Ho, Benjamin; Abisheganaden, John

    2016-01-01

    Abstract To study the performance of serum and pleural lactate dehydrogenase (LDH) level in predicting survival in patients with adenocarcinoma lung presenting with malignant pleural effusions (MPE) at initial diagnosis. Retrospective cohort study of the patient hospitalized for adenocarcinoma lung with MPE in year 2012. Univariate analyses showed lower pleural fluid LDH 667 (313–967) versus 971 (214–3800), P = 0.04, female gender 9 (100%) versus 27 (41.5%), P = 0.009, never smoking status 9 ...

  9. Effect of interlayer anions on [NiFe]-LDH nanosheet water oxidation activity

    OpenAIRE

    Hunter, B. M.; Hieringer, W.; Winkler, J.R.; Gray, H B; Müller, A. M.

    2016-01-01

    We synthesized nickel–iron layered double hydroxide ([NiFe]-LDH) nanosheets with different interlayer anions to probe their role in water oxidation catalysis. In alkaline electrolyte in ambient air, carbonate rapidly replaced other interlayer anions and catalytic activity was highest. Electrocatalytic water oxidation in virtually carbonate-free alkaline electrolyte revealed that activity was a function of anion basicity. Our [NiFe]-LDH nanosheets, prepared by pulsed laser ablation in liquids,...

  10. Protein engineering applications of industrially exploitable enzymes: Geobacillus stearothermophilus LDH and Candida methylica FDH.

    Science.gov (United States)

    Karagüler, N G; Sessions, R B; Binay, B; Ordu, E B; Clarke, A R

    2007-12-01

    Enzymes have become important tools in several industries due to their ability to produce chirally pure and complex molecules with interesting biological properties. The NAD(+)-dependent LDH (lactate dehydrogenase) [bsLDH [Geobacillus stearothermophilus (formerly Bacillus stearothermophilus) LDH] from G. stearothermophilus and the NAD(+)-dependent FDH (formate dehydrogenase) [cmFDH (Candida methylica FDH)] enzyme from C. methylica are particularly crucial enzymes in the pharmaceutical industry and are related to each other in terms of NADH use and regeneration. LDH catalyses the interconversion of pyruvate (oxo acid) and lactate (alpha-hydroxy acid) using the NADH/NAD(+) pair as a redox cofactor. Employing LDH to reduce other oxo acids can generate chirally pure alpha-hydroxy acids of use in the production of pharmaceuticals. One important use of FDH is to regenerate the relatively expensive NADH cofactor that is used by NAD(+)-dependent oxidoreductases such as LDH. Both LDH and FDH from organisms of interest were previously cloned and overproduced. Therefore they are available at a low cost. However, both of these enzymes show disadvantages in the large-scale production of chirally pure compounds. We have applied two routes of protein engineering studies to improve the properties of these two enzymes, namely DNA shuffling and site-directed mutagenesis. Altering the substrate specificity of bsLDH by DNA shuffling and changing the coenzyme specificity of cmFDH by site-directed mutagenesis are the most successful examples of our studies. The present paper will also include the details of these examples together with some other applications of protein engineering regarding these enzymes.

  11. Three different [NiFe] hydrogenases confer metabolic flexibility in the obligate aerobe Mycobacterium smegmatis.

    Science.gov (United States)

    Berney, Michael; Greening, Chris; Hards, Kiel; Collins, Desmond; Cook, Gregory M

    2014-01-01

    Mycobacterium smegmatis is an obligate aerobe that harbours three predicted [NiFe] hydrogenases, Hyd1 (MSMEG_2262–2263), Hyd2 (MSMEG_2720-2719) and Hyd3 (MSMEG_3931-3928). We show here that these three enzymes differ in their phylogeny, regulation and catalytic activity. Phylogenetic analysis revealed that Hyd1 groups with hydrogenases that oxidize H2 produced by metabolic processes, and Hyd2 is homologous to a novel group of putative high-affinity hydrogenases. Hyd1 and Hyd2 respond to carbon and oxygen limitation, and, in the case of Hyd1, hydrogen supplementation. Hydrogen consumption measurements confirmed that both enzymes can oxidize hydrogen. In contrast, the phylogenetic analysis and activity measurements of Hyd3 are consistent with the enzyme evolving hydrogen. Hyd3 is controlled by DosR, a regulator that responds to hypoxic conditions. The strict dependence of hydrogen oxidation of Hyd1 and Hyd2 on oxygen suggests that the enzymes are oxygen tolerant and linked to the respiratory chain. This unique combination of hydrogenases allows M. smegmatis to oxidize hydrogen at high (Hyd1) and potentially tropospheric (Hyd2) concentrations, as well as recycle reduced equivalents by evolving hydrogen (Hyd3). The distribution of these hydrogenases throughout numerous soil and marine species of actinomycetes suggests that oxic hydrogen metabolism provides metabolic flexibility in environments with changing nutrient fluxes.

  12. Hydrogenase Gene Distribution and H2 Consumption Ability within the Thiomicrospira Lineage.

    Science.gov (United States)

    Hansen, Moritz; Perner, Mirjam

    2016-01-01

    Thiomicrospira were originally characterized as sulfur-oxidizing chemolithoautotrophs. Attempts to grow them on hydrogen failed for many years. Only recently we demonstrated hydrogen consumption among two of three tested Thiomicrospira and posited that hydrogen consumption may be more widespread among Thiomicrospira than previously assumed. Here, we investigate and compare the hydrogen consumption ability and the presence of group 1 [NiFe]-hydrogenase genes (enzyme catalyzes H2↔2H(+) + 2e(-)) for sixteen different Thiomicrospira species. Seven of these Thiomicrospira species encoded group 1 [NiFe]-hydrogenase genes and five of these species could also consume hydrogen. All Thiomicrospira species exhibiting hydrogen consumption were from hydrothermal vents along the Mid-Atlantic ridge or Eastern Pacific ridges. The tested Thiomicrospira from Mediterranean and Western Pacific vents could not consume hydrogen. The [NiFe]-hydrogenase genes were categorized into two clusters: those resembling the hydrogenase from Hydrogenovibrio are in cluster I and are related to those from Alpha- and other Gammaproteobacteria. In cluster II, hydrogenases found exclusively in Thiomicrospira crunogena strains are combined and form a monophyletic group with those from Epsilonproteobacteria suggesting they were acquired through horizontal gene transfer. Hydrogen consumption appears to be common among some Thiomicrospira, given that five of the tested sixteen strains carried this trait. The hydrogen consumption ability expands their competitiveness within an environment.

  13. Hydrogenase gene distribution and H2 consumption ability within the Thiomicrospira lineage

    Directory of Open Access Journals (Sweden)

    Moritz eHansen

    2016-02-01

    Full Text Available Thiomicrospira were originally characterized as sulfur-oxidizing chemolithoautotrophs. Attempts to grow them on hydrogen failed for many years. Only recently we demonstrated hydrogen consumption among two of three tested Thiomicrospira and posited that hydrogen consumption may be more widespread among Thiomicrospira than previously assumed. Here, we investigate and compare the hydrogen consumption ability and the presence of group 1 [NiFe]-hydrogenase genes (enzyme catalyzes H22H+ + 2e- for sixteen different Thiomicrospira species. Seven of these Thiomicrospira species encoded group 1 [NiFe]-hydrogenase genes and five of these species could also consume hydrogen. All Thiomicrospira species exhibiting hydrogen consumption were from hydrothermal vents along the Mid-Atlantic ridge or Eastern Pacific ridges. The tested Thiomicrospira from Mediterranean and Western Pacific vents could not consume hydrogen. The [NiFe]-hydrogenase genes were categorized into two clusters: those resembling the hydrogenase from Hydrogenovibrio are in cluster I and are related to those from Alpha- and other Gammaproteobacteria. In cluster II, hydrogenases found exclusively in T. crunogena strains are combined and form a monophyletic group with those from Epsilonproteobacteria suggesting they were acquired through horizontal gene transfer. Hydrogen consumption appears to be common among some Thiomicrospira, given that five of the tested sixteen strains carried this trait. The hydrogen consumption ability expands their competitiveness within an environment.

  14. Characterization of the oxygen tolerance of a hydrogenase linked to a carbon monoxide oxidation pathway in Rubrivivax gelatinosus.

    Science.gov (United States)

    Maness, Pin-Ching; Smolinski, Sharon; Dillon, Anne C; Heben, Michael J; Weaver, Paul F

    2002-06-01

    A hydrogenase linked to the carbon monoxide oxidation pathway in Rubrivivax gelatinosus displays tolerance to O2. When either whole-cell or membrane-free partially purified hydrogenase was stirred in full air (21% O2, 79% N2), its H2 evolution activity exhibited a half-life of 20 or 6 h, respectively, as determined by an anaerobic assay using reduced methyl viologen. When the partially purified hydrogenase was stirred in an atmosphere containing either 3.3 or 13% O2 for 15 min and evaluated by a hydrogen-deuterium (H-D) exchange assay, nearly 80 or 60% of its isotopic exchange rate was retained, respectively. When this enzyme suspension was subsequently returned to an anaerobic atmosphere, more than 90% of the H-D exchange activity was recovered, reflecting the reversibility of this hydrogenase toward O2 inactivation. Like most hydrogenases, the CO-linked hydrogenase was extremely sensitive to CO, with 50% inhibition occurring at 3.9 microM dissolved CO. Hydrogen production from the CO-linked hydrogenase was detected when ferredoxins of a prokaryotic source were the immediate electron mediator, provided they were photoreduced by spinach thylakoid membranes containing active water-splitting activity. Based on its appreciable tolerance to O2, potential applications of this hydrogenase are discussed.

  15. Synthesis and morphological modification of semiconducting Mg(Zn)Al(Ga)–LDH/ITO thin films

    Energy Technology Data Exchange (ETDEWEB)

    Valente, Jaime S., E-mail: jsanchez@imp.mx [Instituto Mexicano del Petróleo, Eje Central # 152, 07730 México D.F. (Mexico); López-Salinas, Esteban [Instituto Mexicano del Petróleo, Eje Central # 152, 07730 México D.F. (Mexico); Prince, Julia [Universidad Anáhuac México Norte, Av. Universidad Anáhuac # 46, Huixquilucan, Edo. de México 52786 (Mexico); González, Ignacio; Acevedo-Peña, Prospero [Universidad Autónoma Metropolitana-Iztapalapa, Departamento de Química, Apdo. Postal 55-534, 09340 México D.F. (Mexico); Ángel, Paz del [Instituto Mexicano del Petróleo, Eje Central # 152, 07730 México D.F. (Mexico)

    2014-09-15

    Layered double hydroxide (LDH) thin films with different chemical compositions (MgZnAl, MgZnGa, MgGaAl) and varying thicknesses were easily prepared by sol–gel method followed by dip-coating. Films were chemically uniform, transparent and well adhered to a conductive indium tin oxide (ITO) substrate. Structure, chemical composition and morphology of the thin films were characterized by XRD-GADDS, SEM-EDS and AFM. Additionally, the semiconducting properties of all the prepared films were studied through the Mott–Schottky relationship; such properties were closely related to the chemical compositions of the film. The films were characterized after electrochemical treatment and important modifications regarding surface morphology, particle and crystal sizes were observed. An in-depth study was conducted in order to investigate the effect of several different electrochemical treatments on the morphology, particle size distribution and crystal size of LDH thin films. Upon electrochemical treatment, the films' surface became smooth and the particles forming the films were transformed from flaky open LDH platelets to uniformly distributed close-packed LDH nanoparticles. - Highlights: • Semiconducting Mg(Zn)Al(Ga)–LDH/ITO thin films prepared by sol–gel. • LDH thin films show a turbostratic morphology made up of porous flakes. • Electrochemical treatments change the flaky structure into a nanoparticle array.

  16. The Actual Role of LDH as Tumor Marker, Biochemical and Clinical Aspects.

    Science.gov (United States)

    Jurisic, Vladimir; Radenkovic, Sandra; Konjevic, Gordana

    2015-01-01

    Lactate dehydrogenase (LDH) among many biochemical parameters represents a very valuable enzyme in patients with cancer with possibility for easy routine measurement in many clinical laboratories. Previous studies where mostly based on investigated LDH in serum of patients with cancer with aims to estimate their clinical significance. The new directions in investigation of LDH where based on the principle that tumor cells release intracellular enzymes trough damaged cell membrane, that is mostly consequence in intracellular mitochondrial machinery alteration, and apoptosis deregulation. This consideration can be used not only in-vitro assays, but also in respect to clinical characteristics of tumor patients. Based on new techniques of molecular biology it is shown that intracellular characteristics of LDH enzyme are very sensitive indicators of the cellular metabolic state, aerobic or anaerobic direction of glycolysis, activation status and malignant transformation. Using different molecular analyses it is very useful to analyzed intracellular LDH activity in different cell line and tumor tissues obtained from patients, not only to understanding complexity in cancer biochemistry but also in early clinical diagnosis. Based on understandings of the LDH altered metabolism, new therapy option is created with aims to blocking certain metabolic pathways and stop tumors growth.

  17. Pleural LDH as a prognostic marker in adenocarcinoma lung with malignant pleural effusion.

    Science.gov (United States)

    Verma, Akash; Phua, Chee Kiang; Sim, Wen Yuan; Algoso, Reyes Elmer; Tee, Kuan Sen; Lew, Sennen J W; Lim, Albert Y H; Goh, Soon Keng; Tai, Dessmon Y H; Kor, Ai Ching; Ho, Benjamin; Abisheganaden, John

    2016-06-01

    To study the performance of serum and pleural lactate dehydrogenase (LDH) level in predicting survival in patients with adenocarcinoma lung presenting with malignant pleural effusions (MPE) at initial diagnosis.Retrospective cohort study of the patient hospitalized for adenocarcinoma lung with MPE in year 2012.Univariate analyses showed lower pleural fluid LDH 667 (313-967) versus 971 (214-3800), P = 0.04, female gender 9 (100%) versus 27 (41.5%), P = 0.009, never smoking status 9 (100%) versus 36 (55.3%), P = 0.009, and epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) therapy 8 (89%) versus 26 (40%), P = 0.009 to correlate with survival of more than 1.7 year versus less than 1.7 year. In multivariate analysis, low pleural fluid LDH and female gender maintained significance. The pleural LDH level of ≤1500 and >1500 U/L discriminated significantly (P = 0.009) between survival.High pleural LDH (>1500 IU/L) predicts shorter survival (less than a year) in patients with adenocarcinoma lung presenting with MPE at the time of initial diagnosis. This marker may be clinically applied for selecting therapeutic modality directed at prevention of reaccumulation of MPE. Patients with low pleural LDH may be considered suitable for measures that provide more sustained effect on prevention of reaccumulation such as chemical pleurodesis or tunneled pleural catheter.

  18. LDH Concentration in Nasal-Wash Fluid as a Biochemical Predictor of Bronchiolitis Severity

    Science.gov (United States)

    Laham, Federico R.; Trott, Amanda A.; Bennett, Berkeley L.; Kozinetz, Claudia A.; Jewell, Alan M.; Garofalo, Roberto P.; Piedra, Pedro A.

    2011-01-01

    Objective Because the decision to hospitalize an infant with bronchiolitis is often supported by subjective criteria and objective indicators of bronchiolitis severity are lacking, we tested the hypothesis that lactate dehydrogenase (LDH), which is released from injured cells, is a useful biochemical indicator of bronchiolitis severity. Patients and Methods We retrospectively analyzed a study of children bronchiolitis. Demographic, clinical information, nasal-wash (NW) and serum specimens were obtained. NW samples were analyzed for respiratory viruses, caspase 3/7 activity and a panel of cytokines and chemokines. Total LDH activity was tested in NW samples and sera. Results Of 101 enrolled children (median age, 5.6 months), 98 had NW specimens available. A viral etiology was found in 82 patients (83.6%), with respiratory syncytial virus (RSV) (66%) and rhinovirus (19%) being the most common viruses detected. Concentrations of LDH in NW specimens were independent from those in sera, and were higher in children with RSV infection or with dual infection. Significant correlations were found between NW LDH and NW cytokines/chemokines. Similarly, NW LDH correlated with NW-caspase 3/7 activity (r=0.75; Pbronchiolitis varied according to viral etiology and disease severity. Values in the upper quartile were associated with ~80% risk reduction in hospitalization, likely reflecting a robust antiviral response. NW LDH may be a useful biomarker to assist the clinician in the decision to hospitalize a child with bronchiolitis. PMID:20100751

  19. Inhibition of hydrogen uptake in Escherichia coli by expressing the hydrogenase from the cyanobacterium Synechocystis sp. PCC 6803

    Directory of Open Access Journals (Sweden)

    Wood Thomas K

    2007-05-01

    Full Text Available Abstract Background Molecular hydrogen is an environmentally-clean fuel and the reversible (bi-directional hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 as well as the native Escherichia coli hydrogenase 3 hold great promise for hydrogen generation. These enzymes perform the simple reaction 2H+ + 2e- ↔ H2 (g. Results Hydrogen yields were enhanced up to 41-fold by cloning the bidirectional hydrogenase (encoded by hoxEFUYH from the cyanobacterium into E. coli. Using an optimized medium, E. coli cells expressing hoxEFUYH also produced twice as much hydrogen as the well-studied Enterobacter aerogenes HU-101, and hydrogen gas bubbles are clearly visible from the cultures. Overexpression of HoxU alone (small diaphorase subunit accounts for 43% of the additional hydrogen produced by HoxEFUYH. In addition, hydrogen production in E. coli mutants with defects in the native formate hydrogenlyase system show that the cyanobacterial hydrogenase depends on both the native E. coli hydrogenase 3 as well as on its maturation proteins. Hydrogen absorption by cells expressing hoxEFUYH was up to 10 times lower than cells which lack the cloned cyanobacterial hydrogenase; hence, the enhanced hydrogen production in the presence of hoxEFUYH is due to inhibition of hydrogen uptake activity in E. coli. Hydrogen uptake by cells expressing hoxEFUYH was suppressed in three wild-type strains and in two hycE mutants but not in a double mutant defective in hydrogenase 1 and hydrogenase 2; hence, the active cyanobacterial locus suppresses hydrogen uptake by hydrogenase 1 and hydrogenase 2 but not by hydrogenase 3. Differential gene expression indicated that overexpression of HoxEFUYH does not alter expression of the native E. coli hydrogenase system; instead, biofilm-related genes are differentially regulated by expression of the cyanobacterial enzymes which resulted in 2-fold elevated biofilm formation. This appears to be the first enhanced hydrogen production

  20. Exfoliation and dispersion of LDH modified with N-tetrabromophthaloyl-glutamic in poly(vinyl alcohol): Morphological and thermal studies

    Indian Academy of Sciences (India)

    Shadpour Mallakpour; Mohammad Dinari; Meysam Talebi

    2015-03-01

    In this study, modified LDH (M-LDH) was synthesized by the co-precipitation reaction of Al(NO3)3.9H2O, Mg(NO3)2.6H2O and N-tetrabromophthaloyl-glutamic (Br-Gl) in short time. M-LDH nanofillers were embedded into poly(vinyl alcohol) (PVA) matrix via ultrasonic irradiation technique which provided PVA/M-LDH NCs. The morphology and structure of the prepared samples were characterized by Fourier transform infrared spectroscopy, thermogravimetric analysis (TGA), field emission scanning electron microscopy and X-ray diffraction techniques TGA of the PVA/M-LDH NCs showed that the thermal stability was improved. The enhancement of thermal properties was ascribed to the homogeneous and good dispersion of M-LDH sheets in the PVA matrix by hydrogen bonding between O–H groups of PVA and the M-LDH.

  1. Flow-FISH analysis and isolation of clostridial strains in an anaerobic semi-solid bio-hydrogen producing system by hydrogenase gene target.

    Science.gov (United States)

    Jen, Chang Jui; Chou, Chia-Hung; Hsu, Ping-Chi; Yu, Sian-Jhong; Chen, Wei-En; Lay, Jiunn-Jyi; Huang, Chieh-Chen; Wen, Fu-Shyan

    2007-04-01

    By using hydrogenase gene-targeted polymerase chain reaction (PCR) and reverse transcriptase PCR (RT-PCR), the predominant clostridial hydrogenase that may have contributed to biohydrogen production in an anaerobic semi-solid fermentation system has been monitored. The results revealed that a Clostridium pasteurianum-like hydrogenase gene sequence can be detected by both PCR and RT-PCR and suggested that the bacterial strain possessing this specific hydrogenase gene was dominant in hydrogenase activity and population. Whereas another Clostridium saccharobutylicum-like hydrogenase gene can be detected only by RT-PCR and suggest that the bacterial strain possessing this specific hydrogenase gene may be less dominant in population. In this study, hydrogenase gene-targeted fluorescence in situ hybridization (FISH) and flow cytometry analysis confirmed that only 6.6% of the total eubacterial cells in a hydrogen-producing culture were detected to express the C. saccharobutylicum-like hydrogenase, whereas the eubacteria that expressed the C. pasteurianum-like hydrogenase was 25.6%. A clostridial strain M1 possessing the identical nucleotide sequences of the C. saccharobutylicum-like hydrogenase gene was then isolated and identified as Clostridium butyricum based on 16S rRNA sequence. Comparing to the original inoculum with mixed microflora, either using C. butyricum M1 as the only inoculum or co-culturing with a Bacillus thermoamylovorans isolate will guarantee an effective and even better production of hydrogen from brewery yeast waste.

  2. Improving cyanobacterail O2-tolerance using CBS hydrogenase for hydrogen production

    Energy Technology Data Exchange (ETDEWEB)

    Maness, Pin-Ching [National Renewable Energy Lab. (NREL), Golden, CO (United States); Eckert, Carrie [National Renewable Energy Lab. (NREL), Golden, CO (United States); Wawrousek, Karen [National Renewable Energy Lab. (NREL), Golden, CO (United States); Noble, Scott [National Renewable Energy Lab. (NREL), Golden, CO (United States); Pennington, Grant [National Renewable Energy Lab. (NREL), Golden, CO (United States); Yu, Jianping [National Renewable Energy Lab. (NREL), Golden, CO (United States)

    2016-11-11

    Cyanobacterial H2 production is a viable path to renewable H2 with water serving as the electron donor and sunlight the energy source. A grand challenge is the sensitivity of the underlying hydrogenase to O2, the latter an inherent byproduct of oxygenic photosynthesis. This challenge has been identified as a technical barrier in the Fuel Cell Technologies Office (FCTO) Multi-year Research, Development and Deployment Plan. One solution is to express in cyanobacterium an O2-tolerant hydrogenase to circumvent this barrier. We have uncovered an O2-tolerant hydrogenase from a photosynthetic bacterium Rubrivivax gelatinosus CBS (Casa Bonita Strain; hereafter “CBS”) with a half-life near 21 h when exposed to ambient O2. We sequenced the CBS genome and identified two sets of maturation machineries hyp1 and hyp2. Transcripts expression analysis and mutagenesis revealed that hyp1 is responsible for the assembly of the O2-tolerant CO-oxidation (Coo) hydrogenase and hyp2 is involved in the maturation of a H2-uptake hydrogenase. The structural genes encoding the O2-tolerant hydrogenase (cooLXUH) and maturation genes hyp1FABCDE were therefore cloned and expressed in the model cyanobacterium Synechocystis sp. PCC 6803. We obtained several recombinants displaying hydrogenase activity in a Synechocystis host lacking background activity, suggesting that the CBS hydrogenase is active in Synechocystis. Yet the activity is extremely low. To ensure balanced protein expression, we systematically optimized heterologous expression of 10 CBS genes by using stronger promoters and better ribosome binding site. Moreover we attempted the expression of cooM and cooK genes, verified to be important in CBS to afford activity. CooM is a very large protein and both CooM and CooK are membrane-associated. These properties limited our success in expressing both genes in Synechocystis, although they

  3. Hydrogen production by a hyperthermophilic membrane-bound hydrogenase in water-soluble nanolipoprotein particles.

    Science.gov (United States)

    Baker, Sarah E; Hopkins, Robert C; Blanchette, Craig D; Walsworth, Vicki L; Sumbad, Rhoda; Fischer, Nicholas O; Kuhn, Edward A; Coleman, Matt; Chromy, Brett A; Létant, Sonia E; Hoeprich, Paul D; Adams, Michael W W; Henderson, Paul T

    2009-06-10

    Hydrogenases constitute a promising class of enzymes for ex vivo hydrogen production. Implementation of such applications is currently hindered by oxygen sensitivity and, in the case of membrane-bound hydrogenases (MBHs), poor water solubility. Nanolipoprotein particles (NLPs) formed from apolipoproteins and phospholipids offer a novel means of incorporating MBHs into a well-defined water-soluble matrix that maintains the enzymatic activity and is amenable to incorporation into more complex architectures. We report the synthesis, hydrogen-evolving activity, and physical characterization of the first MBH-NLP assembly. This may ultimately lead to the development of biomimetic hydrogen-production devices.

  4. Hydrogen Production by a Hyperthermophilic Membrane-Bound Hydrogenase in Soluble Nanolipoprotein Particles

    Energy Technology Data Exchange (ETDEWEB)

    Baker, S E; Hopkins, R C; Blanchette, C; Walsworth, V; Sumbad, R; Fischer, N; Kuhn, E; Coleman, M; Chromy, B; Letant, S; Hoeprich, P; Adams, M W; Henderson, P T

    2008-10-22

    Hydrogenases constitute a promising class of enzymes for ex vivo hydrogen production. Implementation of such applications is currently hindered by oxygen sensitivity and, in the case of membrane-bound hydrogenases (MBH), poor water solubility. Nanolipoprotein particles (NLPs), formed from apolipoproteins and phospholipids, offer a novel means to incorporate MBH into in a well-defined water-soluble matrix that maintains the enzymatic activity and is amenable to incorporation into more complex architectures. We report the synthesis, hydrogen-evolving activity and physical characterization of the first MBH-NLP assembly. This may ultimately lead to the development of biomimetic hydrogen production devices.

  5. AMPKα1-LDH pathway regulates muscle stem cell self-renewal by controlling metabolic homeostasis.

    Science.gov (United States)

    Theret, Marine; Gsaier, Linda; Schaffer, Bethany; Juban, Gaëtan; Ben Larbi, Sabrina; Weiss-Gayet, Michèle; Bultot, Laurent; Collodet, Caterina; Foretz, Marc; Desplanches, Dominique; Sanz, Pascual; Zang, Zizhao; Yang, Lin; Vial, Guillaume; Viollet, Benoit; Sakamoto, Kei; Brunet, Anne; Chazaud, Bénédicte; Mounier, Rémi

    2017-07-03

    Control of stem cell fate to either enter terminal differentiation versus returning to quiescence (self-renewal) is crucial for tissue repair. Here, we showed that AMP-activated protein kinase (AMPK), the master metabolic regulator of the cell, controls muscle stem cell (MuSC) self-renewal. AMPKα1(-/-) MuSCs displayed a high self-renewal rate, which impairs muscle regeneration. AMPKα1(-/-) MuSCs showed a Warburg-like switch of their metabolism to higher glycolysis. We identified lactate dehydrogenase (LDH) as a new functional target of AMPKα1. LDH, which is a non-limiting enzyme of glycolysis in differentiated cells, was tightly regulated in stem cells. In functional experiments, LDH overexpression phenocopied AMPKα1(-/-) phenotype, that is shifted MuSC metabolism toward glycolysis triggering their return to quiescence, while inhibition of LDH activity rescued AMPKα1(-/-) MuSC self-renewal. Finally, providing specific nutrients (galactose/glucose) to MuSCs directly controlled their fate through the AMPKα1/LDH pathway, emphasizing the importance of metabolism in stem cell fate. © 2017 The Authors.

  6. Physical and functional association of lactate dehydrogenase (LDH) with skeletal muscle mitochondria.

    Science.gov (United States)

    Elustondo, Pia A; White, Adrienne E; Hughes, Meghan E; Brebner, Karen; Pavlov, Evgeny; Kane, Daniel A

    2013-08-30

    The intracellular lactate shuttle hypothesis posits that lactate generated in the cytosol is oxidized by mitochondrial lactate dehydrogenase (LDH) of the same cell. To examine whether skeletal muscle mitochondria oxidize lactate, mitochondrial respiratory oxygen flux (JO2) was measured during the sequential addition of various substrates and cofactors onto permeabilized rat gastrocnemius muscle fibers, as well as isolated mitochondrial subpopulations. Addition of lactate did not alter JO2. However, subsequent addition of NAD(+) significantly increased JO2, and was abolished by the inhibitor of mitochondrial pyruvate transport, α-cyano-4-hydroxycinnamate. In experiments with isolated subsarcolemmal and intermyofibrillar mitochondrial subpopulations, only subsarcolemmal exhibited NAD(+)-dependent lactate oxidation. To further investigate the details of the physical association of LDH with mitochondria in muscle, immunofluorescence/confocal microscopy and immunoblotting approaches were used. LDH clearly colocalized with mitochondria in intact, as well as permeabilized fibers. LDH is likely localized inside the outer mitochondrial membrane, but not in the mitochondrial matrix. Collectively, these results suggest that extra-matrix LDH is strategically positioned within skeletal muscle fibers to functionally interact with mitochondria.

  7. Physical and Functional Association of Lactate Dehydrogenase (LDH) with Skeletal Muscle Mitochondria*

    Science.gov (United States)

    Elustondo, Pia A.; White, Adrienne E.; Hughes, Meghan E.; Brebner, Karen; Pavlov, Evgeny; Kane, Daniel A.

    2013-01-01

    The intracellular lactate shuttle hypothesis posits that lactate generated in the cytosol is oxidized by mitochondrial lactate dehydrogenase (LDH) of the same cell. To examine whether skeletal muscle mitochondria oxidize lactate, mitochondrial respiratory oxygen flux (JO2) was measured during the sequential addition of various substrates and cofactors onto permeabilized rat gastrocnemius muscle fibers, as well as isolated mitochondrial subpopulations. Addition of lactate did not alter JO2. However, subsequent addition of NAD+ significantly increased JO2, and was abolished by the inhibitor of mitochondrial pyruvate transport, α-cyano-4-hydroxycinnamate. In experiments with isolated subsarcolemmal and intermyofibrillar mitochondrial subpopulations, only subsarcolemmal exhibited NAD+-dependent lactate oxidation. To further investigate the details of the physical association of LDH with mitochondria in muscle, immunofluorescence/confocal microscopy and immunoblotting approaches were used. LDH clearly colocalized with mitochondria in intact, as well as permeabilized fibers. LDH is likely localized inside the outer mitochondrial membrane, but not in the mitochondrial matrix. Collectively, these results suggest that extra-matrix LDH is strategically positioned within skeletal muscle fibers to functionally interact with mitochondria. PMID:23873936

  8. A pH-responsive layered double hydroxide (LDH)-phthalocyanine nanohybrid for efficient photodynamic therapy.

    Science.gov (United States)

    Li, Xing-Shu; Ke, Mei-Rong; Huang, Wei; Ye, Chun-Hong; Huang, Jian-Dong

    2015-02-16

    A pH-responsive nanohybrid (LDH-ZnPcPS4 ), in which a highly hydrophilic zinc(II) phthalocyanine tetra-α-substituted with 4-sulfonatophenoxy groups (ZnPcPS4 ) is incorporated with a cationic layered double hydroxide (LDH) based on electrostatic interaction, has been specially designed and prepared through a facile co-precipitation approach. ZnPcPS4 is an excellent singlet-oxygen generator with strong absorption at the near-infrared region (692 nm) in cellular culture media, whereas the photoactivities of ZnPcPS4 were remarkably inhibited after incorporation with the LDH. The nanohybrid is essentially stable in aqueous media at pH 7.4; nevertheless, in slightly acidic media of pH 6.5 or 5.0, ZnPcPS4 can be efficiently released from the LDH matrix, thus leading to restoration of the photoactivities. The nanohybrid shows a high photocytotoxicity against HepG2 cells as a result of much more efficient cellular uptake and preferential accumulation in lysosomes, whereby the acidic environment leads to the release of ZnPcPS4 . The IC50 value of LDH-ZnPcPS4 is as low as 0.053 μM, which is 24-fold lower than that of ZnPcPS4 . This work provides a facile approach for the fabrication of photosensitizers with high photocytotoxicity, potential tumor selectivity, and rapid clearance character.

  9. Preparation, characterization and antimicrobial applications of Zn-Fe LDH against MRSA.

    Science.gov (United States)

    Moaty, S A Abdel; Farghali, A A; Khaled, Rehab

    2016-11-01

    Facile and simple processes to get Zn-Fe layered double hydroxide (LDH) with nitrate as the interlayer anion are reported. The method of co-precipitation produced high crystallinity LDH that is marked by XRD, SEM, TEM and FT-IR. Results showed that 99.8% of Cd(+2) removals were at pH11 and 4h. To get the adsorption isotherms, the concentration of metal ions extending from 6 to 18mg/L was utilized. Results supported the Langmuir adsorption model. In contrary, the adsorption process followed the pseudo-second-order reaction kinetics. Interestingly, the prepared LDH shows durable antimicrobial activities against Gram-negative (Proteous vulgaris, Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus pyogenes and MRSA) and fungi (Candida albicans, Aspergillus fumigatus, Geotricumcandidum, and Trichophyton mentagrophytes). The minimum inhibitory concentration (MIC) of Zn-Fe LDH varied from 0.49 to 15.60μg/mL according to the types of microorganisms. The prepared LDH achieved 90% at pH8.50 which is the pH of wastewater and at the same time exhibited durable antimicrobial activities against MRSA, Gram-negative, Gram-positive and fungi. Results have significant implications in the field of bioremediation of water with little cost, simple operation, high productivity and easiness of the equipment.

  10. Mutational analysis of the hyc-operon determining the relationship between hydrogenase-3 and NADH pathway in Enterobacter aerogenes.

    Science.gov (United States)

    Pi, Jian; Jawed, Muhammad; Wang, Jun; Xu, Li; Yan, Yunjun

    2016-01-01

    In this study, the hydrogenase-3 gene cluster (hycDEFGH) was isolated and identified from Enterobacter aerogenes CCTCC AB91102. All gene products were highly homologous to the reported bacterial hydrogenase-3 (Hyd-3) proteins. The genes hycE, hycF, hycG encoding the subunits of hydrogenase-3 were targeted for genetic knockout to inhibit the FHL hydrogen production pathway via the Red recombination system, generating three mutant strains AB91102-E (ΔhycE), AB91102-F (ΔhycF) and AB91102-G (ΔhycG). Deletion of the three genes affected the integrity of hydrogenase-3. The hydrogen production experiments with the mutant strains showed that no hydrogen was detected compared with the wild type (0.886 mol/mol glucose), demonstrating that knocking out any of the three genes could inhibit NADH hydrogen production pathway. Meanwhile, the metabolites of the mutant strains were significantly changed in comparison with the wild type, indicating corresponding changes in metabolic flux by mutation. Additionally, the activity of NADH-mediated hydrogenase was found to be nil in the mutant strains. The chemostat experiments showed that the NADH/NAD(+) ratio of the mutant strains increased nearly 1.4-fold compared with the wild type. The NADH-mediated hydrogenase activity and NADH/NAD(+) ratio analysis both suggested that NADH pathway required the involvement of the electron transport chain of hydrogenase-3.

  11. Insights into the synthesis of layered double hydroxide (LDH) nanoparticles: Part 1. Optimization and controlled synthesis of chloride-intercalated LDH.

    Science.gov (United States)

    Sun, Xiaodi; Neuperger, Erica; Dey, Sandwip K

    2015-12-01

    Layered double hydroxide (LDH) nanoparticles have excellent anion-intercalating property, and their potential as theranostic nanovectors is high. However, understanding of the control of the mean particle size (MPS) and achievement of monodispersed particle size distribution (PSD) remains elusive. Herein, with the aid of statistical design of experiments on a model system of Cl(-)-intercalated (Zn, Al)-LDH, controlled synthesis of single crystalline nanoparticles using the coprecipitation method followed by hydrothermal treatment (HT) was achieved in three steps. First, a 2(4-1) design enabled the identification of influential parameters for MPS (i.e., salt concentration, molar ratio of carbonate to aluminum, solution addition rate, and interaction between salt concentration and stirring rate) and PSD (i.e., salt concentration and stirring rate), as well as the optimum coprecipitation conditions that result in a monodispersed PSD (i.e., low salt concentration and high stirring rate). Second, a preliminary explanation of the HT was suggested and the optimum HT conditions for obtaining ideal Gaussian PSD with chi-squared (χ(2))<3 were found to be 85°C for 5 h. Third, using a central composite design, a quantitative MPS model, expressed in terms of the significant factors, was developed and experimentally verified to synthesize nearly monodispersed LDH nanoparticles with MPS ∼200-500 nm.

  12. New way for iron introduction in LDH matrix used as catalysts for Friedel–Crafts reactions

    Directory of Open Access Journals (Sweden)

    S. Kerchiche

    2017-02-01

    Full Text Available The alkylation of toluene, reaction employing benzyl chloride as the alkylating agent over basic hydrotalcite materials: Fe–Mg–Al-LDH prepared by different synthesis methods, including the method of co-precipitation, impregnation and a new method called the method of intercalation by anion exchange in the lamellar space of the host structure LDH. Our prepared solids were characterized by chemical analysis, XRD analysis, BET method and thermogravimetric analysis (TGA and tested in the alkylation of toluene by benzyl chloride reaction. Fe–Mg–Al-LDH clay without or with calcination (at 773 K has been investigated. The catalyst derived from the hydrotalcite by its calcination at 773 K shows high catalytic activity for the alkylation of toluene and other aromatic compounds. The catalytically active species present in the catalyst in its most active form are the oxides of iron on the catalyst surface.

  13. Prognostic impact of LDH levels in patients with relapsed/refractory seminoma.

    Science.gov (United States)

    Powles, Tom; Bascoul-Mollevi, Caroline; Kramar, Andrew; Lorch, Anja; Beyer, Jörg

    2013-08-01

    To evaluate the impact of age and LDH levels in patients with relapsed seminoma. Data on the 204 seminoma from the International Prognostic Factor Study Group (IPFSG) were analyzed. All patients experienced unequivocal relapse/progression after at least three cisplatin-based chemotherapy cycles. Age and LDH at relapse were assessed in addition to previously identified prognostic factors for all germ cell tumor patients from the database (J Clin Oncol 28:4906, 2010). The impact of the IPFSG score remained highly significant in multivariate analysis. In addition, LDH ≥1.5 times the upper limit of normal (ULN) was significant in univariate (HR 1.96; CI 1.06-3.61) and multivariate analysis (HR 1.90; CI 1.00-3.62). Age, however, was not significant. Therefore, LDH was incorporated into a modified new IPFSG seminoma score by moving patients to the next unfavorable group for patients with LDH values ≥1.5 × ULN. Three prognostic groups were thus generated, which better subdivided seminoma patients than the original IPFSG score. Progression-free survival at 2 years: "very low risk" (n = 23) 85.7% (95% CI 62-95), "low risk" (n = 44) 62.7 % (95% CI 46-75) and "intermediate risk" (n = 36) 35.1% (95% CI 20-51). Overall survival at 3 years: "very low risk" 88.8% (95% CI 62-97), "low risk" 71.3% (95% CI 55-83) and "intermediate risk" 51.3% (95% CI 33-67). The addition of LDH, but not age, improves the impact of the IPFSG prognostic score in seminoma patients relapsing or progressing after cisplatin-based chemotherapy.

  14. Mild hypoxia in vivo regulates cardioprotective SUR2A: A role for Akt and LDH.

    Science.gov (United States)

    Mohammed Abdul, Khaja Shameem; Jovanović, Sofija; Du, Qingyou; Sukhodub, Andriy; Jovanović, Aleksandar

    2015-05-01

    High-altitude residents have lower mortality rates for ischaemic heart disease and this is ascribed to cardiac gene remodelling by chronic hypoxia. SUR2A is a cardioprotective ABC protein serving as a subunit of sarcolemmal ATP-sensitive K(+) channels. The purpose of this study was to determine whether SUR2A is regulated by mild hypoxia in vivo and to elucidate the underlying mechanism. Mice were exposed to either 21% (control) or 18% (mild hypoxia) oxygen for 24h. Exposure to 18% oxygen did not affect partial pressure of O(2) (PO(2)) and CO(2) (PCO(2)) in the blood, haematocrit or level of ATP in the heart. However, hypoxia increased myocardial lactate dehydrogenase (LDH) and lactate as well as NAD(+) without affecting total NAD. SUR2A levels were significantly increased as well as myocardial resistance to ischaemia-reperfusion. Exposure to 18% oxygen did not phosphorylate extracellular signal regulated kinases (ERK1/2) or AMP activated protein kinase (AMPK), but it phosphorylated protein kinase B (Akt). An inhibitor of phosphoinositide 3-kinases (PI3K), LY294002 (0.2mg/mouse), abolished all observed effects of hypoxia. LDH inhibitors, galloflavin (50 μM) and sodium oxamate (80 mM) significantly decreased levels of SUR2A in heart embryonic H9c2 cells, while inactive mutant LDH form, gly193-M-LDH increased cellular sensitivity towards stress induced by 2,4-dinitrophenol (10mM). Treatment of H9c2 cells with sodium lactate (30 mM) increased intracellular lactate, but did not affect LDH activity or SUR2A levels. We conclude that PI3K/Akt signalling pathway and LDH play a crucial role in increase of cardiac SUR2A induced by in vivo exposure to 18% oxygen.

  15. Structural and functional synthetic model of mono-iron hydrogenase featuring an anthracene scaffold

    Science.gov (United States)

    Seo, Junhyeok; Manes, Taylor A.; Rose, Michael J.

    2017-06-01

    Mono-iron hydrogenase was the third type of hydrogenase discovered. Its Lewis acidic iron(II) centre promotes the heterolytic cleavage of the H-H bond and this non-redox H2 activation distinguishes it from the well-studied dinuclear [FeFe] and [NiFe] hydrogenases. Cleavage of the H-H bond is followed by hydride transfer to the enzyme's organic substrate, H4MPT+, which serves as a CO2 'carrier' in methanogenic pathways. Here we report a scaffold-based synthetic approach by which to model mono-iron hydrogenase using an anthracene framework, which supports a biomimetic fac-C,N,S coordination motif to an iron(II) centre. This arrangement includes the biomimetic and organometallic Fe-C σ bond, which enables bidirectional activity reminiscent of the native enzyme: the complex activates H2 under mild conditions, and catalyses C-H hydride abstraction plus H2 generation from a model substrate. Notably, neither H2 activation nor C-H hydride abstraction was observed in the analogous complex with a pincer-type mer-C,N,S ligation, emphasizing the importance of the fac-C,N,S-iron(II) motif in promoting enzyme-like reactivity.

  16. Photocatalytic hydrogen production from a simple water-soluble [FeFe]-hydrogenase model system.

    Science.gov (United States)

    Cao, Wei-Ning; Wang, Feng; Wang, Hong-Yan; Chen, Bin; Feng, Ke; Tung, Chen-Ho; Wu, Li-Zhu

    2012-08-21

    Combined with a simple water soluble [FeFe]-hydrogenase mimic 1, Ru(bpy)(3)(2+) and ascorbic acid enable hydrogen production photocatalytically. More than 88 equivalents of H(2) were achieved in water, which is much better than that obtained in an organic solvent or a mixture of organic solvent and water.

  17. Enzymatic recovery of platinum (IV) from industrial wastewater using a biosulphidogenic hydrogenase

    CSIR Research Space (South Africa)

    Rashamuse, KJ

    2008-04-01

    Full Text Available this treatment process suggesting that platinum sulphide was not formed and supporting the argument that the increased amount (78%) of platinum removal from the industrial wastewater by the growing SRB cells was due to more hydrogenase/cytochrome c3 enzyme...

  18. Aniosotropically organized LDH on PVDF: a geometrically templated electrospun substrate for advanced anion conducting membranes.

    Science.gov (United States)

    Sailaja, G S; Zhang, Peilin; Anilkumar, Gopinathan M; Yamaguchi, Takeo

    2015-04-01

    A bioinspired geometric templating of an electrospun PVDF substrate with hexagonal platelets of Mg-Al layered double hydroxide (LDH), an intrinsic anion conductor, is presented. The distinctive morphology restructures the internal pore geometry and modulates the dynamic wetting profile of PVDF, transforming it into a highly functional substrate for SAFC anion conducting membranes. The membrane fabricated with PVDF-LDH substrate exhibited exceptionally high durability (>140 °C), high anionic conductivity, ion exchange capacity (IEC), restricted swelling, and improved tensile strength, overcoming critical challenges associated with PVDF electrospun substrates and validating its immense potential as a high-temperature-stable and durable substrate for advanced fuel cell membrane applications.

  19. Ageing behaviour of unary hydroxides in trivalent metal salt solutions: Formation of layered double hydroxide (LDH)-like phases

    Indian Academy of Sciences (India)

    Michael Rajamathi; P Vishnu Kamath

    2000-10-01

    The hydroxides of Mg, Ni, Cu and Zn transform into layered double hydroxide (LDH)-like phases on ageing in solutions of Al or Cr salts. This reaction is similar to acid leaching and proceeds by a dissolution–reprecipitation mechanism offering a simple method of LDH synthesis, with implications for the accepted theories of formation of LDH minerals in the earth’s crust.

  20. Evolução molecular adaptativa dos genes da família LDH em teleósteos

    OpenAIRE

    Castro, Nayara Sousa

    2015-01-01

    A L-Lactato Desidrogenase (LDH) é uma enzima chave nos processos do metabolismo glicolítico anaeróbico e possui três genes codificadores em peixes (ldh-a, ldh-b e ldh-c). A regulação desses genes é independente, resultando em especificidade tecidual nos vertebrados. Esses genes foram originados por eventos sucessivos de duplicação do genoma e sua evolução mostra-se intimamente relacionada com o desenvolvimento de mecanismos adaptativos térmicos e de condições fisiológicas distintas, as quais ...

  1. Particle-induced artifacts in the MTT and LDH viability assays.

    Science.gov (United States)

    Holder, Amara L; Goth-Goldstein, Regine; Lucas, Donald; Koshland, Catherine P

    2012-09-17

    In vitro testing is a common first step in assessing combustion-generated and engineered nanoparticle-related health hazards. Commercially available viability assays are frequently used to compare the toxicity of different particle types and to generate dose-response data. Nanoparticles, well-known for having large surface areas and chemically active surfaces, may interfere with viability assays, producing a false assessment of toxicity and making it difficult to compare toxicity data. The objective of this study is to measure the extent of particle interference in two common viability assays, the MTT reduction and the lactate dehydrogenase (LDH) release assays. Diesel particles, activated carbon, flame soot, oxidized flame soot, and titanium dioxide particles are assessed for interactions with the MTT and LDH assay under cell-free conditions. Diesel particles, at concentrations as low as 0.05 μg/mL, reduce MTT. Other particle types reduce MTT only at a concentration of 50 μg/mL and higher. The activated carbon, soot, and oxidized soot particles bind LDH to varying extents, reducing the concentration measured in the LDH assay. The interfering effects of the particles explain in part the different toxicities measured in human bronchial epithelial cells (16HBE14o). We conclude that valid particle toxicity assessments can only be assured after first performing controls to verify that the particles under investigation do not interfere with a specific assay at the expected concentrations.

  2. LDH-C can be differentially expressed during fermentation of CHO cells

    Directory of Open Access Journals (Sweden)

    Szperalski Berthold

    2011-11-01

    Full Text Available Abstract Expression of CHO mRNA was measured with special microarrays from the Consortium for Chinese Hamster Ovary (CHO Cell Genomics led by Prof. Wei-Shou Hu of the University of Minnesota and Prof. Miranda Yap of the Bioprocess Technology Institute of A*STAR, Singapore (http://hugroup.cems.umn.edu/CHO/cho_index.html. Cultivation experiments were performed in small scale 2L stirred tank bioreactors. During fermentation a temperature shift of -3°C was performed. This was accompanied by a reduction of the cell specific lactate production rate. The analysis of transcriptome samples before and after the temperature shift with microarrays showed several changes in the expression of available gene markers. LDH-C expression raised about 2 fold after temperature shift. LDH-A did not change. As LDH-C is known to be a specialized isoenzyme in sperm cells for consuming lactate in a lactate containing milieu, LDH-C could be proposed as a target for genetic engineering, facilitating lactate consumption in the late phase of high cell density cultures and prolonging longevity of CHO production cultures by reducing lactate and base accumulation.

  3. Pathohistological changes in kidney and LDH activity in broiler treated with different doses of ochratoxin A

    Directory of Open Access Journals (Sweden)

    Nedeljković-Trailovć Jelena B.

    2005-01-01

    Full Text Available The three-week long trial was performed on day-old Hybro broilers divided into four groups. After 14 days long preexperimental period, the experimental groups were offered feed contaminated with 0.5, 1.0 and 1.5 ppm OA during 7 respectively. At the end of the trial blood and kidney samples were taken for investigations. In broilers feed with 1.5 ppm of OA histopathological examination of the kidney tissue revealed changes located in proximal tubules. Some cells were dim and swollen. These changes produced particular or total reduction in tubular lumen of kidney. Acute tubular necrosis existed in some of tubulocites in form of small foci. Fragmentation of necrotic mass and presence of fresh red blood cells were also detected. The LDH activity was significantly greater in broilers of experimental groups compared with control group. All presented data indicated that intensity of pathohistological alterations and LDH activity depends upon dietary OTA level. Positive correlation between pathohistological changes and increased LDH activity caused by OTA was noticed. Thus, LDH activity mea- sure could be used as early diagnostic tool in measuring changes caused by OTA.

  4. MMT and LDH organo-modification with surfactants tailored for PLA nanocomposites

    Directory of Open Access Journals (Sweden)

    S. Coiai

    2017-03-01

    Full Text Available Low molecular weight polyesters were end-functionalized with ammonium and carboxylate salts and used in ionic exchange reactions with respectively cationic (MMT and anionic (LDH clays. The hybrid organic-inorganic substrates were structurally analysed to determine the ester oligomers’ modification degree and their thermal behaviour owing to confinement effects. The dispersion of such hybrids in polylactic acid (PLA matrix was performed and the ultimate structural, morphological and thermal properties of the collected nanocomposites were investigated and correlated to the tailored interfacial properties with the different inorganic substrates. While the composites with MMT proved to be stable under thermo-oxidative conditions, the samples obtained by dispersing the LDH hybrid suffered from poor final thermostability owing to molecular weights decrease. Deeper insights about the effect of the interactions at interface (polymer chain-surfactant and polymer chain- inorganic surface evidenced that by promoting an intimate contact between PLA chains and LDH surface (through oligoester used as inorganic substrate modifier a certain extent of PLA hydrolysis triggered by both surfactant and inorganic surface (LDH occurred and cannot completely avoided.

  5. Photosynthetic electron partitioning between [FeFe]-hydrogenase and ferredoxin:NADP⁺-oxidoreductase (FNR) enzymes in vitro

    National Research Council Canada - National Science Library

    Iftach Yacoby; Sergii Pochekailov; Hila Toporik; Maria L. Ghirardi; Paul W. King; Shuguang Zhang

    2011-01-01

    .... To elucidate the basis for competition, we bioengineered a ferredoxin-hydrogenase fusion and characterized hydrogen production kinetics in the presence of Fd, ferredoxin:NADP⁺-oxidoreductase (FNR), and NADP...

  6. A redox hydrogel protects the O2 -sensitive [FeFe]-hydrogenase from Chlamydomonas reinhardtii from oxidative damage.

    Science.gov (United States)

    Oughli, Alaa Alsheikh; Conzuelo, Felipe; Winkler, Martin; Happe, Thomas; Lubitz, Wolfgang; Schuhmann, Wolfgang; Rüdiger, Olaf; Plumeré, Nicolas

    2015-10-12

    The integration of sensitive catalysts in redox matrices opens up the possibility for their protection from deactivating molecules such as O2 . [FeFe]-hydrogenases are enzymes catalyzing H2 oxidation/production which are irreversibly deactivated by O2 . Therefore, their use under aerobic conditions has never been achieved. Integration of such hydrogenases in viologen-modified hydrogel films allows the enzyme to maintain catalytic current for H2 oxidation in the presence of O2 , demonstrating a protection mechanism independent of reactivation processes. Within the hydrogel, electrons from the hydrogenase-catalyzed H2 oxidation are shuttled to the hydrogel-solution interface for O2 reduction. Hence, the harmful O2 molecules do not reach the hydrogenase. We illustrate the potential applications of this protection concept with a biofuel cell under H2 /O2 mixed feed.

  7. Serum LDH and CA-125: Markers for Diagnosis of Ovarian Malignancy.

    Science.gov (United States)

    Deeba, F; Khatun, S; Alam, M M; Shahida, S M

    2015-04-01

    This prospective multi-centre study was carried out in the Department of obstetrics and gynaecology of Bangabandhu Sheikh Mujib Medical University, Dhaka Medical College Hospital and Bangladesh Medical College Hospital, Shaheed Suhrawardy Medical College Hospital, Dhaka, Bangladesh, during the period of January 2008 to December 2009, to establish the raised level of serum LDH and serum CA-125 in pre-operative discrimination of benign and malignant ovarian cancer to be used as a diagnostic marker and its validity by determining sensitivity, specificity, accuracy, positive predictive value (PPV) and negative predictive value (NPP). A total number of 141 consecutive suspected subjects of ovarian tumour admitted in the above mentioned hospitals and enrolled for surgical management were included in this study. Serum LDH was done in all these subjects and they were followed up from the admission upto the postoperative tissue diagnosis of live tumor in respective pathology departments for histopathological correlation. The patients who were diagnosed as malignant placed in Group I and diagnosed benign ovarian tumor placed in Group II. Serous cystadenoma and mucinous cyst adenoma were more common in benign tumors, which were 38.9% and 20.4% respectively. However, more than a half (57.1%) had serous cyst adenocarcinoma in malignant tumors. In LDH for evaluation of malignancy, true positive 16 and false positive 18, false negative 12 and true negative 95 cases. LDH and serum CA-125 level (combined, i.e. both positive) for evaluation of malignancy, true positive 14 and false positive 0, false negative 14 and true negative 113 cases. LDH/serum CA-125 level (anyone positive) for evaluation of malignancy, true positive 25 and false positive 37, false negative 3 and true negative 76 cases. The validity of LDH were sensitivity 57.1%, specificity 84.1%, accuracy 78.7%, positive predictive values 47.1% and negative predictive values 88.8% for malignancy of ovarian tumour. The

  8. The correlation between LDH serum levels and clinical outcome in advanced biliary tract cancer patients treated with first line chemotherapy.

    Science.gov (United States)

    Faloppi, Luca; Del Prete, Michela; Casadei Gardini, Andrea; Santini, Daniele; Silvestris, Nicola; Bianconi, Maristella; Giampieri, Riccardo; Valgiusti, Martina; Brunetti, Oronzo; Bittoni, Alessandro; Andrikou, Kalliopi; Lai, Eleonora; Dessì, Alessandra; Cascinu, Stefano; Scartozzi, Mario

    2016-04-11

    LDH may represent an indirect marker of neo-angiogenesis and worse prognosis in many tumour types. We assessed the correlation between LDH and clinical outcome for biliary tract cancer (BTC) patients treated with first-line chemotherapy. Overall, 114 advanced BTC patients treated with first-line gemcitabine and cisplatin were included. Patients were divided into two groups (low vs. high LDH), according to pre-treatment LDH values. Patients were also classified according to pre- and post-treatment variation in LDH serum levels (increased vs. decreased). Median progression free survival (PFS) was 5.0 and 2.6 months respectively in patients with low and high pre-treatment LDH levels (p = 0.0042, HR = 0.56, 95% CI: 0.37-0.87). Median overall survival (OS) was 7.7 and 5.6 months (low vs. high LDH) (p = 0.324, HR = 0.81, 95% CI: 0.54-1.24). DCR was 71% vs. 43% (low vs. high LDH) (p = 0.002). In 38 patients with decreased LDH values after treatment, PFS and OS were respectively 6.2 and 12.1 months, whereas in 76 patients with post-treatment increased LDH levels, PFS and OS were respectively 3.0 and 5.1 months (PFS: p = 0.0009; HR = 0.49; 95% IC: 0.33-0.74; OS: p < 0.0001; HR = 0.42; 95% IC: 0.27-0.63). Our data seem to suggest that LDH serum level may predict clinical outcome in BTC patients receiving first-line chemotherapy.

  9. LDH inhibition impacts on heat shock response and induces senescence of hepatocellular carcinoma cells.

    Science.gov (United States)

    Manerba, Marcella; Di Ianni, Lorenza; Govoni, Marzia; Roberti, Marinella; Recanatini, Maurizio; Di Stefano, Giuseppina

    2017-07-15

    In normal cells, heat shock response (HSR) is rapidly induced in response to a variety of harmful conditions and represents one of the most efficient defense mechanism. In cancer tissues, constitutive activation converts HSR into a life-threatening process, which plays a major role in helping cell survival and proliferation. Overexpression of heat shock proteins (HSPs) has been widely reported in human cancers and was found to correlate with tumor progression. Hepatocellular carcinoma is one of the conditions in which HSR activation was shown to have the highest clinical significance. Transcription of HSPs is induced by HSF-1, which also activates glycolytic metabolism and increases the expression of LDH-A, the master regulator of the Warburg effect. In this paper, we tried to explore the relationship between HSR and LDH-A. In cultured hepatocellular carcinoma cells, by using two enzyme inhibitors (oxamate and galloflavin), we found that the reduction of LDH-A activity led to decreased level and function of the major HSPs involved in tumorigenesis. Galloflavin (a polyphenol) also inhibited the ATPase activity of two of the examined HSPs. Finally, hindering HSR markedly lowered the alpha-fetoprotein cellular levels and induced senescence. Specific inhibitors of single HSPs are currently under evaluation in different neoplastic diseases. However, one of the effects usually observed during treatment is a compensatory elevation of other HSPs, which decreases treatment efficacy. Our results highlight a connection between LDH and HSR and suggest LDH inhibition as a way to globally impact on this tumor promoting process. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Research data supporting "Photoelectrochemical H2 Evolution with a Hydrogenase Immobilized on a TiO2-protected Silicon Electrode"

    OpenAIRE

    Lee, Chong-Yong; Park, Hyun S.; Fontecilla-Camps, Juan C.; Reisner, Erwin

    2016-01-01

    Experimental Raw data supporting publication: Photoelectrochemical H2 Evolution with a Hydrogenase Immobilized on a TiO2-protected Silicon Electrode This research data supports “Photoelectrochemical H2 Evolution with a Hydrogenase Immobilized on a TiO2-protected Silicon Electrode" which has been published in “Angewandte Chemie international edition (English)”. This work was supported by the BBSRC [grant number BB/K010220/1].

  11. Heterologous expression and maturation of an NADP-dependent [NiFe]-hydrogenase: a key enzyme in biofuel production.

    Directory of Open Access Journals (Sweden)

    Junsong Sun

    Full Text Available Hydrogen gas is a major biofuel and is metabolized by a wide range of microorganisms. Microbial hydrogen production is catalyzed by hydrogenase, an extremely complex, air-sensitive enzyme that utilizes a binuclear nickel-iron [NiFe] catalytic site. Production and engineering of recombinant [NiFe]-hydrogenases in a genetically-tractable organism, as with metalloprotein complexes in general, has met with limited success due to the elaborate maturation process that is required, primarily in the absence of oxygen, to assemble the catalytic center and functional enzyme. We report here the successful production in Escherichia coli of the recombinant form of a cytoplasmic, NADP-dependent hydrogenase from Pyrococcus furiosus, an anaerobic hyperthermophile. This was achieved using novel expression vectors for the co-expression of thirteen P. furiosus genes (four structural genes encoding the hydrogenase and nine encoding maturation proteins. Remarkably, the native E. coli maturation machinery will also generate a functional hydrogenase when provided with only the genes encoding the hydrogenase subunits and a single protease from P. furiosus. Another novel feature is that their expression was induced by anaerobic conditions, whereby E. coli was grown aerobically and production of recombinant hydrogenase was achieved by simply changing the gas feed from air to an inert gas (N2. The recombinant enzyme was purified and shown to be functionally similar to the native enzyme purified from P. furiosus. The methodology to generate this key hydrogen-producing enzyme has dramatic implications for the production of hydrogen and NADPH as vehicles for energy storage and transport, for engineering hydrogenase to optimize production and catalysis, as well as for the general production of complex, oxygen-sensitive metalloproteins.

  12. Impact of the chemicals, essential for the purification process of strict Fe-hydrogenase, on the corrosion of mild steel.

    Science.gov (United States)

    Rouvre, Ingrid; Gauquelin, Charles; Meynial-Salles, Isabelle; Basseguy, Régine

    2016-06-01

    The influence of additional chemical molecules, necessary for the purification process of [Fe]-hydrogenase from Clostridium acetobutylicum, was studied on the anaerobic corrosion of mild steel. At the end of the purification process, the pure [Fe-Fe]-hydrogenase was recovered in a Tris-HCl medium containing three other chemicals at low concentration: DTT, dithionite and desthiobiotin. Firstly, mild steel coupons were exposed in parallel to a 0.1 M pH7 Tris-HCl medium with or without pure hydrogenase. The results showed that hydrogenase and the additional molecules were in competition, and the electrochemical response could not be attributed solely to hydrogenase. Then, solutions with additional chemicals of different compositions were studied electrochemically. DTT polluted the electrochemical signal by increasing the Eoc by 35 mV 24 h after the injection of 300 μL of control solutions with DTT, whereas it drastically decreased the corrosion rate by increasing the charge transfer resistance (Rct 10 times the initial value). Thus, DTT was shown to have a strong antagonistic effect on corrosion and was removed from the purification process. An optimal composition of the medium was selected (0.5 mM dithionite, 7.5 mM desthiobiotin) that simultaneously allowed a high activity of hydrogenase and a lower impact on the electrochemical response for corrosion tests.

  13. Light-driven hydrogen production by a hybrid complex of a [NiFe]-hydrogenase and the cyanobacterial photosystem I.

    Science.gov (United States)

    Ihara, Masaki; Nishihara, Hirofumi; Yoon, Ki-Seok; Lenz, Oliver; Friedrich, Bärbel; Nakamoto, Hitoshi; Kojima, Kouji; Honma, Daisuke; Kamachi, Toshiaki; Okura, Ichiro

    2006-01-01

    In order to generate renewable and clean fuels, increasing efforts are focused on the exploitation of photosynthetic microorganisms for the production of molecular hydrogen from water and light. In this study we engineered a 'hard-wired' protein complex consisting of a hydrogenase and photosystem I (hydrogenase-PSI complex) as a direct light-to-hydrogen conversion system. The key component was an artificial fusion protein composed of the membrane-bound [NiFe] hydrogenase from the beta-proteobacterium Ralstonia eutropha H16 and the peripheral PSI subunit PsaE of the cyanobacterium Thermosynechococcus elongatus. The resulting hydrogenase-PsaE fusion protein associated with PsaE-free PSI spontaneously, thereby forming a hydrogenase-PSI complex as confirmed by sucrose-gradient ultracentrifuge and immunoblot analysis. The hydrogenase-PSI complex displayed light-driven hydrogen production at a rate of 0.58 mumol H(2).mg chlorophyll(-1).h(-1). The complex maintained its accessibility to the native electron acceptor ferredoxin. This study provides the first example of a light-driven enzymatic reaction by an artificial complex between a redox enzyme and photosystem I and represents an important step on the way to design a photosynthetic organism that efficiently converts solar energy and water into hydrogen.

  14. Roles of HynAB and Ech, the only two hydrogenases found in the model sulfate reducer Desulfovibrio gigas.

    Science.gov (United States)

    Morais-Silva, Fabio O; Santos, Catia I; Rodrigues, Rute; Pereira, Inês A C; Rodrigues-Pousada, Claudina

    2013-10-01

    Sulfate-reducing bacteria are characterized by a high number of hydrogenases, which have been proposed to contribute to the overall energy metabolism of the cell, but exactly in what role is not clear. Desulfovibrio spp. can produce or consume H2 when growing on organic or inorganic substrates in the presence or absence of sulfate. Because of the presence of only two hydrogenases encoded in its genome, the periplasmic HynAB and cytoplasmic Ech hydrogenases, Desulfovibrio gigas is an excellent model organism for investigation of the specific function of each of these enzymes during growth. In this study, we analyzed the physiological response to the deletion of the genes that encode the two hydrogenases in D. gigas, through the generation of ΔechBC and ΔhynAB single mutant strains. These strains were analyzed for the ability to grow on different substrates, such as lactate, pyruvate, and hydrogen, under respiratory and fermentative conditions. Furthermore, the expression of both hydrogenase genes in the three strains studied was assessed through quantitative reverse transcription-PCR. The results demonstrate that neither hydrogenase is essential for growth on lactate-sulfate, indicating that hydrogen cycling is not indispensable. In addition, the periplasmic HynAB enzyme has a bifunctional activity and is required for growth on H2 or by fermentation of pyruvate. Therefore, this enzyme seems to play a dominant role in D. gigas hydrogen metabolism.

  15. Analysis of Quaternary Structure of a [LDH-like] Malate Dehydrogenase of Plasmodium falciparum with Oligomeric Mutants

    Science.gov (United States)

    L-Malate dehydrogenase (PfMDH) from Plasmodium falciparum, the causative agent for the most severe form of malaria, has shown remarkable similarities to L-lactate dehydrogenase (PfLDH). PfMDH is more closely related to [LDH-like] MDHs characterized in archea and other prokaryotes. Initial sequence a...

  16. Diversity and transcription of proteases involved in the maturation of hydrogenases in Nostoc punctiforme ATCC 29133 and Nostoc sp. strain PCC 7120

    Directory of Open Access Journals (Sweden)

    Lindblad Peter

    2009-03-01

    Full Text Available Abstract Background The last step in the maturation process of the large subunit of [NiFe]-hydrogenases is a proteolytic cleavage of the C-terminal by a hydrogenase specific protease. Contrary to other accessory proteins these hydrogenase proteases are believed to be specific whereby one type of hydrogenases specific protease only cleaves one type of hydrogenase. In cyanobacteria this is achieved by the gene product of either hupW or hoxW, specific for the uptake or the bidirectional hydrogenase respectively. The filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Nostoc sp strain PCC 7120 may contain a single uptake hydrogenase or both an uptake and a bidirectional hydrogenase respectively. Results In order to examine these proteases in cyanobacteria, transcriptional analyses were performed of hupW in Nostoc punctiforme ATCC 29133 and hupW and hoxW in Nostoc sp. strain PCC 7120. These studies revealed numerous transcriptional start points together with putative binding sites for NtcA (hupW and LexA (hoxW. In order to investigate the diversity and specificity among hydrogeanse specific proteases we constructed a phylogenetic tree which revealed several subgroups that showed a striking resemblance to the subgroups previously described for [NiFe]-hydrogenases. Additionally the proteases specificity was also addressed by amino acid sequence analysis and protein-protein docking experiments with 3D-models derived from bioinformatic studies. These studies revealed a so called "HOXBOX"; an amino acid sequence specific for protease of Hox-type which might be involved in docking with the large subunit of the hydrogenase. Conclusion Our findings suggest that the hydrogenase specific proteases are under similar regulatory control as the hydrogenases they cleave. The result from the phylogenetic study also indicates that the hydrogenase and the protease have co-evolved since ancient time and suggests that at least one major horizontal gene transfer

  17. Diversity and transcription of proteases involved in the maturation of hydrogenases in Nostoc punctiforme ATCC 29133 and Nostoc sp. strain PCC 7120

    Science.gov (United States)

    2009-01-01

    Background The last step in the maturation process of the large subunit of [NiFe]-hydrogenases is a proteolytic cleavage of the C-terminal by a hydrogenase specific protease. Contrary to other accessory proteins these hydrogenase proteases are believed to be specific whereby one type of hydrogenases specific protease only cleaves one type of hydrogenase. In cyanobacteria this is achieved by the gene product of either hupW or hoxW, specific for the uptake or the bidirectional hydrogenase respectively. The filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Nostoc sp strain PCC 7120 may contain a single uptake hydrogenase or both an uptake and a bidirectional hydrogenase respectively. Results In order to examine these proteases in cyanobacteria, transcriptional analyses were performed of hupW in Nostoc punctiforme ATCC 29133 and hupW and hoxW in Nostoc sp. strain PCC 7120. These studies revealed numerous transcriptional start points together with putative binding sites for NtcA (hupW) and LexA (hoxW). In order to investigate the diversity and specificity among hydrogeanse specific proteases we constructed a phylogenetic tree which revealed several subgroups that showed a striking resemblance to the subgroups previously described for [NiFe]-hydrogenases. Additionally the proteases specificity was also addressed by amino acid sequence analysis and protein-protein docking experiments with 3D-models derived from bioinformatic studies. These studies revealed a so called "HOXBOX"; an amino acid sequence specific for protease of Hox-type which might be involved in docking with the large subunit of the hydrogenase. Conclusion Our findings suggest that the hydrogenase specific proteases are under similar regulatory control as the hydrogenases they cleave. The result from the phylogenetic study also indicates that the hydrogenase and the protease have co-evolved since ancient time and suggests that at least one major horizontal gene transfer has occurred. This co

  18. Comparison of carbon materials as electrodes for enzyme electrocatalysis: hydrogenase as a case study.

    Science.gov (United States)

    Quinson, Jonathan; Hidalgo, Ricardo; Ash, Philip A; Dillon, Frank; Grobert, Nicole; Vincent, Kylie A

    2014-01-01

    We present a study of electrocatalysis by an enzyme adsorbed on a range of carbon materials, with different size, surface area, morphology and graphitic structure, which are either commercially available or prepared via simple, established protocols. We choose as our model enzyme the hydrogenase I from E. coli (Hyd-1), which is an active catalyst for H2 oxidation, is relatively robust and has been demonstrated in H2 fuel cells and H2-driven chemical synthesis. The carbon materials were characterised according to their surface area, surface morphology and graphitic character, and we use the electrocatalytic H2 oxidation current for Hyd-1 adsorbed on these materials to evaluate their effectiveness as enzyme electrodes. Here, we show that a variety of carbon materials are suitable for adsorbing hydrogenases in an electroactive configuration. This unified study provides insight into selection and design of carbon materials for study of redox enzymes and different applications of enzyme electrocatalysis.

  19. Isolation, purification and characterization of the hydrogen evolution promoting factor of hydrogenase of Spirulina platensis

    Science.gov (United States)

    Gu, Tian-Qing; Zhang, Hui-Miao; Sun, Shi-Hua

    1996-03-01

    A component (s-factor) with obvious promoting effect on hydrogen evolution of hydrogenase has been isolated and extracted from a cell-free preparation of Spirulina platensis. The effect of the s-factor in the reaction system is similar to that of Na2S2O4, but is coupled with light. The s-factor has the maximum absorption peak at 620 nm in the oxidized state, at 590 nm in the reduced state. The partially purified s-factor showed two bands by SDS-PAGE and is distinctly different from phycocyanin, which has no change of oxidized state and reduced state absorption spectra, and also has no promoting effect on hydrogenase of Spirulina platensis under the light.

  20. Mechanism of O2 diffusion and reduction in FeFe hydrogenases

    Science.gov (United States)

    Kubas, Adam; Orain, Christophe; de Sancho, David; Saujet, Laure; Sensi, Matteo; Gauquelin, Charles; Meynial-Salles, Isabelle; Soucaille, Philippe; Bottin, Hervé; Baffert, Carole; Fourmond, Vincent; Best, Robert B.; Blumberger, Jochen; Léger, Christophe

    2017-01-01

    FeFe hydrogenases are the most efficient H2-producing enzymes. However, inactivation by O2 remains an obstacle that prevents them being used in many biotechnological devices. Here, we combine electrochemistry, site-directed mutagenesis, molecular dynamics and quantum chemical calculations to uncover the molecular mechanism of O2 diffusion within the enzyme and its reactions at the active site. We propose that the partial reversibility of the reaction with O2 results from the four-electron reduction of O2 to water. The third electron/proton transfer step is the bottleneck for water production, competing with formation of a highly reactive OH radical and hydroxylated cysteine. The rapid delivery of electrons and protons to the active site is therefore crucial to prevent the accumulation of these aggressive species during prolonged O2 exposure. These findings should provide important clues for the design of hydrogenase mutants with increased resistance to oxidative damage.

  1. Hydrogenase Enzymes and Their Synthetic Models: The Role of Metal Hydrides.

    Science.gov (United States)

    Schilter, David; Camara, James M; Huynh, Mioy T; Hammes-Schiffer, Sharon; Rauchfuss, Thomas B

    2016-08-10

    Hydrogenase enzymes efficiently process H2 and protons at organometallic FeFe, NiFe, or Fe active sites. Synthetic modeling of the many H2ase states has provided insight into H2ase structure and mechanism, as well as afforded catalysts for the H2 energy vector. Particularly important are hydride-bearing states, with synthetic hydride analogues now known for each hydrogenase class. These hydrides are typically prepared by protonation of low-valent cores. Examples of FeFe and NiFe hydrides derived from H2 have also been prepared. Such chemistry is more developed than mimicry of the redox-inactive monoFe enzyme, although functional models of the latter are now emerging. Advances in physical and theoretical characterization of H2ase enzymes and synthetic models have proven key to the study of hydrides in particular, and will guide modeling efforts toward more robust and active species optimized for practical applications.

  2. ZnCr layered double hydroxide (LDH) nanosheets assisted formation of hierarchical flower-like CdZnS@LDH microstructures with improved visible-light-driven H2 production.

    Science.gov (United States)

    Yao, Lihua; Wei, Ding; Yan, Dongpeng; Hu, Changwen

    2015-03-01

    The development of new semiconductor photocatalysts toward splitting water has supplied a promising way to obtain sustainable and clean hydrogen energy. Herein, CdZnS@layered double hydroxide (LDH) composites with a hierarchical flower-like microstructure have been fabricated with the aid of ZnCr-LDH nanosheets as templates. XRD, SEM and HRTEM show that the ZnCr-LDH nanosheets are uniformly dispersed within the composites. The surface of the hierarchical structures is rough and composed of numerous nanocrystals of CdZnS. The HRTEM images indicate that the surface of CdZnS nanocrystals is mainly composed of the (111) plane. Moreover, the visible-light-driven H2 production performance of the CdZnS in the presence and absence of ZnCr-LDH nanosheets has been measured. The results show that ZnCr-LDH nanosheets play an important role in the hierarchical morphology and photocatalytic activity of the as-prepared samples. In the water-splitting process, the visible-light-driven H2 -production rate of hierarchical flower-like CdZnS@LDH is 4.03 times and nearly 10 times higher than that of pristine CdZnS microsphere and pure commercial CdS, respectively. Therefore, this work not only achieves enhanced catalytic performance of the CdZnS by the introduction of ZnCr-LDH nanosheets, but also supplies an insight into the relationship between the hierarchical morphology and the semiconductor photocatalytic activity.

  3. Lyophilization protects [FeFe]-hydrogenases against O2-induced H-cluster degradation

    OpenAIRE

    Jens Noth; Ramona Kositzki; Kathrin Klein; Martin Winkler; Michael Haumann; Thomas Happe

    2015-01-01

    Nature has developed an impressive repertoire of metal-based enzymes that perform complex chemical reactions under moderate conditions. Catalysts that produce molecular hydrogen (H2) are particularly promising for renewable energy applications. Unfortunately, natural and chemical H2-catalysts are often irreversibly degraded by molecular oxygen (O2). Here we present a straightforward procedure based on freeze-drying (lyophilization), that turns [FeFe]-hydrogenases, which are excellent H2-produ...

  4. Flexibility in Anaerobic Metabolism as Revealed in a Mutant of Chlamydomonas reinhardtii Lacking Hydrogenase Activity

    Energy Technology Data Exchange (ETDEWEB)

    Dubini, A.; Mus, F.; Seibert, M.; Grossman, A. R.; Posewitz, M. C.

    2009-03-13

    The green alga Chlamydomonas reinhardtii has a network of fermentation pathways that become active when cells acclimate to anoxia. Hydrogenase activity is an important component of this metabolism, and we have compared metabolic and regulatory responses that accompany anaerobiosis in wild-type C. reinhardtii cells and a null mutant strain for the HYDEF gene (hydEF-1 mutant), which encodes an [FeFe] hydrogenase maturation protein. This mutant has no hydrogenase activity and exhibits elevated accumulation of succinate and diminished production of CO2 relative to the parental strain during dark, anaerobic metabolism. In the absence of hydrogenase activity, increased succinate accumulation suggests that the cells activate alternative pathways for pyruvate metabolism, which contribute to NAD(P)H reoxidation, and continued glycolysis and fermentation in the absence of O2. Fermentative succinate production potentially proceeds via the formation of malate, and increases in the abundance of mRNAs encoding two malateforming enzymes, pyruvate carboxylase and malic enzyme, are observed in the mutant relative to the parental strain following transfer of cells from oxic to anoxic conditions. Although C. reinhardtii has a single gene encoding pyruvate carboxylase, it has six genes encoding putative malic enzymes. Only one of the malic enzyme genes, MME4, shows a dramatic increase in expression (mRNA abundance) in the hydEF-1 mutant during anaerobiosis. Furthermore, there are marked increases in transcripts encoding fumarase and fumarate reductase, enzymes putatively required to convert malate to succinate. These results illustrate the marked metabolic flexibility of C. reinhardtii and contribute to the development of an informed model of anaerobic metabolism in this and potentially other algae.

  5. LDH dye hybrid material as coloured filler into polystyrene: Structural characterization and rheological properties

    Science.gov (United States)

    Taviot-Gueho, C.; Illaik, A.; Vuillermoz, C.; Commereuc, S.; Verney, V.; Leroux, F.

    2007-05-01

    The organic inorganic hybrid assembly composed of a dye molecule of large size, direct yellow®50, as interleaved anionic molecule and layered double hydroxide host was investigated by X-ray diffraction. Upon hydrothermal post-synthesis treatment, the basal spacing is strongly decreased, explained by a drastic change in the orientation of the organic molecule against the LDH sheets, from perpendicular to parallel. The interactions were studied by 13C CPMAS NMR technique. Dispersed into polystyrene, the coloured filler was found to behave better in the viscoelastic domain than conventional surfactant LDH filler, maintaining similar rheological properties to filler-free PS. We demonstrate here that an intercalated nanocomposite polymer structure, providing an additional function as colour, is not preposterous.

  6. Intercalation of sulfonated melamine formaldehyde polycondensates into a hydrocalumite LDH structure

    Science.gov (United States)

    von Hoessle, F.; Plank, J.; Leroux, F.

    2015-05-01

    A series of sulfonated melamine formaldehyde (SMF) polycondensates possessing different anionic charge amounts and molecular weights was synthesized and incorporated into a hydrocalumite type layered double hydroxide structure using the rehydration method. For this purpose, tricalcium aluminate was dispersed in water and hydrated in the presence of these polymers. Defined inorganic-organic hybrid materials were obtained as reaction products. All SMF polymers tested intercalated readily into the hydrocalumite structure, independent of their different molecular weights (chain lengths) and anionic charge amounts. X-ray diffraction revealed typical patterns for weakly ordered, highly polymer loaded LDH materials which was confirmed via elemental analysis and thermogravimetry. IR spectroscopy suggests that the SMF polymers are interleaved between the [Ca2Al(OH)6]+ main sheets via electrostatic interaction, and that no chemical bond between the host matrix and the guest anion is formed. The SMF polymers well ensconced within the LDH structure exhibit significantly slower thermal degradation.

  7. Hepatocyte damage induced by lymphocytes from patients with chronic liver diseases, as detected by LDH release.

    Science.gov (United States)

    Fernandez-Cruz, E; Escartin, P; Bootello, A; Kreisler, M; Segovia de Arana, J M

    1978-01-01

    We have used a cytoplasmic enzyme system in the study of the in vitro cytotoxic activity of human peripheral blood leucocytes against isolated liver cells in patients with chronic liver diseases. Lymphocytes from primary biliary cirrhosis and chronic active liver disease patients were shown to have an in vitro capacity to induce a cytolitic effect on isolated hepatocytes, as demonstrated by the enhanced release of lactate dehydrogenase (LDH), a cytoplasmic marker enzyme. No significant LDH release was seen with control lymphocytes of normal persons or with lymphocytes from patients with alcoholic cirrhosis. Our results corroborate, in a different assay system, by a simple, reproducible and different method, that lymphocyte-mediated liver cell damage "in vitro" occurs in both primary biliary cirrhosis and chronic active liver disease. PMID:657588

  8. MOF membrane synthesis in the confined space of a vertically aligned LDH network.

    Science.gov (United States)

    Liu, Yi; Wang, Nanyi; Diestel, Lisa; Steinbach, Frank; Caro, Jürgen

    2014-04-25

    MOF membranes have gained widespread attention due to their unprecedented gas separation performance. Relying on physical interactions, we successfully deposited MOF seeds on a substrate modified with a network of vertically aligned LDH walls before secondary growth of the MOF layer. ZIF-8 membranes thus prepared show considerable H2 permeance with high H2-CH4 selectivity. This approach is in general suitable for the deposition of nanoparticles on solid surface and their subsequent growth into a dense layer.

  9. Perbandingan Kadar Ldh Antara Penderita Preeklampsia Berat/Eklampsia Dengan Kehamilan Normal

    OpenAIRE

    Asroel, Edwin Martin

    2016-01-01

    Purpose of study :to analyze level of LDH between patients with preeclampsia / severe preeclampsia and normal pregnancy. Study design :this study is held with analytical observation study with cross sectional study to pregnant woman with preeclampsia / severe preeclampsia and pregnant woman with normal blood pressure. Results :this study was held since March 2014, and got 30 pastients with severe preeclampsia and eclampsia with 5 persons dead (16,7%).Character of pregnant mother mostl...

  10. MMT and LDH organo-modification with surfactants tailored for PLA nanocomposites

    OpenAIRE

    S. Coiai; F. Cicogna; Santi, A.; L. Perez Amaro; R. Spiniello; F. Signori; Fiori, S.; W. Oberhauser; Passaglia, E.

    2017-01-01

    Low molecular weight polyesters were end-functionalized with ammonium and carboxylate salts and used in ionic exchange reactions with respectively cationic (MMT) and anionic (LDH) clays. The hybrid organic-inorganic substrates were structurally analysed to determine the ester oligomers’ modification degree and their thermal behaviour owing to confinement effects. The dispersion of such hybrids in polylactic acid (PLA) matrix was performed and the ultimate structural, morphological and thermal...

  11. Proton-coupled electron transfer dynamics in the catalytic mechanism of a [NiFe]-hydrogenase.

    Science.gov (United States)

    Greene, Brandon L; Wu, Chang-Hao; McTernan, Patrick M; Adams, Michael W W; Dyer, R Brian

    2015-04-08

    The movement of protons and electrons is common to the synthesis of all chemical fuels such as H2. Hydrogenases, which catalyze the reversible reduction of protons, necessitate transport and reactivity between protons and electrons, but a detailed mechanism has thus far been elusive. Here, we use a phototriggered chemical potential jump method to rapidly initiate the proton reduction activity of a [NiFe] hydrogenase. Coupling the photochemical initiation approach to nanosecond transient infrared and visible absorbance spectroscopy afforded direct observation of interfacial electron transfer and active site chemistry. Tuning of intramolecular proton transport by pH and isotopic substitution revealed distinct concerted and stepwise proton-coupled electron transfer mechanisms in catalysis. The observed heterogeneity in the two sequential proton-associated reduction processes suggests a highly engineered protein environment modulating catalysis and implicates three new reaction intermediates; Nia-I, Nia-D, and Nia-SR(-). The results establish an elementary mechanistic understanding of catalysis in a [NiFe] hydrogenase with implications in enzymatic proton-coupled electron transfer and biomimetic catalyst design.

  12. Synthesis, structure and reactivity of Ni site models of [NiFeSe] hydrogenases.

    Science.gov (United States)

    Wombwell, Claire; Reisner, Erwin

    2014-03-21

    A series of structural models of the Ni centre in [NiFeSe] hydrogenases has been developed which exhibits key structural features of the Ni site in the H2 cycling enzyme. Specifically, two complexes with a hydrogenase-analogous four-coordinate 'NiS3Se' primary coordination sphere and complexes with a 'NiS2Se2' and a 'NiS4' core are reported. The reactivity of the complexes towards oxygen and protons shows some relevance to the chemistry of [NiFeSe] hydrogenases. Exposure of a 'NiS3Se' complex to atmospheric oxygen results in the oxidation of the selenolate group in the complex to a diselenide, which is released from the nickel site. Oxidation of the selenolate ligand on Ni occurs approximately four times faster than oxidation with the analogous sulfur complex. Reaction of the complexes with one equivalent of HBF4 results in protonation of the monodentate chalcogenolate and the release of this ligand from the metal centre as a thiol or selenol. Unrelated to their biomimetic nature, the complexes serve also as molecular precursors to modify electrodes with Ni-S-Se containing particles by electrochemical deposition. The activated electrodes evolve H2 in pH neutral water with an electrocatalytic onset potential of -0.6 V and a current density of 15 μA cm(-2) at -0.75 V vs. NHE.

  13. Cyanobacterial Hydrogenases and Hydrogen Metabolism Revisited: Recent Progress and Future Prospects

    Directory of Open Access Journals (Sweden)

    Namita Khanna

    2015-05-01

    Full Text Available Cyanobacteria have garnered interest as potential cell factories for hydrogen production. In conjunction with photosynthesis, these organisms can utilize inexpensive inorganic substrates and solar energy for simultaneous biosynthesis and hydrogen evolution. However, the hydrogen yield associated with these organisms remains far too low to compete with the existing chemical processes. Our limited understanding of the cellular hydrogen production pathway is a primary setback in the potential scale-up of this process. In this regard, the present review discusses the recent insight around ferredoxin/flavodoxin as the likely electron donor to the bidirectional Hox hydrogenase instead of the generally accepted NAD(PH. This may have far reaching implications in powering solar driven hydrogen production. However, it is evident that a successful hydrogen-producing candidate would likely integrate enzymatic traits from different species. Engineering the [NiFe] hydrogenases for optimal catalytic efficiency or expression of a high turnover [FeFe] hydrogenase in these photo-autotrophs may facilitate the development of strains to reach target levels of biohydrogen production in cyanobacteria. The fundamental advancements achieved in these fields are also summarized in this review.

  14. Enhancing hydrogen production of Enterobacter aerogenes by heterologous expression of hydrogenase genes originated from Synechocystis sp.

    Science.gov (United States)

    Song, Wenlu; Cheng, Jun; Zhao, Jinfang; Zhang, Chuanxi; Zhou, Junhu; Cen, Kefa

    2016-09-01

    The hydrogenase genes (hoxEFUYH) of Synechocystis sp. PCC 6803 were cloned and heterologously expressed in Enterobacter aerogenes ATCC13408 for the first time in this study, and the hydrogen yield was significantly enhanced using the recombinant strain. A recombinant plasmid containing the gene in-frame with Glutathione-S-Transferase (GST) gene was transformed into E. aerogenes ATCC13408 to produce a GST-fusion protein. SDS-PAGE and western blot analysis confirm the successful expression of the hox genes. The hydrogenase activity of the recombinant strain is 237.6±9.3ml/(g-DW·h), which is 152% higher than the wild strain. The hydrogen yield of the recombinant strain is 298.3ml/g-glucose, which is 88% higher than the wild strain. During hydrogen fermentation, the recombinant strain produces more acetate and butyrate, but less ethanol. This is corresponding to the NADH metabolism in the cell due to the higher hydrogenase activity with the heterologous expression of hox genes.

  15. Proton Reduction Using a Hydrogenase-Modified Nanoporous Black Silicon Photoelectrode

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Yixin; Anderson, Nicholas C.; Ratzloff, Michael W.; Mulder, David W.; Zhu, Kai; Turner, John A.; Neale, Nathan R.; King, Paul W.; Branz, Howard M.

    2016-06-15

    Metalloenzymes featuring earth-abundant metal-based cores exhibit rates for catalytic processes such as hydrogen evolution comparable to those of noble metals. Realizing these superb catalytic properties in artificial systems is challenging owing to the difficulty of effectively interfacing metalloenzymes with an electrode surface in a manner that supports efficient charge-transfer. Here, we demonstrate that a nanoporous 'black' silicon (b-Si) photocathode provides a unique interface for binding an adsorbed [FeFe]-hydrogenase enzyme ([FeFe]-H2ase). The resulting [FeFe]-H2ase/b-Si photoelectrode displays a 280 mV more positive onset potential for hydrogen generation than bare b-Si without hydrogenase, similar to that observed for a b-Si/Pt photoelectrode at the same light intensity. Additionally, we show that this H2ase/b-Si electrode exhibits a turnover frequency of >/=1300 s-1 and a turnover number above 107 and sustains current densities of at least 1 mA/cm2 based on the actual surface area of the electrode (not the smaller projected geometric area), orders of magnitude greater than that observed for previous enzyme-catalyzed electrodes. While the long-term stability of hydrogenase on the b-Si surface remains too low for practical applications, this work extends the proof-of-concept that biologically derived metalloenzymes can be interfaced with inorganic substrates to support technologically relevant current densities.

  16. [Fe]-hydrogenases in green algae: photo-fermentation and hydrogen evolution under sulfur deprivation

    Energy Technology Data Exchange (ETDEWEB)

    Winkler, M.; Hemschemeier, A.; Happe, T. [Botanisches Institut der Universitat Bonn (Germany); Gotor, C. [CSIC y Universidad de Sevilla (Spain). Instituto de Bioquimica Vegetal y Fotosintesis; Melis, A. [University of California, Berkeley, CA (United States). Department of Plant and Microbial Biology

    2002-12-01

    Recent studies indicate that [Fe]-hydrogenases and H{sub 2} metabolism are widely distributed among green algae. The enzymes are simple structured and catalyze H{sub 2} evolution with similar rates than the more complex [Fe]-hydrogenases from bacteria. Different green algal species developed diverse strategies to survive under sulfur deprivation. Chlamydomonas reinhardtii evolves large quantities of hydrogen gas in the absence of sulfur. In a sealed culture of C. reinhardtii, the photosynthetic O{sub 2} evolution rate drops below the rate of respiratory O{sub 2} consumption due to a reversible inhibition of photosystem II, thus leading to an intracellular anaerobiosis. The algal cells survive under these anaerobic conditions by switching their metabolism to a kind of photo-fermentation. Although possessing a functional [Fe]-hydrogenase gene, the cells of Scenedesmus obliquus produce no significant amounts of H{sub 2} under S-depleted conditions. Biochemical analyses indicate that S. obliquus decreases almost the complete metabolic activities while maintaining a low level of respiratory activity. (author)

  17. Reduced grain chalkiness and its possible physiological mechanism in transgenic rice overexpressing l-GalLDH

    Directory of Open Access Journals (Sweden)

    Le Yu

    2015-04-01

    Full Text Available Chalkiness is one of the key factors determining rice quality and price. Ascorbic acid (Asc is a major plant antioxidant that performs many functions in plants. l-Galactono-1,4-lactone dehydrogenase (l-GalLDH, EC1.3.2.3 is an enzyme that catalyzes the final step of Asc biosynthesis in plants. Here we show that the l-GalLDH-overexpressing transgenic rice, GO-2, which has constitutively higher leaf Asc content than wild-type (WT plants, exhibits significantly reduced grain chalkiness. Higher foliar ascorbate/dehydroascorbate (Asc/DHA ratios at 40, 60, 80, and 100 days of plant age were observed in GO-2. Further investigation showed that the enhanced level of Asc resulted in a significantly higher ribulose-1,5-bisphosphate (RuBP carboxylase/oxygenase (Rubisco protein level in GO-2 at 80 days. In addition, levels of abscisic acid (ABA and jasmonic acid (JA were lower in GO-2 at 60, 80, and 100 days. The results we present here indicate that the enhanced level of Asc is likely responsible for changing redox homeostasis in key developmental stages associated with grain filling and alters grain chalkiness in the l-GalLDH-overexpressing transgenic by maintaining photosynthetic function and affecting phytohormones associated with grain filling.

  18. Reduced grain chalkiness and its possible physiological mechanism in transgenic rice overexpressing L-GalLDH

    Institute of Scientific and Technical Information of China (English)

    Le; Yu; Yonghai; Liu; Jianhua; Tong; Junhui; Ding; Ruozhong; Wang; Changlian; Peng; Langtao; Xiao

    2015-01-01

    Chalkiness is one of the key factors determining rice quality and price. Ascorbic acid(Asc) is a major plant antioxidant that performs many functions in plants. L-Galactono-1,4-lactone dehydrogenase(L-Gal LDH, EC1.3.2.3) is an enzyme that catalyzes the final step of Asc biosynthesis in plants. Here we show that the L-Gal LDH-overexpressing transgenic rice, GO-2,which has constitutively higher leaf Asc content than wild-type(WT) plants, exhibits significantly reduced grain chalkiness. Higher foliar ascorbate/dehydroascorbate(Asc/DHA)ratios at 40, 60, 80, and 100 days of plant age were observed in GO-2. Further investigation showed that the enhanced level of Asc resulted in a significantly higher ribulose-1,5-bisphosphate(Ru BP) carboxylase/oxygenase(Rubisco) protein level in GO-2 at 80 days. In addition, levels of abscisic acid(ABA) and jasmonic acid(JA) were lower in GO-2 at 60, 80, and100 days. The results we present here indicate that the enhanced level of Asc is likely responsible for changing redox homeostasis in key developmental stages associated with grain filling and alters grain chalkiness in the L-Gal LDH-overexpressing transgenic by maintaining photosynthetic function and affecting phytohormones associated with grain filling.

  19. What is the acceptable hemolysis index for the measurements of plasma potassium, LDH and AST?

    Science.gov (United States)

    Rousseau, Nathalie; Pige, Raphaëlle; Cohen, Richard; Pecquet, Matthieu

    2016-06-01

    Hemolysis is a cause of variability in test results for plasma potassium, LDH and AST and is a non-negligible part of measurement uncertainty. However, allowable levels of hemolysis provided by reagent suppliers take neither analytical variability (trueness and precision) nor the measurand into account. Using a calibration range of hemolysis, we measured the plasma concentrations of potassium, LDH and AST, and hemolysis indices with a Cobas C501 analyzer (Roche Diagnostics(®), Meylan, France). Based on the allowable total error (according to Ricós et al.) and the expanded measurement uncertainty equation we calculated the maximum allowable bias for two concentrations of each measurand. Finally, we determined the allowable hemolysis indices for all three measurands. We observed a linear relationship between the observed increases of concentration and hemolysis indices. The LDH measurement was the most sensitive to hemolysis, followed by AST and potassium measurements. The determination of the allowable hemolysis index depends on the targeted measurand, its concentration and the chosen level of requirement of allowable total error.

  20. Glyphosate and glufosinate detection at electrogenerated NiAl-LDH thin films

    Energy Technology Data Exchange (ETDEWEB)

    Khenifi, Aicha [Laboratoire des Materiaux Inorganiques, UMR CNRS 6002, Universite Blaise Pascal, Clermont-Ferrand (France); Laboratoire de physico-chimie des materiaux, catalyse et environnement Usto, Oran, El M' nouar (Algeria); Derriche, Zoubir [Laboratoire de physico-chimie des materiaux, catalyse et environnement Usto, Oran, El M' nouar (Algeria); Forano, Claude; Prevot, Vanessa [Laboratoire des Materiaux Inorganiques, UMR CNRS 6002, Universite Blaise Pascal, Clermont-Ferrand (France); Mousty, Christine, E-mail: Christine.Mousty@univ-bpclermont.fr [Laboratoire des Materiaux Inorganiques, UMR CNRS 6002, Universite Blaise Pascal, Clermont-Ferrand (France); Scavetta, Erika, E-mail: scavetta@fci.unibo.it [Laboratorio di Chimica Analitica, Dipartimento di Chimica Fisica ed Inorganica, Universita degli Studi di Bologna (Italy); Ballarin, Barbara; Guadagnini, Lorella; Tonelli, Domenica [Laboratorio di Chimica Analitica, Dipartimento di Chimica Fisica ed Inorganica, Universita degli Studi di Bologna (Italy)

    2009-11-10

    An amperometric sensor based on Ni{sub 1-x}Al{sub x}(OH){sub 2}NO{sub 3x}.nH{sub 2}O layered double hydroxide (LDH) has been developed for the electrochemical analysis in one step of two herbicides: glyphosate (N-(phosphonomethyl)glycine, Glyp) and glufosinate ((DL-homoalanine-4-yl)-methylphosphinic acid, Gluf). NiAl-LDH was prepared by coprecipitation or by electrodeposition at the Pt electrode surface. Inorganic films were fully characterized by X-ray diffraction, Raman spectroscopy and scanning electron microscopy. Adsorption isotherms of Glyp onto this inorganic lamellar material have been established. Electrocatalytic oxidation of Glyp and Gluf is possible at the Ni{sup 3+} centres of the structure. The electrochemical responses of the NiAl-LDH modified electrode were obtained by cyclic voltammetry and chronoamperometry at 0.49 V/SCE as a function of herbicide concentration in 0.1 M NaOH solution. The electrocatalytic response showed a linear dependence on the Glyp concentration ranging between 0.01 and 0.9 mM with a detection limit of 1 {mu}M and sensitivity 287 mA/M cm{sup 2}. The sensitivity found for Gluf was lower (178 mA/M cm{sup 2}).

  1. Synthesis of new Tb-doped Zn-Al LDH/tryptophan hybrids and their fluorescent property

    Institute of Scientific and Technical Information of China (English)

    陈玉凤; 王肖庆; 罗世地; 鲍垚

    2016-01-01

    A series of hybrids based on Tb-doped Zn-Al layered double hydroxides (Tb-LDHs) combined with tryptophan (hereafter shortened as Try) were synthesized by soft-chemical method. The composition, structure, and fluorescence of the Tb-LDH/Try hy-brids were analyzed by various characterizations. Compositional analysis indicated that the content of tryptophan present in the hybrids gradually increased while the Tb-LDH reacted with 0.05, 0.1, and 0.25 mol/L Try solution, respectively. XRD results revealed that new reflections appeared in the Tb-LDH/Try hybrids. TGA curves of the Tb-LDH/Try hybrids were different from that of Tb-LDH and Try. IR spectra manifested that the IR spectra of the hybrids were characteristic of the Try and Tb-LDH. Fluorescent spectra sug-gested that the green emission due to5D4→7F5 transition of Tb3+ greatly decreased but not quenched, and the emission attributed to Try obviously increased. Meanwhile the fluorescent spectra of Tb-LDH/Try hybrids presented broad continuous bands in visible region.

  2. Assessment of three new parasite lactate dehydrogenase (pan-pLDH) tests for diagnosis of uncomplicated malaria.

    Science.gov (United States)

    Fogg, Carole; Twesigye, Rogers; Batwala, Vincent; Piola, Patrice; Nabasumba, Carolyn; Kiguli, James; Mutebi, Frederick; Hook, Christa; Guillerm, Martine; Moody, Anthony; Guthmann, Jean-Paul

    2008-01-01

    A study to assess the diagnostic capabilities of three parasite lactate dehydrogenase (pan-pLDH) tests, Vistapan), Carestart and Parabank), was conducted in Uganda. An HRP2 test, Paracheck-Pf), and a Giemsa-stained blood film were performed with the pLDH tests for outpatients with suspected malaria. In total, 460 subjects were recruited: 248 with positive blood films and 212 with negative blood films. Plasmodium falciparum was present in 95% of infections. Sensitivity above 90% was shown by two pLDH tests, Carestart (95.6%) and Vistapan (91.9%), and specificity above 90% by Parabank (94.3%) and Carestart (91.5%). Sensitivity decreased with low parasitaemia (chi(2) trend, P90% with parasitaemia > or =100/microl. All tests had good inter-reader reliability (kappa>0.95). Two weeks after diagnosis, 4-10% of pLDH tests were still positive compared with 69.7% of the HRP2 tests. All tests had similar ease of use. In conclusion, two pLDH tests performed well in diagnosing P. falciparum malaria, and all pLDH tests became negative after treatment more quickly than the HRP2. Therefore the rapid test of choice for use with artemisinin-combination therapies in this area would be one of these new pLDH tests.

  3. Self-assembling organomodified Co/Al based layered double hydroxides (LDH) via one-step route

    Institute of Scientific and Technical Information of China (English)

    WANG De-yi; A.LEUTERITZ; U.WAGENKNECHT; G.HEINRICH

    2009-01-01

    The preparation of self-assembling organomodified Co/Al-layered double hydroxide (LDH) via one-step route was studied.A common surfactant,sodium dodecylbenzenesulfonate (DBS),was employed as an organic modifier.The behavior and structure of self-assembled intercalated organic Co/Al-LDH were investigated by FTIR,SEM,WAXS,element analysis and TGA.Based upon the WAXS results and calculation by Bragg equation,the interlayer distance (d value) for organic Co/Al-LDH is enlarged from 0.75 nm to 3.10 nm,showing that the self-assembling behavior has been carried out successfully.Considering the observation from SEM,the product shows the morphology of organic Co/Al-LDH of a layered structure.In addition,FTIR,element analysis and TGA analysis show that the modifier is intercalated into the gallery of the Co/Al-LDH.Since organic modification for nanofiller is deemed to be necessary before applying it into polymer,the successful preparation of organomodified Co/Al-LDH will be significantly beneficial to the preparation and investigation of novel polymer/LDH nanocomposite.

  4. Cloning and Characterization of Multiple RNA Splicing Variants of LDH-C Gene in Human and Rat

    Directory of Open Access Journals (Sweden)

    Qinglian Zhang

    2013-06-01

    Full Text Available The expression of LDH-C (Lactate dehydrogenase C gene is restricted in mature germ cells; however multiple splice variants of LDH-C expressed in human cancers and yak normal testes were reported recently. In order to know if there are any LDH-C splice variants in human normal testes, we set out to clone the putative variants in human and rat. Four splicing variants in human testes, 1 splicing variant in human spermatozoa, 6 splicing variants in rat testes and 1 splicing variant in rat non-testes tissues (liver, heart and muscle were cloned. The putative polypeptides encoded by these variants were compared with the full-length LDH-C protein, the results showed that these putative polypeptides were truncated LDH-C proteins or truncated LDH-C proteins with a few amino acid residues different at N or C terminal. This suggested that these variants are possibly not used for translation, but targets of nonsense-mediated mRNA decay. Western blotting did not detect any bands with similar molecular weight as the putative polypeptides. RT-PCR showed that the expression levels of the splicing variants were significant during development of rat testes. The results indicate that LDH-C was not silenced by transcriptional repression in non-mature germ cells, but significantly transcripted and alternatively spliced.

  5. Degradability Enhancement of Poly(Lactic Acid by Stearate-Zn3Al LDH Nanolayers

    Directory of Open Access Journals (Sweden)

    Mahboobeh Eili

    2012-06-01

    Full Text Available Recent environmental problems and societal concerns associated with the disposal of petroleum based plastics throughout the world have triggered renewed efforts to develop new biodegradable products compatible with our environment. This article describes the preparation, characterization and biodegradation study of poly(lactic acid/layered double hydroxide (PLA/LDH nanocomposites from PLA and stearate-Zn3Al LDH. A solution casting method was used to prepare PLA/stearate-Zn3Al LDH nanocomposites. The anionic clay Zn3Al LDH was firstly prepared by co-precipitation method from a nitrate salt solution at pH 7.0 and then modified by stearate anions through an ion exchange reaction. This modification increased the basal spacing of the synthetic clay from 8.83 Å to 40.10 Å. The morphology and properties of the prepared PLA/stearate-Zn3Al LDH nanocomposites were studied by X-ray diffraction (XRD, transmission electron microscope (TEM, scanning electron microscope (SEM, thermogravimetric analysis (TGA, tensile tests as well as biodegradation studies. From the XRD analysis and TEM observation, the stearate-Zn3Al LDH lost its ordered stacking-structure and was greatly exfoliated in the PLA matrix. Tensile test results of PLA/stearate-Zn3Al LDH nanocomposites showed that the presence of around 1.0–3.0 wt % of the stearate-Zn3Al LDH in the PLA drastically improved its elongation at break. The biodegradation studies demonstrated a significant biodegradation rate improvement of PLA in the presence of stearate-Zn3Al LDH nanolayers. This effect can be caused by the catalytic role of the stearate groups in the biodegradation mechanism leading to much faster disintegration of nanocomposites than pure PLA.

  6. LDH-A promotes malignant progression via activation of epithelial-to-mesenchymal transition and conferring stemness in muscle-invasive bladder cancer

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Fujin [Department of Urinary Surgery, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu (China); Department of Urinary Surgery, Huai' an Hospital to Xuzhou Medical University, Huai' an, Jiangsu (China); Ma, Song [Department of Urinary Surgery, Huai' an Hospital to Xuzhou Medical University, Huai' an, Jiangsu (China); Xue, Yubao [Department of Medical Oncology, Huai' an Hospital to Xuzhou Medical University, Huai' an, Jiangsu (China); Hou, Jianquan, E-mail: Jianquanhou@aliyun.com [Department of Urinary Surgery, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu (China); Zhang, Yongjie, E-mail: zhangyj0818@126.com [Department of Medical Oncology, Huai' an Hospital to Xuzhou Medical University, Huai' an, Jiangsu (China)

    2016-01-22

    Lactate dehydrogenase-A(LDH-A) is an important rate-limiting enzyme in the Warburg effect. Survival analysis indicated poor clinical outcomes in MIBC with high LDH-A expression. The results of in vitro experiment indicated that LDH-A promotes MIBC cells proliferation, invasion and migration. The positive relationship between LDH-A expression and CSC/EMT markers was confirmed both in invasive bladder cell line and in 136 MIBC specimens. Thus, we conclude that LDH-A may be a promising target for MIBC. - Highlights: • Survival analysis indicated poor clinical outcomes in MIBC with high LDH-A expression. • IHC analysis of 136 MIBC specimens revealed increased LDH-A is correlated with positive Oct4 and negative E-cadherin. • In vitro experiments demonstrated LDH-A promotes MIBC progression by positive regulation of EMT/CSC.

  7. The role of LDH serum levels in predicting global outcome in HCC patients undergoing TACE: implications for clinical management.

    Directory of Open Access Journals (Sweden)

    Mario Scartozzi

    Full Text Available In many tumor types serum lactate dehydrogenase (LDH levels is an indirect marker of tumor hypoxia, neo-angiogenesis and worse prognosis. However data about hepatocellular carcinoma (HCC are lacking in the clinical setting of patients undergoing transarterial-chemoembolization (TACE in whom hypoxia and neo-angiogenesis may represent a molecular key to treatment failure. Aim of our analysis was to evaluate the role of LDH pre-treatment levels in determining clinical outcome for patients with HCC receiving TACE. One hundred and fourteen patients were available for our analysis. For all patients LDH values were collected within one month before the procedure. We divided our patients into two groups, according to LDH serum concentration registered before TACE (first: LDH≤450 U/l 84 patients; second: LDH>450 U/l 30 patients. Patients were classified according to the variation in LDH serum levels pre- and post-treatment (increased: 62 patients vs. decreased 52 patients. No statistically significant differences were found between the groups for all clinical characteristics analyzed (gender, median age, performance status ECOG, staging systems. In patients with LDH values below 450 U/l median time to progression (TTP was 16.3 months, whereas it was of 10.1 months in patients above the cut-off (p = 0.0085. Accordingly median overall survival (OS was 22.4 months and 11.7 months (p = 0.0049. In patients with decreased LDH values after treatment median TTP was 12.4 months, and median OS was 22.1 months, whereas TTP was 9.1 months and OS was 9.5 in patients with increased LDH levels (TTP: p = 0.0087; OS: p<0.0001. In our experience, LDH seemed able to predict clinical outcome for HCC patients undergoing TACE. Given the correlation between LDH levels and tumor angiogenesis we can speculate that patients with high LDH pretreatment levels may be optimal candidates for clinical trial exploring a multimodality treatment approach with TACE and anti

  8. Identification, cloning and heterologous expression of active [NiFe]-hydrogenase 2 from Citrobacter sp. SG in Escherichia coli.

    Science.gov (United States)

    Maier, Johannes A H; Ragozin, Sergey; Jeltsch, Albert

    2015-04-10

    Hydrogen (H2) is a potential alternative energy carrier which only produces water and heat upon combustion. Today, industrial hydrogen production mainly uses thermochemical processes based on fossil fuels or electrolysis of water. Therefore, biotechnological approaches to produce H2 from biomass are an interesting alternative. We introduce here a novel direct hydrogen measurement system using a semiconducting device specific for hydrogen detection. Using this device, a bacterium producing considerable amounts of hydrogen under aerobic cultivation was isolated and identified by 16S ribosomal DNA sequencing as Citrobacter sp. The enzyme responsible for the observed hydrogenase activity was partially purified by 3 chromatographic purification steps and could be identified by peptide mass fingerprinting to be a type 2 [NiFe]-hydrogenase. Expression of the [NiFe]-hydrogenase 2 containing operon from Citrobacter sp. SG in Escherichia coli allowed recombinant hydrogen production. The [NiFe]-hydrogenase 2 identified here may be useful for biotechnological hydrogen production. We speculate that the expression of the hydrogenase in Citrobacter may be an adaptation to growth in acidic conditions.

  9. Study of hydrogenases activity inhibition by O{sub 2} and direct electrochemistry; Etude de l'inhibition par O{sub 2} de l'activite d'hydrogenases par electrochimie directe

    Energy Technology Data Exchange (ETDEWEB)

    Baffert, C.; Leger, Ch.; Leroux, F.; Bertrand, P.; Guigliarelli, B. [Laboratoire de Bioenergetique et d' Ingenierie des proteines, BIP-CNRS, 13 - Marseille (France)

    2007-07-01

    At the present time, a great effort of research is made on the identification and the design of enzymes insensitive or low sensitive to O{sub 2}. In parallel, it seems important to understand the inhibition mechanisms in order to propose mutations able to limit this inhibition. The Protein Film Voltametry allows to obtain data which can not be observed or quantitatively obtained by other techniques. The enzyme is immobilized directly on the electrode and the electronic transfer is direct. The redox state of the enzyme depends on the potential of the electrode and the catalytic current is proportional to the enzyme activity. The data obtained for the Ni-Fe hydrogenase (Desulfovibrio fructosovorans) and for the Fe hydrogenase (Clostridium Acetobutylicum) will be compared to the data obtained for hydrogenases of other organisms, by Protein Film Voltametry as well as by other techniques. (O.M.)

  10. PREPARATION OF IgA MONOCLONAL ANTIBODIES TO MURINE LACTIC DEHYDROGENASE-C4 (LDH-C4)

    Institute of Scientific and Technical Information of China (English)

    FENGShu-Lin; BENKun-Long; LIANGZhi-Guo

    1989-01-01

    LDH--C4 is a sperm specific lactic dehydrogcnase in mammals and human, and is consid ered as a model molecule for contraceptive vaccine research. Significant contraceptive effects were observed in female mice, rabbits and baboons immunized with purified

  11. Advanced treatment of stabilized landfill leachate after biochemical process with hydrocalumite chloride (Ca/Al-Cl LDH).

    Science.gov (United States)

    Chen, Hua; Sun, Ying; Ruan, Xiuxiu; Yu, Ying; Zhu, Minying; Zhang, Jia; Zhou, Jizhi; Xu, Yunfeng; Liu, Jianyong; Qian, Guangren

    2016-06-01

    This study investigated the effectiveness of Ca/Al-Cl LDH for the treatment of stabilized landfill leachate. Experiments were performed including different dosage of Ca/Al-Cl LDH and comparison with different reagents, such as CaCl2 and AlCl3. As a result, Ca/Al-Cl LDH efficiently removed organic matters in stabilized landfill leachate with the maximum removal (59.41% COD, 62.06% DOC and 70.56% UV254) at the dose of 30g/L. According to UV254 and EEM, it is remarkable that the formation of Ca/Al-LDH has a greater beneficial to organic removal than other reagents, especially for fulvic acid-like and humic acid-like compounds. Moreover, the removal of fulvic acid-like compounds was much better than humic acid-like compounds. The previous compounds had more carboxylic groups, thus had a better removal selectivity.

  12. Formation of Hierarchical Structure Composed of (Co/Ni)Mn-LDH Nanosheets on MWCNT Backbones for Efficient Electrocatalytic Water Oxidation.

    Science.gov (United States)

    Jia, Gan; Hu, Yingfei; Qian, Qinfeng; Yao, Yingfang; Zhang, Shiying; Li, Zhaosheng; Zou, Zhigang

    2016-06-15

    Active, stable, and cost-effective electrocatalysts are attractive alternatives to the noble metal oxides that have been used in water splitting. The direct nucleation and growth of electrochemically active LDH materials on chemically modified MWCNTs exhibit considerable electrocatalytic activity toward oxygen evolution from water oxidation. CoMn-based and NiMn-based hybrids were synthesized using a facile chemical bath deposition method and the as-synthesized materials exhibited three-dimensional hierarchical configurations with tunable Co/Mn and Ni/Mn ratio. Benefiting from enhanced electrical conductivity with MWCNT backbones and LDH lamellar structure, the Co5Mn-LDH/MWCNT and Ni5Mn-LDH/MWCNT could generated a current density of 10 mA cm(-2) at overpotentials of ∼300 and ∼350 mV, respectively, in 1 M KOH. In addition, the materials also exhibited outstanding long-term electrocatalytic stability.

  13. Local diffusion homogeneity (LDH): an inter-voxel diffusion MRI metric for assessing inter-subject white matter variability.

    Science.gov (United States)

    Gong, Gaolang

    2013-01-01

    Many diffusion parameters and indices (e.g., fractional anisotropy [FA] and mean diffusivity [MD]) have been derived from diffusion magnetic resonance imaging (MRI) data. These parameters have been extensively applied as imaging markers for localizing white matter (WM) changes under various conditions (e.g., development, degeneration and disease). However, the vast majority of the existing parameters is derived from intra-voxel analyses and represents the diffusion properties solely within the voxel unit. Other types of parameters that characterize inter-voxel relationships have been largely overlooked. In the present study, we propose a novel inter-voxel metric referred to as the local diffusion homogeneity (LDH). This metric quantifies the local coherence of water molecule diffusion in a model-free manner. It can serve as an additional marker for evaluating the WM microstructural properties of the brain. To assess the distinguishing features between LDH and FA/MD, the metrics were systematically compared across space and subjects. As an example, both the LDH and FA/MD metrics were applied to measure age-related WM changes. The results indicate that LDH reveals unique inter-subject variability in specific WM regions (e.g., cerebral peduncle, internal capsule and splenium). Furthermore, there are regions in which measurements of age-related WM alterations with the LDH and FA/MD metrics yield discrepant results. These findings suggest that LDH and FA/MD have different sensitivities to specific WM microstructural properties. Taken together, the present study shows that LDH is complementary to the conventional diffusion-MRI markers and may provide additional insights into inter-subject WM variability. Further studies, however, are needed to uncover the neuronal mechanisms underlying the LDH.

  14. High sensitive C-reactive protein as a systemic inflammatory marker and LDH-3 isoenzyme in chronic obstructive pulmonary disease.

    Science.gov (United States)

    Nillawar, Anup N; Bardapurkar, J S; Bardapurkar, S J

    2012-01-01

    Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease, mainly due to tobacco smoke. Pulmonary function tests (PFTs) are mandatory to diagnose COPD which shows irreversible airway obstruction. This study was aimed at understanding the behavior of biochemical parameters such as high sensitive C-reactive protein (hs-CRP) and lactate dehydrogenase (LDH) isoenzymes in COPD. Cytoplasmic cellular enzymes, such as LDH in the extracellular space, although of no further metabolic function in this space, are of benefit because they serve as indicators suggestive of disturbances of the cellular integrity induced by pathological conditions. The lung pattern is characterized by proportional increases in isoenzymes 3, 4, and 5. Hs-CRP indicates low grade of systemic inflammation. Total (n = 45) patients of COPD (diagnosed on PFTs) were included. We followed the guidelines laid by the institute ethical committee. Investigations performed on the serum were the serum for hs-CRP, LDH isoenzymes on agarose gel electrophoresis. The results obtained showed that the value of hs-CRP was 4.6 ± 0.42 mg/L. The isoenzymes pattern was characterized by an increase in LDH-3 and LDH-4 fractions. This is evident even in those patients with normal LDH (n = 13) levels. This study states that there is a moderate positive correlation in between CRP and LDH-3 (r = 0.33; P = 0.01). Raised LDH-3 levels do not correlate with FEV(1) % (forced expiratory volume in first second) predicted. Moreover, it associates positively with hs-CRP and smoking status and negatively with body mass index. This underlines the potential of these parameters to complement the present system of staging which is solely based upon FEV(1) % predicted.

  15. Local diffusion homogeneity (LDH: an inter-voxel diffusion MRI metric for assessing inter-subject white matter variability.

    Directory of Open Access Journals (Sweden)

    Gaolang Gong

    Full Text Available Many diffusion parameters and indices (e.g., fractional anisotropy [FA] and mean diffusivity [MD] have been derived from diffusion magnetic resonance imaging (MRI data. These parameters have been extensively applied as imaging markers for localizing white matter (WM changes under various conditions (e.g., development, degeneration and disease. However, the vast majority of the existing parameters is derived from intra-voxel analyses and represents the diffusion properties solely within the voxel unit. Other types of parameters that characterize inter-voxel relationships have been largely overlooked. In the present study, we propose a novel inter-voxel metric referred to as the local diffusion homogeneity (LDH. This metric quantifies the local coherence of water molecule diffusion in a model-free manner. It can serve as an additional marker for evaluating the WM microstructural properties of the brain. To assess the distinguishing features between LDH and FA/MD, the metrics were systematically compared across space and subjects. As an example, both the LDH and FA/MD metrics were applied to measure age-related WM changes. The results indicate that LDH reveals unique inter-subject variability in specific WM regions (e.g., cerebral peduncle, internal capsule and splenium. Furthermore, there are regions in which measurements of age-related WM alterations with the LDH and FA/MD metrics yield discrepant results. These findings suggest that LDH and FA/MD have different sensitivities to specific WM microstructural properties. Taken together, the present study shows that LDH is complementary to the conventional diffusion-MRI markers and may provide additional insights into inter-subject WM variability. Further studies, however, are needed to uncover the neuronal mechanisms underlying the LDH.

  16. High sensitive C-reactive protein as a systemic inflammatory marker and LDH-3 isoenzyme in chronic obstructive pulmonary disease

    Directory of Open Access Journals (Sweden)

    Anup N Nillawar

    2012-01-01

    Full Text Available Background and Objectives: Chronic obstructive pulmonary disease (COPD is a chronic inflammatory disease, mainly due to tobacco smoke. Pulmonary function tests (PFTs are mandatory to diagnose COPD which shows irreversible airway obstruction. This study was aimed at understanding the behavior of biochemical parameters such as high sensitive C-reactive protein (hs-CRP and lactate dehydrogenase (LDH isoenzymes in COPD. Cytoplasmic cellular enzymes, such as LDH in the extracellular space, although of no further metabolic function in this space, are of benefit because they serve as indicators suggestive of disturbances of the cellular integrity induced by pathological conditions. The lung pattern is characterized by proportional increases in isoenzymes 3, 4, and 5. Hs-CRP indicates low grade of systemic inflammation. Materials and Methods: Total (n = 45 patients of COPD (diagnosed on PFTs were included. We followed the guidelines laid by the institute ethical committee. Investigations performed on the serum were the serum for hs-CRP, LDH isoenzymes on agarose gel electrophoresis. Results: The results obtained showed that the value of hs-CRP was 4.6 ΁ 0.42 mg/L. The isoenzymes pattern was characterized by an increase in LDH-3 and LDH-4 fractions. This is evident even in those patients with normal LDH (n = 13 levels. Interpretation and Conclusion: This study states that there is a moderate positive correlation in between CRP and LDH-3 (r = 0.33; P = 0.01. Raised LDH-3 levels do not correlate with FEV 1 % (forced expiratory volume in first second predicted. Moreover, it associates positively with hs-CRP and smoking status and negatively with body mass index. This underlines the potential of these parameters to complement the present system of staging which is solely based upon FEV 1 % predicted.

  17. Genomic and metagenomic surveys of hydrogenase distribution indicate H2 is a widely utilised energy source for microbial growth and survival.

    Science.gov (United States)

    Greening, Chris; Biswas, Ambarish; Carere, Carlo R; Jackson, Colin J; Taylor, Matthew C; Stott, Matthew B; Cook, Gregory M; Morales, Sergio E

    2016-03-01

    Recent physiological and ecological studies have challenged the long-held belief that microbial metabolism of molecular hydrogen (H2) is a niche process. To gain a broader insight into the importance of microbial H2 metabolism, we comprehensively surveyed the genomic and metagenomic distribution of hydrogenases, the reversible enzymes that catalyse the oxidation and evolution of H2. The protein sequences of 3286 non-redundant putative hydrogenases were curated from publicly available databases. These metalloenzymes were classified into multiple groups based on (1) amino acid sequence phylogeny, (2) metal-binding motifs, (3) predicted genetic organisation and (4) reported biochemical characteristics. Four groups (22 subgroups) of [NiFe]-hydrogenase, three groups (6 subtypes) of [FeFe]-hydrogenases and a small group of [Fe]-hydrogenases were identified. We predict that this hydrogenase diversity supports H2-based respiration, fermentation and carbon fixation processes in both oxic and anoxic environments, in addition to various H2-sensing, electron-bifurcation and energy-conversion mechanisms. Hydrogenase-encoding genes were identified in 51 bacterial and archaeal phyla, suggesting strong pressure for both vertical and lateral acquisition. Furthermore, hydrogenase genes could be recovered from diverse terrestrial, aquatic and host-associated metagenomes in varying proportions, indicating a broad ecological distribution and utilisation. Oxygen content (pO2) appears to be a central factor driving the phylum- and ecosystem-level distribution of these genes. In addition to compounding evidence that H2 was the first electron donor for life, our analysis suggests that the great diversification of hydrogenases has enabled H2 metabolism to sustain the growth or survival of microorganisms in a wide range of ecosystems to the present day. This work also provides a comprehensive expanded system for classifying hydrogenases and identifies new prospects for investigating H2

  18. Adsorption study of anionic reactive dye from aqueous solution to Mg-Fe-CO3 layered double hydroxide (LDH)

    Science.gov (United States)

    Ahmed, I. M.; Gasser, M. S.

    2012-10-01

    Mg-Fe-Cl Layered double hydroxides (LDHs) have been prepared using a method involving separate nucleation and aging steps with Mg/Fe = 3. The interlayer anions readily replaced by carbonate are characterized by X-ray diffraction (XRD) and FTIR. The effects of different parameters, such as pH, contact time, concentration of dye and temperature on the capacity and adsorption mechanism of Mg-Fe-CO3-LDH in removing an anionic dye (congo red, CR) from aqueous solution were separately investigated. The results show that Mg-Fe-CO3-LDH is particularly efficient in removing CR and the dye removal increases with decreasing pH. The adsorption of CR on Mg-Fe-CO3-LDH reached equilibrium after 15 min where 100 mg/L CR was removed. The equilibrium isotherm indicates that the adsorption of CR onto Mg-Fe-CO3-LDH fits to Langmuir and Freundlich equation as well. The adsorption data obtained from the Langmuir model gave good values of the determination coefficient and the saturated adsorption capacity of Mg-Fe-CO3-LDH for CR was found to be 104.6 mg/g. The regeneration study indicates that the prepared LDH could be used for several cycles. The thermodynamic parameters have been calculated, and the adsorption process was found to be spontaneous, endothermic in nature and follows a pseudo-second-order kinetic model.

  19. High-performance hydrogen production and oxidation electrodes with hydrogenase supported on metallic single-wall carbon nanotube networks.

    Science.gov (United States)

    Svedružić, Draženka; Blackburn, Jeffrey L; Tenent, Robert C; Rocha, John-David R; Vinzant, Todd B; Heben, Michael J; King, Paul W

    2011-03-30

    We studied the electrocatalytic activity of an [FeFe]-hydrogenase from Clostridium acetobutylicum (CaH2ase) immobilized on single-wall carbon nanotube (SWNT) networks. SWNT networks were prepared on carbon cloth by ultrasonic spraying of suspensions with predetermined ratios of metallic and semiconducting nanotubes. Current densities for both proton reduction and hydrogen oxidation electrocatalytic activities were at least 1 order of magnitude higher when hydrogenase was immobilized onto SWNT networks with high metallic tube (m-SWNT) content in comparison to hydrogenase supported on networks with low metallic tube content or when SWNTs were absent. We conclude that the increase in electrocatalytic activities in the presence of SWNTs was mainly due to the m-SWNT fraction and can be attributed to (i) substantial increases in the active electrode surface area, and (ii) improved electronic coupling between CaH2ase redox-active sites and the electrode surface.

  20. Purification, crystallization and preliminary X-ray analysis of the membrane-bound [NiFe] hydrogenase from Allochromatium vinosum

    Energy Technology Data Exchange (ETDEWEB)

    Kellers, Petra; Ogata, Hideaki; Lubitz, Wolfgang, E-mail: lubitz@mpi-muelheim.mpg.de [Max-Planck-Institut für Bioanorganische Chemie, Stiftstrasse 34-36, D-45470 Mülheim an der Ruhr (Germany)

    2008-08-01

    This article describes the first successful crystallization of a membrane-bound [NiFe] hydrogenase isolated from a photosynthetic organism (A. vinosum). The crystals obtained produced diffraction patterns up to 2.5 Å resolution. The membrane-bound [NiFe] hydrogenase is a unique metalloprotein that is able to catalyze the reversible oxidation of hydrogen to protons and electrons during a complex reaction cycle. The [NiFe] hydrogenase was isolated from the photosynthetic purple sulfur bacterium Allochromatium vinosum and its crystallization and preliminary X-ray analysis are reported. It was crystallized by the hanging-drop vapour-diffusion method using sodium citrate and imidazole as crystallization agents. The crystals belong to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 205.00, b = 217.42, c = 120.44 Å. X-ray diffraction data have been collected to 2.5 Å resolution.

  1. Deposition of LDH on plasma treated polylactic acid to reduce water permeability

    KAUST Repository

    Bugatti, Valeria

    2013-04-01

    A simple and scalable deposition process was developed to prepare polylactic acid (PLA) coatings with enhanced water barrier properties for food packaging applications. This method based on electrostatic interactions between the positively charged layers of layered double hydroxides (LDHs) modified with ionic liquids (ILs) and the negatively charged plasma treated polylactic acid leads to homogeneous, stable, and highly durable coatings. Deposition of the LDH coatings increases the surface hydrophobicity of the neat PLA, which results to a decrease in water permeability by about 35%. © 2013 Elsevier Inc.

  2. Effect of Ultraviolet Aging on Rheology and Chemistry of LDH-Modified Bitumen

    OpenAIRE

    Xing Liu; Shaopeng Wu; Gang Liu; Liping Li

    2015-01-01

    Layered double hydroxides (LDHs) are an ultravioletlight (UV)-resistant material. In this study, two types of LDHs (Mg-Al-LDHs and Zn-Al-LDHs) were applied to modify bitumen by melt-blending. The effect of ultraviolet aging on the rheology and chemistry of LDH-modified bitumen was studied by means of dynamic shear rheometer (DSR), thin-layer chromatography with flame ionization detection (TLC-FID), Fourier transform infrared spectroscopy (FTIR), and Ultraviolet-Visible (UV-Vis) spectrophotome...

  3. LDH-A promotes malignant progression via activation of epithelial-to-mesenchymal transition and conferring stemness in muscle-invasive bladder cancer.

    Science.gov (United States)

    Jiang, Fujin; Ma, Song; Xue, Yubao; Hou, Jianquan; Zhang, Yongjie

    2016-01-22

    Lactate dehydrogenase-A(LDH-A) is an important rate-limiting enzyme in the Warburg effect. Survival analysis indicated poor clinical outcomes in MIBC with high LDH-A expression. The results of in vitro experiment indicated that LDH-A promotes MIBC cells proliferation, invasion and migration. The positive relationship between LDH-A expression and CSC/EMT markers was confirmed both in invasive bladder cell line and in 136 MIBC specimens. Thus, we conclude that LDH-A may be a promising target for MIBC.

  4. Guiding Principles of Hydrogenase Catalysis Instigated and Clarified by Protein Film Electrochemistry.

    Science.gov (United States)

    Armstrong, Fraser A; Evans, Rhiannon M; Hexter, Suzannah V; Murphy, Bonnie J; Roessler, Maxie M; Wulff, Philip

    2016-05-17

    Protein film electrochemistry (PFE) is providing cutting-edge insight into the chemical principles underpinning biological hydrogen. Attached to an electrode, many enzymes exhibit "reversible" electrocatalytic behavior, meaning that a catalyzed redox reaction appears reversible or quasi-reversible when viewed by cyclic voltammetry. This efficiency is most relevant for enzymes that are inspiring advances in renewable energy, such as hydrogen-activating and CO2-reducing enzymes. Exploiting the rich repertoire of available instrumental methods, PFE experiments yield both a general snapshot and fine detail, all from tiny samples of enzyme. The dynamic electrochemical investigations blaze new trails and add exquisite detail to the information gained from structural and spectroscopic studies. This Account describes recent investigations of hydrogenases carried out in Oxford, including ideas initiated with PFE and followed through with complementary techniques, all contributing to an eventual complete picture of fast and efficient H2 activation without Pt. By immobilization of an enzyme on an electrode, catalytic electron flow and the chemistry controlling it can be addressed at the touch of a button. The buried nature of the active site means that structures that have been determined by crystallography or spectroscopy are likely to be protected, retained, and fully relevant in a PFE experiment. An electrocatalysis model formulated for the PFE of immobilized enzymes predicts interesting behavior and gives insight into why some hydrogenases are H2 producers and others are H2 oxidizers. Immobilization also allows for easy addition and removal of inhibitors along with precise potential control, one interesting outcome being that formaldehyde forms a reversible complex with reduced [FeFe]-hydrogenases, thereby providing insight into the order of electron and proton transfers. Experiments on O2-tolerant [NiFe]-hydrogenases show that O2 behaves like a reversible inhibitor: it

  5. Equating salivary lactate dehydrogenase (LDH) with LDH-5 expression in patients with oral squamous cell carcinoma: An insight into metabolic reprogramming of cancer cell as a predictor of aggressive phenotype.

    Science.gov (United States)

    Saluja, Tajindra Singh; Spadigam, Anita; Dhupar, Anita; Syed, Shaheen

    2016-04-01

    Oral squamous cell carcinoma (OSCC) is the sixth most common human malignancy. According to World Health Organization, oral cancer has been reported to have the highest morbidity and mortality and a survival rate of approximately 50 % at 5 years from diagnosis. This is attributed to the subjectivity in TNM staging and histological grading which may result in less than optimum treatment outcomes including tumour recurrence. One of the hallmarks of cancer is aerobic glycolysis also known as the Warburg effect. This glycolytic phenotype (hypoxic state) not only confers immortality to cancer cells, but also correlates with the belligerent behaviour of various malignancies and is reflected as an increase in the expression of lactate dehydrogenase 5 (LDH-5), the main isoform of LDH catalysing the conversion of pyruvate to lactate during glycolysis. The diagnostic role of salivary LDH in assessing the metabolic phenotype of oral cancer has not been studied. Since salivary LDH is mainly sourced from oral epithelial cells, any pathological changes in the epithelium should reflect diagnostically in saliva. Thus in our current research, we made an attempt to ascertain the biological behaviour and aggressiveness of OSCC by appraising its metabolic phenotype as indirectly reflected in salivary LDH activity. We found that salivary LDH can be used to assess the aggressiveness of different histological grades of OSCC. For the first time, an evidence of differing metabolic behaviour in similar histologic tumour grade is presented. Taken together, our study examines the inclusion of salivary LDH as potential diagnostic parameter and therapeutic index in OSCC.

  6. 层状双羟基氢氧化物(LDH)的表面有机化研究%Surface Organizing of Layered Double Hydroxide (LDH)

    Institute of Scientific and Technical Information of China (English)

    杨巧珍; 李峰; 段雪

    2003-01-01

    采用成核晶化隔离法合成MgAl-CO32-型层状双羟基氢氧化物(LDH),并用不同偶联剂对其进行了表面有机化改性研究.通过X射线衍射(XRD)、红外光谱(IR)、X光电子能谱(XPS)、激光粒度分析和透射电镜(TEM)等手段探讨了偶联剂与LDH表面作用的原理.结果表明:硅烷偶联剂和铝钛复合偶联剂均可实现LDH的表面有机化,其中硅烷偶联剂AG-102的改性效果最好,而且用它改性的LDH在PVC中的团聚小,并具有较好的分散性,是一种理想的LDH表面改性剂.

  7. Development of a Rhodobacter capsulatus self-reporting model system for optimizing light-dependent, [FeFe]-hydrogenase-driven H 2 production: A Model System for Optimizing H 2 Production

    Energy Technology Data Exchange (ETDEWEB)

    Wecker, Matt S. A. [GeneBiologics, LLC, Boulder Colorado; Beaton, Stephen E. [United States Air Force Academy, Department of Chemistry, Colorado Springs Colorado; Chado, Robert A. [United States Air Force Academy, Department of Chemistry, Colorado Springs Colorado; Ghirardi, Maria L. [National Renewable Energy Laboratory, MS 3313, 15013 Denver West Parkway Golden Colorado 80401

    2016-08-23

    The photosynthetic bacterium Rhodobacter capsulatus normally photoproduces H2 as a by-product of its nitrogenase-catalyzed nitrogen-fixing activity. Such H2 production, however, is expensive from a metabolic perspective, requiring nearly four times as many photons as the equivalent algal hydrogenase-based system (Ghirardi et al. 2009). Here we report the insertion of a Clostridium acetobutylicum [FeFe]-hydrogenase and its three attendant hydrogenase assembly proteins into an R. capsulatus strain lacking its native uptake hydrogenase. Further, this strain is modified to fluoresce upon sensing H2. The resulting strain photoproduces H2 and self-reports its own H2 production through fluorescence. This model system represents a unique method of developing hydrogenase-based H2 production in R. capsulatus, may serve as a powerful system for in vivo directed evolution of hydrogenases and hydrogenase-associated genes, and provides a means of screening for increased metabolic production of H2.

  8. Hydrogenase activity in Azospirillum brasilense is inhibited by nitrite, nitric oxide, carbon monoxide, and acetylene

    Energy Technology Data Exchange (ETDEWEB)

    Tibelius, K.H.; Knowles, R.

    1984-10-01

    Nitrite, NO, CO, and C/sub 2/H/sub 2/ inhibited O/sub 2/-dependent H/sub 2/ uptake (H/sup 3/H oxidation) in denitrifying Azospirillum brasilense Sp7 grown anaerobically on N/sub 2/O or NO/sub 3//sup -/. The apparent K/sub i/ values for inhibition of O/sub 2/-dependent H/sub 2/ uptake were 20 ..mu..M for NO/sub 2//sup -/, 0.4 ..mu..M for NO, 28 ..mu..M for CO, and 88 ..mu..M for C/sub 2/H/sub 2/. These inhibitors also affected methylene blue-dependent H/sub 2/ uptake, presumably by acting directly on the hydrogenase. Nitrite and NO inhibited H/sub 2/ uptake irreversibly, whereas inhibition due to CO was easily reversed by repeatedly evacuating and backfilling with N/sub 2/. The C/sub 2/H/sub 2/ inhibition was not readily reversed, partly due to difficulty in removing the last traces of this gas from solution. The NO/sub 2//sup -/ inhibition of malate-dependent respiration was readily reversed by repeatedly washing the cells, in contrast to the effect of NO/sub 2//sup -/ on H/sub 2/-dependent respiration. These results suggest that the low hydrogenase activities observed in NO/sub 3//sup -/-grown cultures of A. brasilense may be due to the irreversible inhibition of hydrogenase by NO/sub 2//sup -/ and NO produced by NO/sub 3//sup -/ reduction.

  9. Transcription and Regulation of the Bidirectional Hydrogenase in the Cyanobacterium Nostoc sp. Strain PCC 7120▿

    Science.gov (United States)

    Sjöholm, Johannes; Oliveira, Paulo; Lindblad, Peter

    2007-01-01

    The filamentous, heterocystous cyanobacterium Nostoc sp. strain PCC 7120 (Anabaena sp. strain PCC 7120) possesses an uptake hydrogenase and a bidirectional enzyme, the latter being capable of catalyzing both H2 production and evolution. The completely sequenced genome of Nostoc sp. strain PCC 7120 reveals that the five structural genes encoding the bidirectional hydrogenase (hoxEFUYH) are separated in two clusters at a distance of approximately 8.8 kb. The transcription of the hox genes was examined under nitrogen-fixing conditions, and the results demonstrate that the cluster containing hoxE and hoxF can be transcribed as one polycistronic unit together with the open reading frame alr0750. The second cluster, containing hoxU, hoxY, and hoxH, is transcribed together with alr0763 and alr0765, located between the hox genes. Moreover, alr0760 and alr0761 form an additional larger operon. Nevertheless, Northern blot hybridizations revealed a rather complex transcription pattern in which the different hox genes are expressed differently. Transcriptional start points (TSPs) were identified 66 and 57 bp upstream from the start codon of alr0750 and hoxU, respectively. The transcriptions of the two clusters containing the hox genes are both induced under anaerobic conditions concomitantly with the induction of a higher level of hydrogenase activity. An additional TSP, within the annotated alr0760, 244 bp downstream from the suggested translation start codon, was identified. Electrophoretic mobility shift assays with purified LexA from Nostoc sp. strain PCC 7120 demonstrated specific interactions between the transcriptional regulator and both hox promoter regions. However, when LexA from Synechocystis sp. strain PCC 6803 was used, the purified protein interacted only with the promoter region of the alr0750-hoxE-hoxF operon. A search of the whole Nostoc sp. strain PCC 7120 genome demonstrated the presence of 216 putative LexA binding sites in total, including recA and rec

  10. Modelling NiFe hydrogenases: nickel-based electrocatalysts for hydrogen production

    Energy Technology Data Exchange (ETDEWEB)

    Canaguier, S.; Artero, V.; Fontecave, M. [CEA, DSV, iRTSV, Lab Chim Biol Metaux, CEA-CNRS-Univ Grenoble 1, UMR 5249, F-38054 Grenoble 9 (France)

    2008-07-01

    NiFe hydrogenases are unique enzymes that catalyze the H{sup +}/H{sub 2} interconversion with remarkable efficiency. The determination of the tridimensional structure of their active site (a sulfur-rich dinuclear nickel-iron cluster with diatomic cyanide and carbonyl ligands) has stimulated the synthesis of a variety of nickel-based complexes as potential electrocatalysts for hydrogen production. These catalysts may provide an adequate alternative to platinum. This paper gives an historical perspective of this biomimetic structural approach and then focusses on recently reported bio-inspired functional mimics displaying electrocatalytic activity for hydrogen production. (authors)

  11. Effect of Ultraviolet Aging on Rheology and Chemistry of LDH-Modified Bitumen

    Directory of Open Access Journals (Sweden)

    Xing Liu

    2015-08-01

    Full Text Available Layered double hydroxides (LDHs are an ultravioletlight (UV-resistant material. In this study, two types of LDHs (Mg-Al-LDHs and Zn-Al-LDHs were applied to modify bitumen by melt-blending. The effect of ultraviolet aging on the rheology and chemistry of LDH-modified bitumen was studied by means of dynamic shear rheometer (DSR, thin-layer chromatography with flame ionization detection (TLC-FID, Fourier transform infrared spectroscopy (FTIR, and Ultraviolet-Visible (UV-Vis spectrophotometry to reveal the mechanisms of action for LDHs and bitumen. The results showed that within the UV spectra (220–400 nm, the reflectance of Zn-Al-LDHs was larger than that of Mg-Al-LDHs. These two LDHs have different influences on the performance of bitumen. Mg-Al-LDHs had a more obvious influence on the physical and dynamic rheological properties of bitumen than Zn-Al-LDHs. Zn-Al-LDHs improved the UV-aging resistance of bitumen more. The reason can be that the reflectance of the Zn-Al-LDHs to the UV light is larger than that of the Mg-Al-LDHs. The Zn-Al-LDH-modified bitumen had more potential to improve the UV-aging resistance during the service life of asphalt pavement.

  12. Molecular detection of the clostridia in an anaerobic biohydrogen fermentation system by hydrogenase mRNA-targeted reverse transcription-PCR.

    Science.gov (United States)

    Chang, Jui-Jen; Chen, Wei-En; Shih, Shiou-Yun; Yu, Sian-Jhong; Lay, Jiunn-Jyi; Wen, Fu-Shyan; Huang, Chieh-Chen

    2006-05-01

    Molecular biological approaches were developed to monitor the potential biohydrogen-producing clostridia in an anaerobic semisolid fermentation system that used brewery yeast waste as the fermentation substrate. The denaturing gradient gel electrophoresis with 16S rDNA gene-targeted polymerase chain reaction (PCR) analysis was employed to confirm the existence of clostridia in the system. Remarkably, reproducible nucleotide sequences of clostridia were obtained from different hydrogen production stages by using hydrogenase gene-targeted reverse transcription (RT)-PCR. These RNA-based information suggested that the predominant hydrogen-producing strains possess either a specific Clostridium pasteurianum-like or a specific Clostridium saccharobutylicum-like hydrogenase sequence. Comparison of the hydrogenase gene-targeted sequence profiles between PCR and RT-PCR revealed that the specific C. pasteurianum-like hydrogenase harboring bacterial strains were dominant in both mRNA and bacterial population level. On the other hand, the specific C. saccharobutylicum-like hydrogenase harboring strains expressed high level of hydrogenase mRNA but may not be dominant in population. Furthermore, quantitative real-time RT-PCR analysis showed the expression pattern of the clostridial hydrogenase mRNA and may serve as an activity index for the system.

  13. Deletion of a gene cluster for [Ni-Fe] hydrogenase maturation in the anaerobic hyperthermophilic bacterium Caldicellulosiruptor bescii identifies its role in hydrogen metabolism.

    Science.gov (United States)

    Cha, Minseok; Chung, Daehwan; Westpheling, Janet

    2016-02-01

    The anaerobic, hyperthermophlic, cellulolytic bacterium Caldicellulosiruptor bescii grows optimally at ∼80 °C and effectively degrades plant biomass without conventional pretreatment. It utilizes a variety of carbohydrate carbon sources, including both C5 and C6 sugars, released from plant biomass and produces lactate, acetate, CO2, and H2 as primary fermentation products. The C. bescii genome encodes two hydrogenases, a bifurcating [Fe-Fe] hydrogenase and a [Ni-Fe] hydrogenase. The [Ni-Fe] hydrogenase is the most widely distributed in nature and is predicted to catalyze hydrogen production and to pump protons across the cellular membrane creating proton motive force. Hydrogenases are the key enzymes in hydrogen metabolism and their crystal structure reveals complexity in the organization of their prosthetic groups suggesting extensive maturation of the primary protein. Here, we report the deletion of a cluster of genes, hypABFCDE, required for maturation of the [Ni-Fe] hydrogenase. These proteins are specific for the hydrogenases they modify and are required for hydrogenase activity. The deletion strain grew more slowly than the wild type or the parent strain and produced slightly less hydrogen overall, but more hydrogen per mole of cellobiose. Acetate yield per mole of cellobiose was increased ∼67 % and ethanol yield per mole of cellobiose was decreased ∼39 %. These data suggest that the primary role of the [Ni-Fe] hydrogenase is to generate a proton gradient in the membrane driving ATP synthesis and is not the primary enzyme for hydrogen catalysis. In its absence, ATP is generated from increased acetate production resulting in more hydrogen produced per mole of cellobiose.

  14. Can elevated lactate and LDH produce a false positive enzymatic ethanol result in live patients presenting to the emergency department?

    Science.gov (United States)

    Nacca, Nicholas; Hodgman, Michael J; Lao, Kirselle; Elkins, Matthew; Holland, Michael G

    2017-08-16

    There have been allegations in the courtroom that elevated serum lactic acid in trauma victims can yield a falsely elevated serum ethanol assay. Most hospitals utilize an indirect method of ethanol measurement where a serum sample is added to a mix of alcohol dehydrogenase and oxidized nicotinamide adenine dinucleotide (NAD+). This allows any ethanol in the patient's serum to be metabolized to acetaldehyde, and in the process results in the reduction of NAD + to NADH. NADH is then measured using spectrophotometry. The courtroom allegation stems from the concept that oxidation of lactate to pyruvate by lactate dehydrogenase (LDH) results in the same molar-for-molar reduction of NAD + to NADH, and could therefore theoretically cause patients with elevated lactate and LDH to have a falsely elevated ethanol concentration. Patients with elevated lactic acid and LDH concentrations who presented to a university hospital from 20 April 2015 to 13 December 2015 were identified to provide possible test specimens. If a sufficient amount of serum was available, the sample was used to re-run the lactate and LDH concentration simultaneously with an enzymatic ethanol assay. Any samples that had elevated lactic acid and LDH concentrations on this retesting, and also yielded a positive ethanol concentration, were sent for confirmatory gas chromatography testing of ethanol concentrations. A control group of 20 samples with normal lactate and LDH were included. A total of 37 samples were included in the final analysis. Only 4 patients had an elevated enzymatic ethanol concentration, and all 4 also had a measurable GC ethanol concentration. The lactate in this dataset ranged from 2.4 to 24.2 mmol/L, with a mean of 6.53 mmol/L (normal value 0.5-2.2). The LDH ranged from 242 to 8838 U/L with a mean of 1695 U/L (normal value 122-225 U/L). Twenty control samples were run on patients with normal lactate and LDH, none of which yielded a positive enzymatic ethanol result

  15. Ni adsorption and Ni-Al LDH precipitation in a sandy aquifer: an experimental and mechanistic modeling study.

    Science.gov (United States)

    Regelink, Inge C; Temminghoff, Erwin J M

    2011-03-01

    Mining activities and industries have created nickel (Ni) contaminations in many parts of the world. The objective of this study is to increase our understanding of Ni adsorption and Nickel-Aluminium Layered Double Hydroxide (Ni-Al LDH) precipitation to reduce Ni mobility in a sandy soil aquifer. At pH ≥ 7.2 both adsorption and Ni-Al LDH precipitation occurred. In batch experiments with the sandy soil up to 70% of oxalate-extractable Al was taken up in LDH formation during 56 days. In a long term column experiment 99% of influent Ni was retained at pH 7.5 due to Ni adsorption (≈ 34%) and Ni-Al LDH precipitation (≈ 66%) based on mechanistic reactive transport modeling. The subsequent leaching at pH 6.5 could be largely attributed to desorption. Our results show that even in sandy aquifers with relatively low Al content, Ni-Al LDH precipitation is a promising mechanism to immobilize Ni.

  16. 弓形虫乳酸脱氢酶LDH2基因启动子的克隆及鉴定%Cloning of Toxoplasma gondii LDH2 gene promoter and evaluation of its promoting activity

    Institute of Scientific and Technical Information of China (English)

    张加勤; 侯香华; 宋秀宇

    2013-01-01

    To clone promoter fragment of Toxoplasma gondii LDH2 gene and to evaluate the promoting activity, a series of fragments of 5 UTR of Toxoplasma gondii LDH2 gene were amplified by PCR, and then inserted into pTX3-basic via Kpn I and Xhol I to construct luciferase reporter plasmids. Forty eight hours after tachyzoites being co-transfected by luciferase reporter plasmids and reference plasmid pRL-Tg, dual-lucif erase assays were carried out to determinate activities of the fragments to initiate luciferase transcription. Results showed that the sequence of the 5 UTR of Toxoplasma gondii LDH2 gene fragments was proved to be correct by sequencing, and a series of luciferase reporter plasmids were verified by PCR, restrictive endonuclease digest, and sequencing. The luciferase activities of all the luciferase reporter plasmids with 5 UTR of Toxoplasma gondii LDH2 in tachyzoites were similar with the negative control pTX3-basic. While in bradyzoites, all the plasmids yielded 40 to 50 folds of luciferase activity to the negative control, and 1. 2 to 1. 5 folds to the positive control of pTX3-basic, excepting for the plasmids pTX3-293 and pTX3-193. In conclusion, the promoter region of Toxoplasma gondii LDH2 was located and cloned successfully. Our results provide evidence for investigation of the mechanism on regulation of the LDH2 gene.%目的 克隆弓形虫乳酸脱氢酶LDH2基因启动子并进行初步鉴定.方法 PCR扩增出LDH2基因5′侧翼序列系列截短突变体,定向插入载体pTX3–basic的Kpn I / Xhol I酶切位点之间,转化大肠埃希菌Top 10,氨苄西林筛选,建立重组LDH2基因5′侧翼序列系列截短突变体荧光素酶表达载体.转染刚地弓形虫速殖子,利用荧光素酶检测试剂盒测定所克隆片段的启动转录活性.结果 成功扩增出LDH2基因5′侧翼序列系列突变体;系列重组荧光素酶表达质粒经PCR、酶切及测序鉴定正确无误;系列重组荧光素酶表达质粒在弓形虫速殖

  17. Isolation of genes (nif/hup cosmids) involved in hydrogenase and nitrogenase activities in Rhizobium japonicum.

    Science.gov (United States)

    Hom, S S; Graham, L A; Maier, R J

    1985-03-01

    Recombinant cosmids containing a Rhizobium japonicum gene involved in both hydrogenase (Hup) and nitrogenase (Nif) activities were isolated. An R. japonicum gene bank utilizing broad-host-range cosmid pLAFR1 was conjugated into Hup- Nif- R. japonicum strain SR139. Transconjugants containing the nif/hup cosmid were identified by their resistance to tetracycline (Tcr) and ability to grow chemoautotrophically (Aut+) with hydrogen. All Tcr Aut+ transconjugants possessed high levels of H2 uptake activity, as determined amperometrically. Moreover, all Hup+ transconjugants tested possessed the ability to reduce acetylene (Nif+) in soybean nodules. Cosmid DNAs from 19 Hup+ transconjugants were transferred to Escherichia coli by transformation. When the cosmids were restricted with EcoRI, 15 of the 19 cosmids had a restriction pattern with 13.2-, 4.0-, 3.0-, and 2.5-kilobase DNA fragments. Six E. coli transformants containing the nif/hup cosmids were conjugated with strain SR139. All strain SR139 transconjugants were Hup+ Nif+. Moreover, one nif/hup cosmid was transferred to 15 other R. japonicum Hup- mutants. Hup+ transconjugants of six of the Hup- mutants appeared at a frequency of 1.0, whereas the transconjugants of the other nine mutants remained Hup-. These results indicate that the nif/hup gene cosmids contain a gene involved in both nitrogenase and hydrogenase activities and at least one and perhaps other hup genes which are exclusively involved in H2 uptake activity.

  18. Distal [FeS]-Cluster Coordination in [NiFe]-Hydrogenase Facilitates Intermolecular Electron Transfer.

    Science.gov (United States)

    Petrenko, Alexander; Stein, Matthias

    2017-01-05

    Biohydrogen is a versatile energy carrier for the generation of electric energy from renewable sources. Hydrogenases can be used in enzymatic fuel cells to oxidize dihydrogen. The rate of electron transfer (ET) at the anodic side between the [NiFe]-hydrogenase enzyme distal iron-sulfur cluster and the electrode surface can be described by the Marcus equation. All parameters for the Marcus equation are accessible from Density Functional Theory (DFT) calculations. The distal cubane FeS-cluster has a three-cysteine and one-histidine coordination [Fe₄S₄](His)(Cys)₃ first ligation sphere. The reorganization energy (inner- and outer-sphere) is almost unchanged upon a histidine-to-cysteine substitution. Differences in rates of electron transfer between the wild-type enzyme and an all-cysteine mutant can be rationalized by a diminished electronic coupling between the donor and acceptor molecules in the [Fe₄S₄](Cys)₄ case. The fast and efficient electron transfer from the distal iron-sulfur cluster is realized by a fine-tuned protein environment, which facilitates the flow of electrons. This study enables the design and control of electron transfer rates and pathways by protein engineering.

  19. Electrochemistry of Simple Organometallic Models of Iron-Iron Hydrogenases in Organic Solvent and Water.

    Science.gov (United States)

    Gloaguen, Frederic

    2016-01-19

    Synthetic models of the active site of iron-iron hydrogenases are currently the subjects of numerous studies aimed at developing H2-production catalysts based on cheap and abundant materials. In this context, the present report offers an electrochemist's view of the catalysis of proton reduction by simple binuclear iron(I) thiolate complexes. Although these complexes probably do not follow a biocatalytic pathway, we analyze and discuss the interplay between the reduction potential and basicity and how these antagonist properties impact the mechanisms of proton-coupled electron transfer to the metal centers. This question is central to any consideration of the activity at the molecular level of hydrogenases and related enzymes. In a second part, special attention is paid to iron thiolate complexes holding rigid and unsaturated bridging ligands. The complexes that enjoy mild reduction potentials and stabilized reduced forms are promising iron-based catalysts for the photodriven evolution of H2 in organic solvents and, more importantly, in water.

  20. Hydride bridge in [NiFe]-hydrogenase observed by nuclear resonance vibrational spectroscopy

    Science.gov (United States)

    Ogata, Hideaki; Krämer, Tobias; Wang, Hongxin; Schilter, David; Pelmenschikov, Vladimir; van Gastel, Maurice; Neese, Frank; Rauchfuss, Thomas B.; Gee, Leland B.; Scott, Aubrey D.; Yoda, Yoshitaka; Tanaka, Yoshihito; Lubitz, Wolfgang; Cramer, Stephen P.

    2015-08-01

    The metabolism of many anaerobes relies on [NiFe]-hydrogenases, whose characterization when bound to substrates has proven non-trivial. Presented here is direct evidence for a hydride bridge in the active site of the 57Fe-labelled fully reduced Ni-R form of Desulfovibrio vulgaris Miyazaki F [NiFe]-hydrogenase. A unique `wagging' mode involving H- motion perpendicular to the Ni(μ-H)57Fe plane was studied using 57Fe-specific nuclear resonance vibrational spectroscopy and density functional theory (DFT) calculations. On Ni(μ-D)57Fe deuteride substitution, this wagging causes a characteristic perturbation of Fe-CO/CN bands. Spectra have been interpreted by comparison with Ni(μ-H/D)57Fe enzyme mimics [(dppe)Ni(μ-pdt)(μ-H/D)57Fe(CO)3]+ and DFT calculations, which collectively indicate a low-spin Ni(II)(μ-H)Fe(II) core for Ni-R, with H- binding Ni more tightly than Fe. The present methodology is also relevant to characterizing Fe-H moieties in other important natural and synthetic catalysts.

  1. Optimization of detecting hydrogenase activity for acidogenic fermentation of activated sludge

    Institute of Scientific and Technical Information of China (English)

    ZHENG Guo-chen; HE Jun-guo; LI Jian-zheng; AJAY Kumar Jha; ZHANG Li-guo

    2010-01-01

    In order to evaluate the hydrogen-producing efficiency of anaerobic activated sludge in Anaerobic Baffled Reactor(ABR)fermentation processes,the optimal conditions for hydrogen producing hydrogenase method on methyl viologen(MV)assay was used to detect the hydrogen production activity of the activated sludge.The most favorable parameters such as 0.6 mL sodium acetate buffer(pH 5.0),100 μL lysozyme,0.2 mL sodium di bromoethane(9.0 mmol/L)and 0.7 mmol/L iron added into 1 mL activated sludge(2.66~26.64 gMLVSS/L)were found.Furthermore,reaction temperature and culture time were detected as 40 ℃ and 30 min respectively.Sodium thiosulfate and sodium sulfides were taken as the reducing agent while trichloroacetic acid as terminator.Under the MV optimal conditions,micro-texic Dimethyl sulfoxide(DMSO)get higher security and better accuracy.The sensitivity of the detection methods(DMSO as electron carrier)was increased by more than30%.The results show that the optimal conditions can be applied to measure hydrogenase activity correlating with its specific hydrogen production rate in a hydrogen-producing anaerobic activated sludge system.

  2. Using in vitro maturation and cell-free expression to explore [FeFe] hydrogenase activation and protein scaffolding requirements

    Energy Technology Data Exchange (ETDEWEB)

    Swartz, James [Stanford Univ., CA (United States)

    2017-01-25

    Final Project Report describing work to elucidate mechanisms for the activation of [FeFe]-hydrogenases and to explore the impact of the polypeptide scaffolding on the function of the Fe-S redox and catalytic centers with emphasis on improving oxygen tolerance.

  3. O2-stable membrane-bound [NiFe]hydrogenase from a newly isolated Citrobacter sp. S-77.

    Science.gov (United States)

    Eguchi, Shigenobu; Yoon, Ki-Seok; Ogo, Seiji

    2012-11-01

    Hydrogenases are of great interest due to their potential use in H(2)-based technology. However, most hydrogenases are highly sensitive to O(2), which have been the major bottleneck in hydrogenase studies. Here we report an O(2)-stable membrane-bound [NiFe]hydrogenase (MBH) purified from a newly isolated strain, S-77. According to the 16S rRNA gene sequence and phylogenetic analysis of the strain S-77, it belongs to the genus of Citrobacter. In vitro experiments using the cytoplasmic membrane of strain S-77 suggested that a cytochrome b acts as the physiological electron acceptor of the MBH. The purified MBH was composed of a dimer of heterodimers, consisting of two distinct subunits with the molecular weights of 58.5 and 38.5 kDa. The enzyme showed a specific activity for H(2)-oxidation of 661 U/mg, which is 35-fold greater than that for H(2)-production of 18.7 U/mg. Notably, the MBH showed a remarkable O(2)-stability, maintaining almost 95% of its original activity even after incubation for 30 h in air at 4°C. These results suggest that the O(2)-stable MBH may play an important role in the H(2)-metabolic pathway under the aerobic conditions of Citrobacter sp. S-77. This is the first report of the purification and biochemical characterization of an O(2)-stable MBH from the genus of Citrobacter.

  4. Patterns of [FeFe] hydrogenase diversity in the gut microbial communities of lignocellulose-feeding higher termites.

    Science.gov (United States)

    Ballor, Nicholas R; Leadbetter, Jared R

    2012-08-01

    Hydrogen is the central free intermediate in the degradation of wood by termite gut microbes and can reach concentrations exceeding those measured for any other biological system. Degenerate primers targeting the largest family of [FeFe] hydrogenases observed in a termite gut metagenome have been used to explore the evolution and representation of these enzymes in termites. Sequences were cloned from the guts of the higher termites Amitermes sp. strain Cost010, Amitermes sp. strain JT2, Gnathamitermes sp. strain JT5, Microcerotermes sp. strain Cost008, Nasutitermes sp. strain Cost003, and Rhyncotermes sp. strain Cost004. Each gut sample harbored a more rich and evenly distributed population of hydrogenase sequences than observed previously in the guts of lower termites and Cryptocercus punctulatus. This accentuates the physiological importance of hydrogen for higher termite gut ecosystems and may reflect an increased metabolic burden, or metabolic opportunity, created by a lack of gut protozoa. The sequences were phylogenetically distinct from previously sequenced [FeFe] hydrogenases. Phylogenetic and UniFrac comparisons revealed congruence between host phylogeny and hydrogenase sequence library clustering patterns. This may reflect the combined influences of the stable intimate relationship of gut microbes with their host and environmental alterations in the gut that have occurred over the course of termite evolution. These results accentuate the physiological importance of hydrogen to termite gut ecosystems.

  5. Structure of an Actinobacterial-Type [NiFe]-Hydrogenase Reveals Insight into O2-Tolerant H2 Oxidation.

    Science.gov (United States)

    Schäfer, Caspar; Bommer, Martin; Hennig, Sandra E; Jeoung, Jae-Hun; Dobbek, Holger; Lenz, Oliver

    2016-02-01

    A novel group of bacterial [NiFe]-hydrogenases is responsible for high-affinity H2 uptake from the troposphere, and is therefore thought to play an important role in the global H2 cycle. Here we present the first crystal structure at 2.85-Å resolution of such an actinobacterial-type hydrogenase (AH), which was isolated from the dihydrogen oxidizing bacterium, Ralstonia eutropha. The enzyme has a dimeric structure carrying two active [NiFe] sites that are interconnected by six [4Fe4S] clusters over a range of approximately 90 Å. Unlike most other [NiFe]-hydrogenases, the [4Fe4S] cluster proximal to the [NiFe] site is coordinated by three cysteines and one aspartate. Mutagenesis experiments revealed that this aspartate residue is related to the apparent O2 insensitivity of the AH. Our data provide first structural insight into specialized hydrogenases that are supposed to consume atmospheric H2 under challenging conditions, i.e. at high O2 concentration and wide temperature and pH ranges.

  6. Structural and gene expression analyses of uptake hydrogenases and other proteins involved in nitrogenase protection in Frankia

    Indian Academy of Sciences (India)

    K H Richau; R L Kudahettige; P Pujic; N P Kudahettige; A Sellstedt

    2013-11-01

    The actinorhizal bacterium Frankia expresses nitrogenase and can therefore convert molecular nitrogen into ammonia and the by-product hydrogen. However, nitrogenase is inhibited by oxygen. Consequently, Frankia and its actinorhizal hosts have developed various mechanisms for excluding oxygen from their nitrogen-containing compartments. These include the expression of oxygen-scavenging uptake hydrogenases, the formation of hopanoid-rich vesicles, enclosed by multi-layered hopanoid structures, the lignification of hyphal cell walls, and the production of haemoglobins in the symbiotic nodule. In this work, we analysed the expression and structure of the so-called uptake hydrogenase (Hup), which catalyses the in vivo dissociation of hydrogen to recycle the energy locked up in this ‘waste’ product. Two uptake hydrogenase syntons have been identified in Frankia: synton 1 is expressed under free-living conditions while synton 2 is expressed during symbiosis. We used qPCR to determine synton 1 hup gene expression in two Frankia strains under aerobic and anaerobic conditions. We also predicted the 3D structures of the Hup protein subunits based on multiple sequence alignments and remote homology modelling. Finally, we performed BLAST searches of genome and protein databases to identify genes that may contribute to the protection of nitrogenase against oxygen in the two Frankia strains. Our results show that in Frankia strain ACN14a, the expression patterns of the large (HupL1) and small (HupS1) uptake hydrogenase subunits depend on the abundance of oxygen in the external environment. Structural models of the membrane-bound hydrogenase subunits of ACN14a showed that both subunits resemble the structures of known [NiFe] hydrogenases (Volbeda et al. 1995), but contain fewer cysteine residues than the uptake hydrogenase of the Frankia DC12 and Eu1c strains. Moreover, we show that all of the investigated Frankia strains have two squalene hopane cyclase genes (shc1 and shc2

  7. Optical and UV-Aging Properties of LDH-Modified Bitumen

    Directory of Open Access Journals (Sweden)

    Xing Liu

    2015-07-01

    Full Text Available Layered double hydroxides (LDHs are an ultraviolet-light (UV resistant material. In this study, LDHs were used to modify bitumen. The optical and UV aging properties of LDHs modified bitumen were investigated. Firstly, the thin films of bitumen, with and without LDHs, were prepared. By using the UV-Vis spectrophotometer, absorbance, reflectance, and transmittance of bituminous thin film were evaluated. The morphology of LDHs-modified bitumen was observed by using fluorescence microscopy (FM. Finally, the aging resistance of LDH-modified bitumen was investigated by using the UV-aging oven. Results indicated that the LDHs, especially with 5 wt % in the bitumen, can effectively absorb and reflect the UV light and improve the UV-aging resistance of bitumen. This implied that the addition of LDHs into bitumen had the potential to prolong the service life of asphalt pavement.

  8. In situ synthesis of MOF membranes on ZnAl-CO3 LDH buffer layer-modified substrates.

    Science.gov (United States)

    Liu, Yi; Wang, Nanyi; Pan, Jia Hong; Steinbach, Frank; Caro, Jürgen

    2014-10-15

    We develop here a urea hydrolysis method to in situ prepare asymmetric ZnAl-CO3 layered double hydroxide (LDH) buffer layers with various stable equilibrium morphology on porous Al2O3 substrates. In particular it is found that well-intergrown ZIF-8 membranes can be directly synthesized on the ZnAl-CO3 LDH buffer layer-modified substrates, owing to the specific metal-imidazole interaction between ZnAl-CO3 LDHs and ZIF-8. Other Zn-based MOF membranes, like ZIF-7 and ZIF-90, can also be synthesized with this method. Our finding demonstrates that LDH buffer layer represents a new concept for substrate modification.

  9. Characterization of Hydrogen Metabolism in the Multicellular Green Alga Volvox carteri.

    Directory of Open Access Journals (Sweden)

    Adam J Cornish

    Full Text Available Hydrogen gas functions as a key component in the metabolism of a wide variety of microorganisms, often acting as either a fermentative end-product or an energy source. The number of organisms reported to utilize hydrogen continues to grow, contributing to and expanding our knowledge of biological hydrogen processes. Here we demonstrate that Volvox carteri f. nagariensis, a multicellular green alga with differentiated cells, evolves H2 both when supplied with an abiotic electron donor and under physiological conditions. The genome of Volvox carteri contains two genes encoding putative [FeFe]-hydrogenases (HYDA1 and HYDA2, and the transcripts for these genes accumulate under anaerobic conditions. The HYDA1 and HYDA2 gene products were cloned, expressed, and purified, and both are functional [FeFe]-hydrogenases. Additionally, within the genome the HYDA1 and HYDA2 genes cluster with two putative genes which encode hydrogenase maturation proteins. This gene cluster resembles operon-like structures found within bacterial genomes and may provide further insight into evolutionary relationships between bacterial and algal [FeFe]-hydrogenase genes.

  10. ANTS-anchored Zn-Al-CO3-LDH particles as fluorescent probe for sensing of folic acid

    Science.gov (United States)

    Liu, Pengfei; Liu, Dan; Liu, Yanhuan; Li, Lei

    2016-09-01

    A novel fluorescent nanosensor for detecting folic acid (FA) in aqueous media has been developed based on 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) anchored to the surface of Zn-Al-CO3-layered double hydroxides (LDH) particles. The nanosensor showed high fluorescence intensity and good photostability due to a strong coordination interaction between surface Zn2+ ions of Zn-Al-CO3-LDH and N atoms of ANTS, which were verified by result of X-ray photoelectron spectroscopy (XPS). ANTS-anchored on the surface of Zn-Al-CO3-LDH restricted the intra-molecular rotation leading to ANTS-anchored J-type aggregation emission enhancement. ANTS-anchored Zn-Al-CO3-LDH particles exhibited highly sensitive and selective response to FA over other common metal ions and saccharides present in biological fluids. The proposed mechanism was that oxygen atoms of -SO3 groups in ANTS-anchored on the surface of Zn-Al-CO3-LDH were easily collided by FA molecules to form potential hydrogen bonds between ANTS-anchored and FA molecules, which could effectively quench the ANTS-anchored fluorescence. Under the simulated physiological conditions (pH of 7.4), the fluorescence quenching was fitted to Stern-Volmer equation with a linear response in the concentration range of 1 μM to 200 μM with a limit of detection of 0.1 μM. The results indicate that ANTS-anchored Zn-Al-CO3-LDH particles can afford a very sensitive system for the sensing FA in aqueous solution.

  11. Technical note: comparison of the PrestoBlue and LDH release assays with the MTT assay for skin viability assessment.

    Science.gov (United States)

    Gaucher, Sonia; Jarraya, Mohamed

    2015-09-01

    MTT assay is the gold standard for assessing skin sample viability but it is time-consuming. Here we compared the MTT test with two other assays for the assessment of skin viability. The MTT, PrestoBlue (colorimetric method) and LDH release assays were applied to fresh and cryopreserved skin. Skin viability was considered proportional to the optical density values of the relevant analytes. PrestoBlue did not reliably distinguish between fresh and cryopreserved skin. The LDH release assay did not allow us to establish a viability index. We recommend the MTT assay for assessing skin viability.

  12. Is engineering O{sub 2}-tolerant hydrogenases just a matter of reproducing the active sites of the naturally occurring O{sub 2}-resistant enzymes?

    Energy Technology Data Exchange (ETDEWEB)

    Leroux, Fanny; Liebgott, Pierre-Pol; Kpebe, Arlette; Leger, Christophe; Rousset, Marc; Dementin, Sebastien [CNRS, Laboratoire de Bioenergetique et Ingenierie des Proteines, Institut de Microbiologie de la Mediterranee, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20 (France); Cournac, Laurent; Richaud, Pierre [CEA, DSV, IBEB, Laboratoire de Bioenergetique et Biotechnologie des Bacteries et Microalgues, 13108 Saint-Paul-lez-Durance (France); Aix-Marseille Universite, 3 place Victor-Hugo, 13331 Marseille (France); CNRS, UMR Biologie Vegetale et Microbiologie Environnementales, 13108 Saint-Paul-lez-Durance (France); Burlat, Benedicte; Guigliarelli, Bruno; Bertrand, Patrick [CNRS, Laboratoire de Bioenergetique et Ingenierie des Proteines, Institut de Microbiologie de la Mediterranee, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20 (France); Aix-Marseille Universite, 3 place Victor-Hugo, 13331 Marseille (France)

    2010-10-15

    Reproducing the naturally occurring O{sub 2}-tolerant hydrogenases is a potential strategy to make the oxygen sensitive enzymes, produced by organisms of biotechnological interest, more resistant. The search for resistance ''hotspots'' that could be transposed into sensitive hydrogenases is underway. Here, we replaced two residues (Y77 and V78) of the oxygen sensitive [NiFe] hydrogenase from Desulfovibrio fructosovorans with Gly and with Cys, respectively, to copy the active site pocket of the resistant membrane-bound [NiFe] enzyme from Ralstonia eutropha and we examined how this affected oxygen sensitivity. The results are discussed in the light of a short review of the recent results dealing with the reactivity of hydrogenases towards oxygen. (author)

  13. Testis-Specific Lactate Dehydrogenase (LDH-C4) in Skeletal Muscle Enhances a Pika’s Sprint-Running Capacity in Hypoxic Environment

    Science.gov (United States)

    Wang, Yang; Wei, Lian; Wei, Dengbang; Li, Xiao; Xu, Lina; Wei, Linna

    2015-01-01

    LDH-C4 is a lactate dehydrogenase that catalyzes the conversion of pyruvate to lactate. In mammals, ldh-c was originally thought to be expressed only in testis and spermatozoa. Plateau pika (Ochotona curzoniae), which belongs to the genus Ochotona of the Ochotonidea family, is a hypoxia tolerant mammal living 3000–5000 m above sea level on the Qinghai-Tibet Plateau, an environment which is strongly hypoxic. Ldh-c is expressed not only in testis and sperm but also in somatic tissues of plateau pika. In this study, the effects of N-propyl oxamate and N-isopropyl oxamate on LDH isozyme kinetics were compared to screens for a selective inhibitor of LDH-C4. To reveal the role and physiological mechanism of LDH-C4 in skeletal muscle of plateau pika, we investigated the effect of N-isopropyl oxamate on the pika exercise tolerance as well as the physiological mechanism. Our results show that Ki of N-propyl oxamate and N-isopropyl oxamate for LDH-A4, LDH-B4, and LDH-C4 were 0.094 mmol/L and 0.462 mmol/L, 0.119 mmol/L and 0.248 mmol/L, and 0.015 mmol/L and 0.013 mmol/L, respectively. N-isopropyl oxamate is a powerful selective inhibitor of plateau pika LDH-C4. In our exercise tolerance experiment, groups treated with inhibitors had significantly lower swimming times than the uninhibited control group. The inhibition rates of LDH, LD, and ATP were 37.12%, 66.27%, and 32.42%, respectively. Our results suggested that ldh-c is expressed in the skeletal muscle of plateau pika, and at least 32.42% of ATP in the skeletal muscle is catalyzed by LDH-C4 by anaerobic glycolysis. This suggests that pika has reduced dependence on oxygen and enhanced adaptation to hypoxic environment due to increased anaerobic glycolysis by LDH-C4 in skeletal muscle. LDH-C4 in plateau pika plays the crucial role in anaerobic glycolysis and generates ATP rapidly since this is the role of LDH-A4 in most species on plain land, which provide evidence that the native humans and animals in Qinghai

  14. Testis-Specific Lactate Dehydrogenase (LDH-C4 in Skeletal Muscle Enhances Apika’s Sprint-Running Capacity in Hypoxic Environment

    Directory of Open Access Journals (Sweden)

    Yang Wang

    2015-08-01

    Full Text Available LDH-C4 is a lactate dehydrogenase that catalyzes the conversion of pyruvate to lactate. In mammals, ldh-c was originally thought to be expressed only in testis and spermatozoa. Plateau pika (Ochotona curzoniae, which belongs to the genus Ochotona of the Ochotonidea family, is a hypoxia tolerant mammal living 3000–5000 m above sea level on the Qinghai-Tibet Plateau, an environment which is strongly hypoxic. Ldh-c is expressed not only in testis and sperm but also in somatic tissues of plateau pika. In this study, the effects of N-propyl oxamate and N-isopropyl oxamate on LDH isozyme kinetics were compared to screens for a selective inhibitor of LDH-C4. To reveal the role and physiological mechanism of LDH-C4 in skeletal muscle of plateau pika, we investigated the effect of N-isopropyl oxamate on the pika exercise tolerance as well as the physiological mechanism. Our results show that Ki of N-propyl oxamate and N-isopropyl oxamate for LDH-A4, LDH-B4, and LDH-C4 were 0.094 mmol/L and 0.462 mmol/L, 0.119 mmol/L and 0.248 mmol/L, and 0.015 mmol/L and 0.013 mmol/L, respectively. N-isopropyl oxamate is a powerful selective inhibitor of plateau pika LDH-C4. In our exercise tolerance experiment, groups treated with inhibitors had significantly lower swimming times than the uninhibited control group. The inhibition rates of LDH, LD, and ATP were 37.12%, 66.27%, and 32.42%, respectively. Our results suggested that ldh-c is expressed in the skeletal muscle of plateau pika, and at least 32.42% of ATP in the skeletal muscle is catalyzed by LDH-C4 by anaerobic glycolysis. This suggests that pika has reduced dependence on oxygen and enhanced adaptation to hypoxic environment due to increased anaerobic glycolysis by LDH-C4 in skeletal muscle. LDH-C4 in plateau pika plays the crucial role in anaerobic glycolysis and generates ATP rapidly since this is the role of LDH-A4 in most species on plain land, which provide evidence that the native humans and animals in

  15. Testis-Specific Lactate Dehydrogenase (LDH-C4) in Skeletal Muscle Enhances a Pika's Sprint-Running Capacity in Hypoxic Environment.

    Science.gov (United States)

    Wang, Yang; Wei, Lian; Wei, Dengbang; Li, Xiao; Xu, Lina; Wei, Linna

    2015-08-07

    LDH-C4 is a lactate dehydrogenase that catalyzes the conversion of pyruvate to lactate. In mammals, ldh-c was originally thought to be expressed only in testis and spermatozoa. Plateau pika (Ochotona curzoniae), which belongs to the genus Ochotona of the Ochotonidea family, is a hypoxia tolerant mammal living 3000-5000 m above sea level on the Qinghai-Tibet Plateau, an environment which is strongly hypoxic. Ldh-c is expressed not only in testis and sperm but also in somatic tissues of plateau pika. In this study, the effects of N-propyl oxamate and N-isopropyl oxamate on LDH isozyme kinetics were compared to screens for a selective inhibitor of LDH-C4. To reveal the role and physiological mechanism of LDH-C4 in skeletal muscle of plateau pika, we investigated the effect of N-isopropyl oxamate on the pika exercise tolerance as well as the physiological mechanism. Our results show that Ki of N-propyl oxamate and N-isopropyl oxamate for LDH-A4, LDH-B4, and LDH-C4 were 0.094 mmol/L and 0.462 mmol/L, 0.119 mmol/L and 0.248 mmol/L, and 0.015 mmol/L and 0.013 mmol/L, respectively. N-isopropyl oxamate is a powerful selective inhibitor of plateau pika LDH-C4. In our exercise tolerance experiment, groups treated with inhibitors had significantly lower swimming times than the uninhibited control group. The inhibition rates of LDH, LD, and ATP were 37.12%, 66.27%, and 32.42%, respectively. Our results suggested that ldh-c is expressed in the skeletal muscle of plateau pika, and at least 32.42% of ATP in the skeletal muscle is catalyzed by LDH-C4 by anaerobic glycolysis. This suggests that pika has reduced dependence on oxygen and enhanced adaptation to hypoxic environment due to increased anaerobic glycolysis by LDH-C4 in skeletal muscle. LDH-C4 in plateau pika plays the crucial role in anaerobic glycolysis and generates ATP rapidly since this is the role of LDH-A4 in most species on plain land, which provide evidence that the native humans and animals in Qinghai-Tibet plateau

  16. Preparation and Morphology of Polyvinyl Chloride/LDH Nanocomposites%PVC/LDH纳米复合材料的制备及形貌

    Institute of Scientific and Technical Information of China (English)

    黄年华; 李治华; 王维; 刘肖

    2009-01-01

    Exfoliated polyvinyl chloride(PVC)/MgAl hyered double hydroxide(LDH) nanocomposites were synthesized by a solution delamination-restacking method through tetrahydrofuran(THF) as solvent. The structures of hurylether phosphate modified LDH and PVC/LDH nanocomposites were characterized by FTIR, XRD, TEM and TGA. The effects of preparation conditions including the content of LDH in PVC matix, the concentration and delamination time of LDH in THF on the struc-ture of nanocornposites were studied. The XRD and TEM results show completely exfoliated LDH layers can be achieved by de-creasing the content of LDH and the concentration of LDH in THF, elongating the delamination time of LDH in TFIF, and ex-foliated LDH hyers disorderedly disperse in the PVC matrix. The TGA results show that PVC/LDH nanocomposites have sig-nificandy enhanced thermal stability. When 30% weight loss is selected as a point of comparison, the thermal decomposition temperature of exfoliated PVC/LDH nanocomposite with 2% LDH is 16 ℃ higher than that of pristine PVC. The char residue of these nanocomposites at 650 ℃ is increased by the presence of LDH.%以四氢呋喃为溶剂,通过溶液层离/重组法制备了聚氯乙烯(PVC)/层状双氢氧化物(LDH)纳米复合材料.采用TEM、XRD、FTIR和TGA等手段研究了十二烷基单磷酸根离子(PK)改性的层状双氢氧化物(LDH-PK)及其纳米复合材料的结构与性能,并系统考察了各种制备条件对纳米复合材料结构的影响.XRD和TEM结果表明,剥离型纳米复合材料可通过减少LDH在聚合物中的含量及在四氢呋喃中的浓度和增加层离时间等条件获得;剥离的LDH片层无序的分散在PVC基材中.TGA结果表明,纳米复合材料相比于纯PVC具有更好的热稳定性;以30%失重为比较点,当LDH的质量百分含量为2%时,纳米复合材料的热降解温度比纯PVC高出近16℃,并能显著促进PVC成炭.

  17. Preparation and electrochemical performances of in-situ Ni/Al-LDH/MCNTs composites%Ni/Al-LDH/MCNTs原位复合材料的制备与电化学性能

    Institute of Scientific and Technical Information of China (English)

    李延伟; 梁晓丽; 姚金环; 姜吉琼

    2014-01-01

    Ni/Al-LDH/MCNTs composites with MCNTs contents of 1%, 3%, and 5%were prepared by the in-situ homogeneous precipitation method with Ni(NO3)3·6H2O, Al(NO3)3·9H2O, urea, and MCNTs as raw materials. The microstructure and morphology of the Ni/Al-LDH/MCNTs composites were analyzed with X-ray diffraction (XRD), Fourier transform infrared (FTIR) and field emission scanning electron microscopy (FESEM). The electrochemical performance of the Ni/Al-LDH/MCNTs composites used as cathode materials for nickel-hydrogen battery were characterized with cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and charge-discharge tests. The results demonstrated that the introduction of MCNTs could enhance electrochemical activity, decrease electrochemical reaction resistance, and significantly improve high rate performance of Ni/Al-LDH. In particular, the Ni/Al-LDH/MCNTs composite with MCNTs content of 3% had the best electrochemical performance. For example, the specific discharge capacities of the Ni/Al-LDH/MCNTs composite with MCNTs content of 3%under current densities of 200, 500, 1000, and 2000 mA·g-1 were 330, 321, 307, and 288 mA·h·g-1, respectively. While for the Ni/Al-LDH without MCNTs, the specific discharge capacity was only 205 mA·h·g-1 under the current density of 2000 mA·g-1.%以Ni(NO3)2·6H2O、Al(NO3)3·9H2O、尿素和MCNTs为原料,采用原位均相沉淀法制备了MCNTs含量(质量分数)分别为1%、3%和5%的Ni/Al-LDH/MCNTs复合电极活性材料。采用X射线衍射(XRD)、傅里叶变换红外光谱(FTIR)和场发射扫描电子显微镜(FESEM)表征了材料的微观结构和形貌;采用循环伏安(CV)、电化学交流阻抗(EIS)和充放电测试研究了该复合材料作为镍氢电池正极材料的电化学性能。结果表明,在Ni/Al-LDH 中复合 MCNTs 能够提高材料的电化学活性,降低电化学反应电阻,显著改善材料的大电流充放电性能。其中MCNTs含量为3

  18. Comparison of N(2) Fixation and Yields in Cajanus cajan between Hydrogenase-Positive and Hydrogenase-Negative Rhizobia by In Situ Acetylene Reduction Assays and Direct N Partitioning.

    Science.gov (United States)

    La Favre, J S; Focht, D D

    1983-08-01

    Pigeon peas [Cajanus cajan (L.) Millsp.] were grown in soil columns containing (15)N-enriched organic matter. Seasonal N(2) fixation activity was determined by periodically assaying plants for reduction of C(2)H(2). N(2) fixation rose sharply from the first assay period at 51 days after planting to a peak of activity between floral initiation and fruit set. N(2) fixation (acetylene reduction) activity dropped concomitantly with pod maturation but recovered after pod harvests. Analysis of (15)N content of plant shoots revealed that approximately 91 to 94% of plant N was derived from N(2) fixation. The effect of inoculation with hydrogenase-positive and hydrogenase-negative rhizobia was examined. Pigeon peas inoculated with strain P132 (hydrogenase-positive) yielded significantly more total shoot N than other inoculated or uninoculated treatments. However, two other hydrogenase-positive strains did not yield significantly more total shoot N than a hydrogenase-negative strain. The extent of nodulation by inoculum strains compared to indigenous rhizobia was determined by typing nodules according to intrinsic antibiotic resistance of the inoculum strains. The inoculum strains were detected in almost all typed nodules of inoculated plants.Gas samples were taken from soil columns several times during the growth cycle of the plants. H(2) was never detected, even in columns containing pigeon peas inoculated with hydrogenase-negative rhizobia. This was attributed to H(2) consumption by soil bacteria. Estimation of N(2) fixation by acetylene reduction activity was closest to the direct (15)N method when ethylene concentrations in the gas headspace (between the column lid and soil surface) were extrapolated to include the soil pore space as opposed solely to measurement in the headspace. There was an 8-fold difference between the two acetylene reduction assay methods of estimation. Based on a planting density of 15,000 plants per hectare, the direct (15)N fixation rates ranged

  19. Ni adsorption and Ni-Al LDH precipitation in a sandy aquifer: An experimental and mechanistic modeling study

    NARCIS (Netherlands)

    Regelink, I.C.; Temminghoff, E.J.M.

    2011-01-01

    Mining activities and industries have created nickel (Ni) contaminations in many parts of the world. The objective of this study is to increase our understanding of Ni adsorption and Nickel-Aluminium Layered Double Hydroxide (Ni-Al LDH) precipitation to reduce Ni mobility in a sandy soil aquifer. At

  20. Effects and Mechanism of Atmospheric-Pressure Dielectric Barrier Discharge Cold Plasma on Lactate Dehydrogenase (LDH) Enzyme.

    Science.gov (United States)

    Zhang, Hao; Xu, Zimu; Shen, Jie; Li, Xu; Ding, Lili; Ma, Jie; Lan, Yan; Xia, Weidong; Cheng, Cheng; Sun, Qiang; Zhang, Zelong; Chu, Paul K

    2015-01-01

    Proteins are carriers of biological functions and the effects of atmospheric-pressure non-thermal plasmas on proteins are important to applications such as sterilization and plasma-induced apoptosis of cancer cells. Herein, we report our detailed investigation of the effects of helium-oxygen non-thermal dielectric barrier discharge (DBD) plasmas on the inactivation of lactate dehydrogenase (LDH) enzyme solutions. Circular dichroism (CD) and dynamic light scattering (DLS) indicate that the loss of activity stems from plasma-induced modification of the secondary molecular structure as well as polymerization of the peptide chains. Raising the treatment intensity leads to a reduced alpha-helix content, increase in the percentage of the beta-sheet regions and random sequence, as well as gradually decreasing LDH activity. However, the structure of the LDH plasma-treated for 300 seconds exhibits a recovery trend after storage for 24 h and its activity also increases slightly. By comparing direct and indirect plasma treatments, plasma-induced LDH inactivation can be attributed to reactive species (RS) in the plasma, especially ones with a long lifetime including hydrogen peroxide, ozone, and nitrate ion which play the major role in the alteration of the macromolecular structure and molecular diameter in lieu of heat, UV radiation, and charged particles.

  1. Electrostatic self-assembly of sandwich-like CoAl-LDH/polypyrrole/graphene nanocomposites with enhanced capacitive performance.

    Science.gov (United States)

    Zhang, Yu; Du, Dongfeng; Li, Xuejin; Sun, Hongman; Li, Li; Bai, Peng; Xing, Wei; Xue, Qingzhong; Yan, Zifeng

    2017-09-01

    A novel sandwich-like composite with ultrathin CoAl-LDH nanoplates electrostatically assembled on both sides of 2D polypyrrole/graphene (PG) substrate has been successfully fabricated using facile hydrothermal techniques. The PG not only serves as an excellent conductive and structural scaffold to enhance the transmission of electrons and prevents aggregation of CoAl-LDH nanoplates, but also contributes to the enhancement of the specific capacitance. Owing to the homogeneous dispersion of CoAl-LDH nanoplates and its intimate interaction with PG substrate, the resulting CoAl-LDH/PG nanocomposite material exhibits excellent capacitive performance, e.g. enhanced gravimetric specific capacitance (864 F g-1 at 1 A g-1 ), high rate performance (75% retention at 20 A g-1) and excellent cyclic life (almost no degradation in supercapacitor performance after 5000 cycles) in aqueous KOH solution. Furthermore, the assembled asymmetric capacitor is able to deliver superhigh energy density of 46.8 Wh kg-1 at 1.2 kW kg-1 and maintain 90.1% of its initial capacitance after 10000 cycles. These results indicate a rational assembly strategy towards a high-performance pseudocapacitive electrode material with excellent rate performance, high specific capacitance and outstanding cycle stability.

  2. Co intake mediated formation of ultrathin nanosheets of transition metal LDH-an advanced electrocatalyst for oxygen evolution reaction.

    Science.gov (United States)

    Long, Xia; Xiao, Shuang; Wang, Zilong; Zheng, Xiaoli; Yang, Shihe

    2015-01-21

    By controlling the ratio of tri- and bi-valent ions, multi-transition metal based layered double hydroxide (LDH) ultrathin nanosheets are synthesized. They show advanced OER performance with low overpotentials (∼0.2 V) and decreased Tafel slopes with increasing Co incorporation due to the modulated electronic structures of catalytic centers and the increased surface area and electronic conductivity.

  3. Effects and Mechanism of Atmospheric-Pressure Dielectric Barrier Discharge Cold Plasma on Lactate Dehydrogenase (LDH) Enzyme

    Science.gov (United States)

    Zhang, Hao; Xu, Zimu; Shen, Jie; Li, Xu; Ding, Lili; Ma, Jie; Lan, Yan; Xia, Weidong; Cheng, Cheng; Sun, Qiang; Zhang, Zelong; Chu, Paul K.

    2015-05-01

    Proteins are carriers of biological functions and the effects of atmospheric-pressure non-thermal plasmas on proteins are important to applications such as sterilization and plasma-induced apoptosis of cancer cells. Herein, we report our detailed investigation of the effects of helium-oxygen non-thermal dielectric barrier discharge (DBD) plasmas on the inactivation of lactate dehydrogenase (LDH) enzyme solutions. Circular dichroism (CD) and dynamic light scattering (DLS) indicate that the loss of activity stems from plasma-induced modification of the secondary molecular structure as well as polymerization of the peptide chains. Raising the treatment intensity leads to a reduced alpha-helix content, increase in the percentage of the beta-sheet regions and random sequence, as well as gradually decreasing LDH activity. However, the structure of the LDH plasma-treated for 300 seconds exhibits a recovery trend after storage for 24 h and its activity also increases slightly. By comparing direct and indirect plasma treatments, plasma-induced LDH inactivation can be attributed to reactive species (RS) in the plasma, especially ones with a long lifetime including hydrogen peroxide, ozone, and nitrate ion which play the major role in the alteration of the macromolecular structure and molecular diameter in lieu of heat, UV radiation, and charged particles.

  4. 'Dual hit' metabolic modulator LDCA selectively kills cancer cells by efficient competitive inhibition of LDH-A.

    Science.gov (United States)

    Ghosh, Monisankar; Saha, Suchandrima; Dutta, Samir Kumar

    2016-02-07

    Herein, we synthesize and elucidate the potential of a novel 'dual hit' molecule, LDCA, to constitutively block lactate dehydrogenase isoform-A (LDH-A) to selectively subvert apoptosis and rigorously attenuate breast tumor progression in a mouse model, comprehensively delineating the therapeutic prospectus of LDCA in the field of cancer metabolics.

  5. Chapter Eight - Structural Characterization of Poised States in the Oxygen Sensitive Hydrogenases and Nitrogenases

    Energy Technology Data Exchange (ETDEWEB)

    King, Paul W [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Mulder, David W [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Artz, Jacob H. [Washington State University; Zadvornyy, Oleg A. [Washington State University; Peters, John W. [Washington State University

    2017-08-21

    The crystallization of FeS cluster-containing proteins has been challenging due to their oxygen sensitivity, and yet these enzymes are involved in many critical catalytic reactions. The last few years have seen a wealth of innovative experiments designed to elucidate not just structural but mechanistic insights into FeS cluster enzymes. Here, we focus on the crystallization of hydrogenases, which catalyze the reversible reduction of protons to hydrogen, and nitrogenases, which reduce dinitrogen to ammonia. A specific focus is given to the different experimental parameters and strategies that are used to trap distinct enzyme states, specifically, oxidants, reductants, and gas-treatments. Other themes presented here include the recent use of Cryo-EM, and how coupling various spectroscopies to crystallization is opening up new approaches for structural and mechanistic analysis.

  6. Following [FeFe] Hydrogenase Active Site Intermediates by Time-Resolved Mid-IR Spectroscopy.

    Science.gov (United States)

    Mirmohades, Mohammad; Adamska-Venkatesh, Agnieszka; Sommer, Constanze; Reijerse, Edward; Lomoth, Reiner; Lubitz, Wolfgang; Hammarström, Leif

    2016-08-18

    Time-resolved nanosecond mid-infrared spectroscopy is for the first time employed to study the [FeFe] hydrogenase from Chlamydomonas reinhardtii and to investigate relevant intermediates of the enzyme active site. An actinic 355 nm, 10 ns laser flash triggered photodissociation of a carbonyl group from the CO-inhibited state Hox-CO to form the state Hox, which is an intermediate of the catalytic proton reduction cycle. Time-resolved infrared spectroscopy allowed us to directly follow the subsequent rebinding of the carbonyl, re-forming Hox-CO, and determine the reaction half-life to be t1/2 ≈ 13 ± 5 ms at room temperature. This gives direct information on the dynamics of CO inhibition of the enzyme.

  7. Improved production of biohydrogen in light-powered Escherichia coli by co-expression of proteorhodopsin and heterologous hydrogenase.

    Science.gov (United States)

    Kim, Jaoon Y H; Jo, Byung Hoon; Jo, Younghwa; Cha, Hyung Joon

    2012-01-04

    Solar energy is the ultimate energy source on the Earth. The conversion of solar energy into fuels and energy sources can be an ideal solution to address energy problems. The recent discovery of proteorhodopsin in uncultured marine γ-proteobacteria has made it possible to construct recombinant Escherichia coli with the function of light-driven proton pumps. Protons that translocate across membranes by proteorhodopsin generate a proton motive force for ATP synthesis by ATPase. Excess protons can also be substrates for hydrogen (H(2)) production by hydrogenase in the periplasmic space. In the present work, we investigated the effect of the co-expression of proteorhodopsin and hydrogenase on H(2) production yield under light conditions. Recombinant E. coli BL21(DE3) co-expressing proteorhodopsin and [NiFe]-hydrogenase from Hydrogenovibrio marinus produced ~1.3-fold more H(2) in the presence of exogenous retinal than in the absence of retinal under light conditions (70 μmole photon/(m2·s)). We also observed the synergistic effect of proteorhodopsin with endogenous retinal on H(2) production (~1.3-fold more) with a dual plasmid system compared to the strain with a single plasmid for the sole expression of hydrogenase. The increase of light intensity from 70 to 130 μmole photon/(m(2)·s) led to an increase (~1.8-fold) in H(2) production from 287.3 to 525.7 mL H(2)/L-culture in the culture of recombinant E. coli co-expressing hydrogenase and proteorhodopsin in conjunction with endogenous retinal. The conversion efficiency of light energy to H(2) achieved in this study was ~3.4%. Here, we report for the first time the potential application of proteorhodopsin for the production of biohydrogen, a promising alternative fuel. We showed that H(2) production was enhanced by the co-expression of proteorhodopsin and [NiFe]-hydrogenase in recombinant E. coli BL21(DE3) in a light intensity-dependent manner. These results demonstrate that E. coli can be applied as light

  8. Improved production of biohydrogen in light-powered Escherichia coli by co-expression of proteorhodopsin and heterologous hydrogenase

    Directory of Open Access Journals (Sweden)

    Kim Jaoon YH

    2012-01-01

    Full Text Available Abstract Background Solar energy is the ultimate energy source on the Earth. The conversion of solar energy into fuels and energy sources can be an ideal solution to address energy problems. The recent discovery of proteorhodopsin in uncultured marine γ-proteobacteria has made it possible to construct recombinant Escherichia coli with the function of light-driven proton pumps. Protons that translocate across membranes by proteorhodopsin generate a proton motive force for ATP synthesis by ATPase. Excess protons can also be substrates for hydrogen (H2 production by hydrogenase in the periplasmic space. In the present work, we investigated the effect of the co-expression of proteorhodopsin and hydrogenase on H2 production yield under light conditions. Results Recombinant E. coli BL21(DE3 co-expressing proteorhodopsin and [NiFe]-hydrogenase from Hydrogenovibrio marinus produced ~1.3-fold more H2 in the presence of exogenous retinal than in the absence of retinal under light conditions (70 μmole photon/(m2·s. We also observed the synergistic effect of proteorhodopsin with endogenous retinal on H2 production (~1.3-fold more with a dual plasmid system compared to the strain with a single plasmid for the sole expression of hydrogenase. The increase of light intensity from 70 to 130 μmole photon/(m2·s led to an increase (~1.8-fold in H2 production from 287.3 to 525.7 mL H2/L-culture in the culture of recombinant E. coli co-expressing hydrogenase and proteorhodopsin in conjunction with endogenous retinal. The conversion efficiency of light energy to H2 achieved in this study was ~3.4%. Conclusion Here, we report for the first time the potential application of proteorhodopsin for the production of biohydrogen, a promising alternative fuel. We showed that H2 production was enhanced by the co-expression of proteorhodopsin and [NiFe]-hydrogenase in recombinant E. coli BL21(DE3 in a light intensity-dependent manner. These results demonstrate that E. coli

  9. Isolation and characterization of the small subunit of the uptake hydrogenase from the cyanobacterium Nostoc punctiforme.

    Science.gov (United States)

    Raleiras, Patrícia; Kellers, Petra; Lindblad, Peter; Styring, Stenbjörn; Magnuson, Ann

    2013-06-21

    In nitrogen-fixing cyanobacteria, hydrogen evolution is associated with hydrogenases and nitrogenase, making these enzymes interesting targets for genetic engineering aimed at increased hydrogen production. Nostoc punctiforme ATCC 29133 is a filamentous cyanobacterium that expresses the uptake hydrogenase HupSL in heterocysts under nitrogen-fixing conditions. Little is known about the structural and biophysical properties of HupSL. The small subunit, HupS, has been postulated to contain three iron-sulfur clusters, but the details regarding their nature have been unclear due to unusual cluster binding motifs in the amino acid sequence. We now report the cloning and heterologous expression of Nostoc punctiforme HupS as a fusion protein, f-HupS. We have characterized the anaerobically purified protein by UV-visible and EPR spectroscopies. Our results show that f-HupS contains three iron-sulfur clusters. UV-visible absorption of f-HupS has bands ∼340 and 420 nm, typical for iron-sulfur clusters. The EPR spectrum of the oxidized f-HupS shows a narrow g = 2.023 resonance, characteristic of a low-spin (S = ½) [3Fe-4S] cluster. The reduced f-HupS presents complex EPR spectra with overlapping resonances centered on g = 1.94, g = 1.91, and g = 1.88, typical of low-spin (S = ½) [4Fe-4S] clusters. Analysis of the spectroscopic data allowed us to distinguish between two species attributable to two distinct [4Fe-4S] clusters, in addition to the [3Fe-4S] cluster. This indicates that f-HupS binds [4Fe-4S] clusters despite the presence of unusual coordinating amino acids. Furthermore, our expression and purification of what seems to be an intact HupS protein allows future studies on the significance of ligand nature on redox properties of the iron-sulfur clusters of HupS.

  10. Distribution & diagnostic efficacy of cardiac markers CK-MB & LDH in pericardial fluid for postmortem diagnosis of ischemic heart disease.

    Science.gov (United States)

    Ghormade, Pankaj Suresh; Kumar, Narendra Baluram; Tingne, Chaitanya Vidyadhar; Keoliya, Ajay Narmadaprasad

    2014-11-01

    The aim of the present study is to evaluate the diagnostic efficacy of biochemical markers creatine kinase-MB (CK-MB) and LDH in pericardial fluid for postmortem diagnosis of ischemic heart disease (IHD). We studied 119 medico-legal autopsies selected during a period of 2 years. Subjects were assigned into diagnostic groups upon final cause of death as follows: (1) sudden cardiac death due to IHD's (n = 52), (2) violent asphyxia (n = 24); (3) polytraumatic deaths (n = 20); (4) natural deaths excluding cardiac causes (n = 23). Pericardial fluid samples were tested for estimating enzyme levels. Histological examination was performed with hematoxylin and eosin (H&E) stain on myocardial tissue samples. We observed highest levels of CK-MB & LDH in deaths due to IHD's. Kruskal-Wallis test revels significant differences in activities of CK-MB (P = 0.0001) and LDH (P = 0.0065) amongst all diagnostic groups. Mann-Whitney test showed highly significant (P MB in group 1 as compared to other diagnostic groups. However, LDH levels were non-discriminatory (P = 0.0827) between cases of IHD's and cases of other natural deaths. CK-MB levels were statistically non-significant between cases divided as myocardial infarction (MI) and severe coronary artery disease in group 1, hence its role for postmortem detection of MI is somewhat limiting. However, sensitivity and negative predictive values of its cut off level obtained in cases of IHD's are nearly equal to diagnostic efficacy in clinical settings. Hence, it can be useful additional diagnostic tool for autopsy diagnosis of IHD's. Whereas, LDH is not useful for postmortem diagnosis in these cases. Copyright © 2014 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

  11. Utility of NSE, ProGRP and LDH in Diagnosis and Treatment
in Patients with Small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Yan PENG

    2016-09-01

    Full Text Available Background and objective Small cell lung cancer (SCLC is a rapidly growing tumor with characteristic of neuroendocrine cellular function. Neuron specific enolase (NSE, pro-gastrin-releasing peptide (ProGRP and lactic dehydrogenase (LDH are valuable in diagnosis and treatment of SCLC. By analyzing the variation of NSE, ProGRP and LDH before and after treatment, the aim of this study is to investigate the efficacy of tumor markers in diagnostic staging, therapeutic evaluation and prediction of disease relapsing. Methods Patients with SCLC who receiving the first line chemotherapy in Cancer Hospital, Chinese Academy of Medical Sciences were enrolled and retrospectively analyzed. Clinical characteristic (includes NSE, ProGRP and LDH level before and after 2 cycles chemotherapy, efficacy evaluation, progression-free survival (PFS were analyzed. Results Before treatment, Serum NSE, ProGRP and LDH in patients with extensive disease (ED were significantly higher than those with limited disease (LD(all P4 cycles chemotherapy and with obvious decrease of ProGRP than those who accepted ≤4 cycles chemotherapy and with less obvious decrease of ProGRP in LD group; ED patients with no more than 2 distant metastasis, normal LDH level before treatment and obvious decrease of ProGRP after chemotherapy had lower short term relapse risk. In addition, the types of relapse (sensitive relapse, drug resistance relapse and refractory relapse were negatively correlated with decrease of ProGRP (P=0.044. By multivariate analysis, numbers of chemotherapy cycle was independent prognostic factor for PFS in LD SCLC; numbers of distant metastasis and decrease of ProGRP were independent prognostic factors for PFS in ED SCLC. Conclusion Increase level of serum tumor markers is related to tumor burden. Decrease level of ProGRP after treatment may prognose efficacy and relapse risk.

  12. Synthesis of Co-Al-Cl LDH by cathodic material reprocessing from cellular phone batteries

    Energy Technology Data Exchange (ETDEWEB)

    Amaral, Fabio Augusto do; Machado, Erica Oliveira; Freitas, Leonardo Luis de; Santana, Laiane Kalita; Canobre, Sheila Cristina, E-mail: fabioamaral@yahoo.com.br, E-mail: fabioamaral@iqufu.ufu.br [Universidade Federal de Uberlandia (UFU/LAETE), (Brazil). Inst. de Quimica. Lab. de Armazenamento de Energia e Tratamento de Efluente

    2014-08-15

    The aim of this paper was the recovering of the cathodic material from discarded lithium ion batteries for obtainment of the lamellar double hydroxides (LDHs) by the co-precipitation method at variable pH in HCl and H{sub 2}O{sub 2} 1:1 (v/v) acid solution containing Co and Al (extracted from cathodic material composed of LiCoO{sub 2} and aluminum foil). These metals were precipitated in LiOH at pH 9 or 11, or NH{sub 4}OH at pH 9 and submitted to the hydrothermal treatment (HT) to improve the structural organization of the LDHs lamellae. After precipitation, the resulting solids were structurally characterized by XRD for phase identification and calculation of the unit cell parameter, thermally by TGA for the identification of the mass loss and morphologically by SEM. The sample obtained by precipitation with LiOH at pH 11 / hydrothermal treatment showed diffraction peaks similar to hydrotalcite, morphological and thermal characteristics similar to the pattern Co-Al-Cl LDH obtained by co-precipitation at constant pH 8. (author)

  13. Direct Synthesis of Unilamellar MgAl-LDH Nanosheets and Stacking in Aqueous Solution.

    Science.gov (United States)

    Liang, Dujuan; Yue, Wenbo; Sun, Genban; Zheng, Dong; Ooi, Kenta; Yang, Xiaojing

    2015-11-17

    Two-dimensional (2D) materials, such as graphene, inorganic oxides, and hydroxides, are one of the most extensively studied classes of materials due to their unilamellar crystallites or nanosheet structures. In this study, instead of using the universal exfoliation method of the bulky crystal precursor, 2D crystals/nanosheets of MgAl-layered double hydroxides (LDHs) were synthesized in formamide. We propose that the obtained crystals are unilamellar according to the XRD, TEM, and AFM observations. The HRTEM and fast Fourier transform images confirm that the crystal structures are the same as those of the exfoliated MgAl-LDH nanosheets. The directly synthesized sheets can stack into a 3D crystal structure, which is the same as that of typical LDHs except for the disordered orientation of the a-/b- crystal axis of each sheet. This result provides not only a novel approach to the preparation of 2D crystals but also insight into the formation mechanism of LDHs.

  14. Role of the synthesis route on the properties of hybrid LDH-graphene as basic catalysts

    Science.gov (United States)

    Álvarez, Mayra G.; Tichit, Didier; Medina, Francesc; Llorca, Jordi

    2017-02-01

    Layered double hydroxides (LDH or HT) or their derived mixed oxides present marked acid-base properties useful in catalysis, but they lead to agglomerate inducing a weak accessibility to the active sites. In this study we report the preparation and characterization of HT/Graphene (HT/rGO) nanocomposites as active and selective basic catalysts for the acetone condensation reaction. The graphene high specific surface area and structural compatibility with the HT allowed increasing the number and accessibility of the active sites and activity of this later. Two series of HT/rGO nanocomposites with 0.5 ≤ HT/rGO ≤ 10 mass ratio were prepared by: i) direct HT coprecipitation in the presence of GO; ii) self-assembly of preformed HT with GO. The prepared HT/rGO nanocomposites were dried either in air at 80 °C or freeze-dried. A series of characterizations showed the great influence of the preparation method and HT/rGO mass ratio on both the nanocomposite structure and catalytic activity. An optimum activity was observed for a HT/rGO = 10 catalyst. Particularly, the highest catalytic activity was found in those nanocomposites obtained by coprecipitation and freeze dried (3 times more active than bulk HT) which can be connected to their structure with a better accessibility to the basic sites.

  15. Antibodies against Clonorchis sinensis LDH could cross-react with LDHB localizing on the plasma membrane of human hepatocarcinoma cell SMMC-7721 and induce apoptosis.

    Science.gov (United States)

    Song, Tianzhang; Gan, Wenjia; Chen, Jintao; Huang, Lilin; Yin, Hongling; He, Tailong; Huang, Huaiqiu; Hu, Xuchu

    2016-04-01

    Lactate dehydrogenase (LDH) is a terminal enzyme in anaerobic glycolytic pathway. It widely exists in various organisms and is in charge of converting the glycolysis product pyruvic acid to lactic acid. Most parasites, including Clonorchis sinensis, predominantly depend on glycolysis to provide energy. Bioinformatic analysis predicts that the LDHs from many species have more than one transmembrane region, suggesting that it may be a membrane protein. C. sinensis LDH (CsLDH) has been confirmed as a transmembrane protein mainly located in the tegument. The antibodies against CsLDH can inhibit the worm's energy metabolism, kill the worm, and may have the same effects on human cancer cells. In this study, we cloned and characterized human LDHA (HsLDHA), HsLDHB, and CsLDH. Semi-quantitative real-time RCP showed that HsLDHB only existed in hepatocarcinoma cell SMMC-7721. Confocal microscopy and Western blot experiments revealed that HsLDHB was localized in the plasma membrane of SMMC-7721 cells, and the antibodies against CsLDH could cross-react with it. This cross-reaction could inhibit the enzymatic activity of HsLDHB. The cancer cells co-cultured with anti-CsLDH sera showed a significant decrease in cell proliferation rate and increases in caspase 9 and reactive oxygen species (ROS) levels. Therefore, anti-CsLDH antibodies can induce the apoptosis of cancer cells SMMC-7721 and may serve as a new tool to inhibit tumor.

  16. Anti-Metastatic and Anti-Angiogenic Activities of Core-Shell SiO2@LDH Loaded with Etoposide in Non-Small Cell Lung Cancer.

    Science.gov (United States)

    Zhu, Yanjing; Zhu, Rongrong; Wang, Mei; Wu, Bin; He, Xiaolie; Qian, Yechang; Wang, Shilong

    2016-11-01

    Currently, nanoparticles have gained a great attention in the anti-tumor research area. However, to date, studies on the anti-metastasis action of core-shell SiO2@LDH (LDH: layered double hydroxide) nanoparticles remain untouched. Two emerging aspects considered are establishing research on the controlling delivery effect of SiO2@LDH combined with anti-cancer medicine from a new perspective. The fine properties synthetic SiO2@LDH-VP16 (VP16: etoposide) are practiced to exhibit the nanoparticle's suppression on migration and invasion of non-small cell lung cancer (NSCLC). Both in vitro and in vivo inspection shows that SiO2@LDH can help VP16 better function as an anti-metastasis agent. On the other hand, anti-angiogenic efficiency, co-localization, as well as western blot are investigated to explain the possible mechanism. A clear mergence of SiO2@LDH-VP16 and cytomembrane/microtubule may be observed from co-location images. Results offer evidence that SiO2@LDH-VP16 plays positions on cytomembrane and microtubules. It efficiently inhibits metastasis on NSCLC by reducing vascularization, and eliciting depression of the PI3K-AKT and FAK-Paxillin signaling pathways. SiO2@LDH-VP16, the overall particle morphology, and function on anti-metastasis and anti-angiogenic may be tuned to give new opportunities for novel strategies for cancer therapy.

  17. Regulation of crayfish, Orconectes virilis, tail muscle lactate dehydrogenase (LDH) in response to anoxic conditions is associated with alterations in phosphorylation patterns.

    Science.gov (United States)

    Green, Stuart R; Storey, Kenneth B

    2016-12-01

    Lactate dehydrogenase (LDH), the terminal enzyme of anaerobic glycolysis, has a crucial role in sustaining ATP production by glycolysis during periods of anoxia via regenerating NAD(+) through the production of lactate. The present study examined the effects of prolonged (20h) anoxic submergence on LDH from the tail muscle of an anoxia-tolerant crayfish (Orconectes virilis). LDH was purified to homogeneity from tail muscle of both aerobic control and anoxic crayfish in a three step process. Analysis of the kinetic parameters and the stability of LDH showed that the Vmax in the pyruvate-reducing direction was significantly higher for the enzyme from anoxic crayfish whereas in the lactate-oxidizing direction the Vmax was significantly higher for the control enzyme. Differential scanning fluorimetry was used to assess thermal unfolding of crayfish LDH. The results showed that the enzyme from control muscle had a significantly higher melting temperature (greater thermal stability) than the anoxic enzyme form, suggesting that there was a structural difference between the two enzyme forms. Immunoblotting of purified LDH implicated post-translational modification as the reason for this difference; purified LDH from aerobic control crayfish showed significantly higher amounts of serine/threonine phosphorylation than did the anoxic enzyme form. This study provides evidence for anoxia-induced modifications of crayfish muscle LDH that may contribute significantly to modulating enzyme function under anoxic conditions.

  18. O₂migration rates in [NiFe] hydrogenases. A joint approach combining free-energy calculations and kinetic modeling.

    Science.gov (United States)

    Topin, Jérémie; Diharce, Julien; Fiorucci, Sébastien; Antonczak, Serge; Golebiowski, Jérôme

    2014-01-23

    Hydrogenases are promising candidates for the catalytic production of green energy by means of biological ways. The major impediment to such a production is rooted in their inhibition under aerobic conditions. In this work, we model dioxygen migration rates in mutants of a hydrogenase of Desulfovibrio fructusovorans. The approach relies on the calculation of the whole potential of mean force for O2 migration within the wild-type as well as in V74M, V74F, and V74Q mutant channels. The three free-energy barriers along the entire migration pathway are converted into chemical rates through modeling based on Transition State Theory. The use of such a model recovers the trend of O2 migration rates among the series.

  19. Symbiotic Legume Nodules Employ Both Rhizobial Exo- and Endo-Hydrogenases to Recycle Hydrogen Produced by Nitrogen Fixation

    OpenAIRE

    Ciccolella, Christopher O.; Raynard, Nathan A.; John H-M Mei; Derek C Church; Ludwig, Robert A.

    2010-01-01

    BACKGROUND: In symbiotic legume nodules, endosymbiotic rhizobia (bacteroids) fix atmospheric N(2), an ATP-dependent catalytic process yielding stoichiometric ammonium and hydrogen gas (H(2)). While in most legume nodules this H(2) is quantitatively evolved, which loss drains metabolic energy, certain bacteroid strains employ uptake hydrogenase activity and thus evolve little or no H(2). Rather, endogenous H(2) is efficiently respired at the expense of O(2), driving oxidative phosphorylation, ...

  20. International Hydrogenase Conference (7th) Held at the University of Reading on August 24th to 29th 2004.

    Science.gov (United States)

    2004-08-19

    Homologous/heterologous over-expression in Clostridium p68 acetobutylicum and characterization of purified clostridial and algal Fe-only hydrogenases with...yield from fermentative anaerobes can vary from 1.61-2.36 mol H2/mol glucose in Clostridium species to 3.3 mol H,/mol glucose from the extreme...and purification system functional in Clostridium acetobutylicum . C. acetobutylicum ATCC 824 was selected for the homologous over-expression of its Fe

  1. ANTS-anchored Zn-Al-CO{sub 3}-LDH particles as fluorescent probe for sensing of folic acid

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Pengfei; Liu, Dan; Liu, Yanhuan [State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029 (China); Beijing Key Laboratory of Environmentally Harmful Chemical Analysis, Beijing University of Chemical Technology, Beijing 100029 (China); Li, Lei, E-mail: lilei@mail.buct.edu.cn [State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029 (China); Beijing Key Laboratory of Environmentally Harmful Chemical Analysis, Beijing University of Chemical Technology, Beijing 100029 (China)

    2016-09-15

    A novel fluorescent nanosensor for detecting folic acid (FA) in aqueous media has been developed based on 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) anchored to the surface of Zn-Al-CO{sub 3}-layered double hydroxides (LDH) particles. The nanosensor showed high fluorescence intensity and good photostability due to a strong coordination interaction between surface Zn{sup 2+} ions of Zn-Al-CO{sub 3}-LDH and N atoms of ANTS, which were verified by result of X-ray photoelectron spectroscopy (XPS). ANTS-anchored on the surface of Zn-Al-CO{sub 3}-LDH restricted the intra-molecular rotation leading to ANTS-anchored J-type aggregation emission enhancement. ANTS-anchored Zn-Al-CO{sub 3}-LDH particles exhibited highly sensitive and selective response to FA over other common metal ions and saccharides present in biological fluids. The proposed mechanism was that oxygen atoms of -SO{sub 3} groups in ANTS-anchored on the surface of Zn-Al-CO{sub 3}-LDH were easily collided by FA molecules to form potential hydrogen bonds between ANTS-anchored and FA molecules, which could effectively quench the ANTS-anchored fluorescence. Under the simulated physiological conditions (pH of 7.4), the fluorescence quenching was fitted to Stern-Volmer equation with a linear response in the concentration range of 1 μM to 200 μM with a limit of detection of 0.1 μM. The results indicate that ANTS-anchored Zn-Al-CO{sub 3}-LDH particles can afford a very sensitive system for the sensing FA in aqueous solution. - Highlights: • A novel fluorescent nanosensor has been developed. • The sensor exhibited highly sensitive and selective response to FA. • The fluorescence quenching was fitted to Stern–Volmer equation. • The linear response range was 1–200 μM with a limit of detection of 0.1 μM.

  2. Identification of a Catalytic Iron-Hydride at the H-Cluster of [FeFe]-Hydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Mulder, David W.; Guo, Yisong; Ratzloff, Michael W.; King, Paul W.

    2017-01-11

    Hydrogenases couple electrochemical potential to the reversible chemical transformation of H2 and protons, yet the reaction mechanism and composition of intermediates are not fully understood. In this Communication we describe the biophysical properties of a hydride-bound state (Hhyd) of the [FeFe]-hydrogenase from Chlamydomonas reinhardtii. The catalytic H-cluster of [FeFe]-hydrogenase consists of a [4Fe-4S] subcluster ([4Fe-4S]H) linked by a cysteine thiol to an azadithiolate-bridged 2Fe subcluster ([2Fe]H) with CO and CN- ligands. Mossbauer analysis and density functional theory (DFT) calculations show that Hhyd consists of a reduced [4Fe-4S]H+ coupled to a diferrous [2Fe]H with a terminally bound Fe-hydride. The existence of the Fe-hydride in Hhyd was demonstrated by an unusually low Mossbauer isomer shift of the distal Fe of the [2Fe]H subcluster. A DFT model of Hhyd shows that the Fe-hydride is part of a H-bonding network with the nearby bridging azadithiolate to facilitate fast proton exchange and catalytic turnover.

  3. Using Gas Chromatography/Isotope Ratio Mass Spectrometry to Determine the Fractionation Factor for H2 Production by Hydrogenases

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Hui; Ghandi, H.; Shi, Liang; Kreuzer, Helen W.; Ostrom, Nathaniel; Hegg, Eric L.

    2012-01-15

    Hydrogenases catalyze the reversible formation of H2, and they are key enzymes in the biological cycling of H2. H isotopes should be a very useful tool in quantifying proton trafficking in biological H2 production processes, but there are several obstacles that have thus far limited the use of this tool. In this manuscript, we describe a new method that overcomes some of these barriers and is specifically designed to measure isotopic fractionation during enzyme-catalyzed H2 evolution. A key feature of this technique is that purified hydrogenases are employed, allowing precise control over the reaction conditions and therefore a high level of precision. A custom-designed high-throughput gas chromatography-isotope ratio mass spectrometer is employed to measure the isotope ratio of the H2. Using this method, we determined that the fractionation factor of H2 production by the [NiFe]-hydrogenase from Desulfivibrio fructosovran is 0.27. This result indicates that, as expected, protons are highly favored over deuterons during H2 evolution. Potential applications of this new method are discussed.

  4. 复合水滑石热稳定剂对PVC热稳定性能的影响%Effect of MgAl-CO_3-LDH/ZnAl-CO_3-LDH composites on thermal stability of PVC

    Institute of Scientific and Technical Information of China (English)

    刘庆艳; 杨占红; 易师

    2010-01-01

    采用共沉淀法制备镁铝水滑石和锌铝水滑石,采用XRD、FT-IR对其进行表征,同时将其按不同比例进行复配应用到聚氯乙烯(PVC)中.利用静态热老化法研究复合水滑石稳定剂在聚氯乙烯中的热稳定性能.结果表明:合成的镁铝水滑石和锌铝水滑石结晶度高,结构规整;镁铝水滑石和锌铝水滑石复配物的热稳定性能优于单一镁铝水滑石、锌铝水滑石,其最佳复配摩尔比为x(MgAl-CO_3-LDH)-x(ZnAl-CO_3-LDH)=1-0.7.当该复配物与同样金属含量的ZnMgAl-CO_3-LDH(x(Zn)-x(Mg)-x(Al)=0.7-1-1.7)相比时,前者热稳定性能更好.另外,制备该复配物的工艺较制备ZnMgAl-CO_3-LDH的工艺更简单.

  5. Probing the interaction between di- and tri-functionalized carboxy-phosphonic acid and LDH layer structure

    Science.gov (United States)

    Sabbar, El Mouloudi; de Roy, Marie Elisabeth; Leroux, Fabrice

    2006-11-01

    A series of carboxyphosphonate-derivatives of (Zn, Al) layered double hydroxide (LDH) was prepared and characterized by a combination of techniques including powder X-ray diffraction, FTIR spectroscopy and solid-state 13C CP-MAS and 31P MAS NMR spectroscopies. The number of carbon atoms, n=1 or 2 in HO2C(CH2)nPO3H2, was found to have an influence on the accommodation of the interleaved molecule, the carboxylate function lying perpendicular or parallel to the inorganic sheets of the LDH host material. These observations are in agreement with NMR results which evidence an interaction of the interlayered molecules through either the carbonyl or the phosphonate group or both in the case of the larger molecule, N-(phosphonomethyl)iminodiacetic acid. The variation of the basal spacing of the hybrid materials as a function of the temperature indicates a rearrangement of the molecule within the host material gap.

  6. Adsorption of charged protein residues on an inorganic nanosheet: Computer simulation of LDH interaction with ion channel

    Science.gov (United States)

    Tsukanov, Alexey A.; Psakhie, Sergey G.

    2016-08-01

    Quasi-two-dimensional and hybrid nanomaterials based on layered double hydroxides (LDH), cationic clays, layered oxyhydroxides and hydroxides of metals possess large specific surface area and strong electrostatic properties with permanent or pH-dependent electric charge. Such nanomaterials may impact cellular electrostatics, changing the ion balance, pH and membrane potential. Selective ion adsorption/exchange may alter the transmembrane electrochemical gradient, disrupting potential-dependent cellular processes. Cellular proteins as a rule have charged residues which can be effectively adsorbed on the surface of layered hydroxide based nanomaterials. The aim of this study is to attempt to shed some light on the possibility and mechanisms of protein "adhesion" an LDH nanosheet and to propose a new direction in anticancer medicine, based on physical impact and strong electrostatics. An unbiased molecular dynamics simulation was performed and the combined process free energy estimation (COPFEE) approach was used.

  7. Prognostic significance of pretreatment serum levels of albumin, LDH and total bilirubin in patients with non-metastatic breast cancer.

    Science.gov (United States)

    Liu, Xiaoan; Meng, Qing H; Ye, Yuanqing; Hildebrandt, Michelle A T; Gu, Jian; Wu, Xifeng

    2015-02-01

    Liver function tests (LFTs) have been reported as independent predictors of non-liver disease-related morbidity and mortality in general population and cancer patients. In this study, we evaluated the relationship between pretreatment serum LFTs and overall survival (OS) in non-metastatic Caucasian breast cancer patients. Seven LFTs, including albumin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase (LDH), total bilirubin and total protein, were measured in pretreatment serum from 2425 female Caucasian patients with newly diagnosed, histologically confirmed non-metastatic invasive breast cancer. Multivariate Cox model was used to estimate hazard ratio (HR) and 95% confidence interval (CI) for the association of individual LFTs with 5-year OS while adjusting for age, smoking status, pathological characteristics and treatment regimen. We found that serum albumin, LDH and total bilirubin were significantly associated with 5-year OS in multivariate Cox analyses. Patients with higher albumin level exhibited 45% reduced risk of death (HR = 0.55, 95% CI: 0.40-0.75) compared with those with lower albumin level. Patients with higher total bilirubin level had a nearly 40% reduction in the risk of death (HR = 0.62, 95% CI: 0.45-0.85) and patients with higher LDH levels had a 1.42-fold increased risk of death (HR = 1.42, 95% CI: 1.08-1.88). Furthermore, cumulative analysis showed a significant dose-response trend of significantly increasing risk of death with increasing number of unfavorable LFT levels. Our result highlighted the potential of using pretreatment serum levels of albumin, LDH and total bilirubin as prognostic factors for OS in patients with non-metastatic breast cancer.

  8. Alteration of hydrogen metabolism of ldh-deleted Enterobacter aerogenes by overexpression of NAD+-dependent formate dehydrogenase.

    Science.gov (United States)

    Lu, Yuan; Zhao, Hongxin; Zhang, Chong; Lai, Qiheng; Wu, Xi; Xing, Xin-Hui

    2010-03-01

    The NAD+-dependent formate dehydrogenase FDH1 gene (fdh1), cloned from Candida boidinii, was expressed in the ldh-deleted mutant of Enterobacter aerogenes IAM1183 strain. The plasmid of pCom10 driven by the PalkB promoter was used to construct the fdh1 expression system and thus introduce a new dihydronicotinamide adenine dinucleotide (NADH) regeneration pathway from formate in the ldh-deleted mutant. The knockout of NADH-consuming lactate pathway affected the whole cellular metabolism, and the hydrogen yield increased by 11.4% compared with the wild strain. Expression of fdh1 in the ldh-deleted mutant caused lower final cell concentration and final pH after 16 h cultivation, and finally resulted in 86.8% of increase in hydrogen yield per mole consumed glucose. The analysis of cellular metabolites and estimated redox state balance in the fdhl-expressed strain showed that more excess of reducing power was formed by the rewired NADH regeneration pathway, changing the metabolic distribution and promoting the hydrogen production.

  9. Ascorbic acid deficiency leads to increased grain chalkiness in transgenic rice for suppressed of L-GalLDH.

    Science.gov (United States)

    Yu, Le; Liu, Yonghai; Lu, Lina; Zhang, Qilei; Chen, Yezheng; Zhou, Liping; Chen, Hua; Peng, Changlian

    2017-04-01

    The grain chalkiness of rice (Oryza sativa L.), which determines the rice quality and price, is a major concern in rice breeding. Reactive oxygen species (ROS) plays a critical role in regulating rice endosperm chalkiness. Ascorbic acid (Asc) is a major plant antioxidant, which strictly regulates the levels of ROS. l-galactono-1, 4-lactone dehydrogenase (L-GalLDH, EC 1.3.2.3) is an enzyme that catalyzes the last step of Asc biosynthesis in higher plants. Here we show that the L-GalLDH-suppressed transgenic rice, GI-1 and GI-2, which have constitutively low (between 30% and 50%) leaf and grain Asc content compared with the wild-type (WT), exhibit significantly increased grain chalkiness. Further examination showed that the deficiency of Asc resulted in a higher lipid peroxidation and H2O2 content, accompanied by a lower hydroxyl radical scavenging rate, total antioxidant capacity and photosynthetic ability. In addition, changes of the enzyme activities and gene transcript abundances related to starch synthesis were also observed in GI-1 and GI-2 grains. The results we presented here suggest a close correlation between Asc deficiency and grain chalkiness in the L-GalLDH-suppressed transgenics. Asc deficiency leads to the accumulation of H2O2, affecting antioxidant capacity and photosynthetic function, changing enzyme activities and gene transcript abundances related to starch synthesis, finally leading to the increased grain chalkiness. Copyright © 2017 Elsevier GmbH. All rights reserved.

  10. Selective Oxidation of Glycerol with 3% H2O2 Catalyzed by LDH-Hosted Cr(III Complex

    Directory of Open Access Journals (Sweden)

    Gongde Wu

    2015-11-01

    Full Text Available A series of layered double hydroxides (LDHs –hosted sulphonato-salen Cr(III complexes were prepared and characterized by various physico-chemical measurements, such as Fourier transform infrared spectroscopy (FTIR, ultraviolet-visible spectroscopy (UV-Vis, powder X-ray diffraction (XRD, transmission electron microscope (TEM, scanning electron microscope (SEM and elemental analysis. Additionally, their catalytic performances were investigated in the selective oxidation of glycerol (GLY using 3% H2O2 as an oxidant. It was found that all the LDH-hosted Cr(III complexes exhibited significantly enhanced catalytic performance compared to the homogeneous Cr(III complex. Additionally, it was worth mentioning that the metal composition of LDH plates played an important role in the catalytic performances of LDH-hosted Cr(III complex catalysts. Under the optimal reaction conditions, the highest GLY conversion reached 85.5% with 59.3% of the selectivity to 1,3-dihydroxyacetone (DHA. In addition, the catalytic activity remained after being recycled five times.

  11. Investigation of the stabilizing effects of hydroxyethyl cellulose on LDH during freeze drying and freeze thawing cycles.

    Science.gov (United States)

    Al-Hussein, Anas; Gieseler, Henning

    2015-01-01

    The objective of this study was to investigate both the cryoprotective and lyoprotective effects of the polymer hydroxyethyl cellulose (HEC) on the model protein lactate dehydrogenase (LDH) during freeze thawing and freeze drying cycles. The effect of annealing on both protein stability and the physical state of HEC was evaluated. HEC was used as a sole excipient in the protein formulations, and its stabilizing was compared to that of other excipients which are commonly used in freeze dried protein formulations. Furthermore, other quality aspects of the freeze dried samples containing solely HEC were investigated, such as, reconstitution time and product elegance. Protein stability was evaluated functionally by measuring the activity recovery of the model protein LDH. The physical state of HEC after freeze drying was investigated and compared to this of other studied solutes using differential scanning calorimetry and X-ray powder diffractometry. HEC showed superior cryoprotective effects on LDH during freeze thawing, and considerable lyoprotective effects during the freeze drying process. Annealing had limited influence on the stabilizing effect of HEC. The extensive reconstitution times of the HEC lyophilisates could be greatly improved by incorporation of the surfactant Tween 80 into the formulations prior to freeze drying.

  12. A mitochondria targeting Mn nanoassembly of BODIPY for LDH-A, mitochondria modulated therapy and bimodal imaging of cancer.

    Science.gov (United States)

    Boison, Daniel; Lu, Wen-Long; Xu, Qin-Mei; Yang, Huang; Huang, Tao; Chen, Qiu-Yun; Gao, Jing; Zhao, Yao

    2016-11-01

    HIF-1α and LDH-A are important targets for hypoxia-driven drug resistance. Mitochondria targeted fluorescent manganese(II)-complexes can be used as potential fluorescence imaging agents, MRI contrast agents and HIF-1α and LDH-A involved anticancer complexes. In this study, a fluorescent manganese(II) nanoparticle, labeled as (PEG-Mn-BDA), was synthesized and used as both fluorescent and MRI imaging agents in cancer cells. In vitro bioassay results indicate that PEG-Mn-BDA was able to inhibit LDH-A activity and depolarize mitochondrial membrane potential with the generation of intracellular ROS, which contributed to the induction of apoptosis. Moreover, the pro-apoptotic protein, caspase 3 was highly expressed. In vivo, PEG-Mn-BDA could also exert inhibition on a mouse hepatocellular carcinoma xenograft. These results suggest that mitochondria targeted PEG-Mn-BDA was able to simultaneously induce selective inhibition on cancer cells and a mouse carcinoma xenograft, label cancer cells with fluorescence and enhance MRI contrast. Therefore, PEG-Mn-BDA is a good candidate for cancer treatment and imaging.

  13. ZnO nanoparticles augment ALT, AST, ALP and LDH expressions in C2C12 cells.

    Science.gov (United States)

    Pandurangan, Muthuraman; Kim, Doo Hwan

    2015-11-01

    The present study aimed to investigate the effect of ZnO nanoparticles on alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) enzyme expressions in C2C12 cells. ZnO nanoparticles are widely used in the several cosmetic lotions and other biomedical products. Several studies report on ZnO nanoparticle mediated cytotoxicity. However, there are no reports on the effect of ZnO nanoparticles on ALT, AST, ALP and LDH enzyme expressions in C2C12 cells. A cytotoxicity assay was carried out to determine the effect of ZnO nanoparticles (1-5 mg/ml) on C2C12 cell viability at 48 and 72 h. ZnO nanoparticles increased ALT, AST, ALP and LDH enzyme mRNA expression and their activities in C2C12 cells. In conclusion, the present study showed that ZnO nanoparticles increased these enzyme activities and its mRNA expression in C2C12 cells in a dose-dependent manner.

  14. Molecular Dynamics Study of the Proposed Proton Transport Pathways in [FeFe]-Hydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Ginovska-Pangovska, Bojana; Ho, Ming-Hsun; Linehan, John C.; Cheng, Yuhui; Dupuis, Michel; Raugei, Simone; Shaw, Wendy J.

    2014-01-15

    Possible proton channels in Clostridium pasteurianum [FeFe]-hydrogenase were investigated with molecular dynamics simulations. This study was undertaken to discern proposed channels, compare their properties, evaluate the functional channel, and to provide insight into the features of an active proton channel. Our simulations suggest that protons are not transported through water wires. Instead, a five-residue motif (E282, S319, E279, HOH, C299) was found to be the likely channel, consistent with experimental observations. This channel connects the surface of the enzyme and the di-thiomethylamine bridge of the catalytic H-cluster, permitting the transport of protons. The channel was found to have a persistent hydrogen bonded core (residues E279 to S319), with less persistent hydrogen bonds at the ends of the channel. The hydrogen bond occupancy in this network was found to be sensitive to the protonation state of the residues in the channel, with different protonation states enhancing or stabilizing hydrogen bonding in different regions of the network. Single site mutations to non-hydrogen bonding residues break the hydrogen bonding network at that residue, consistent with experimental observations showing catalyst inactivation. In many cases, these mutations alter the hydrogen bonding in other regions of the channel which may be equally important in catalytic failure. A correlation between the protein dynamics near the proton channel and the redox partner binding regions was also found as a function of protonation state. The likely mechanism of proton movement in [FeFe]-hydrogenases is discussed based on the structural analysis presented here. This work was funded by the DOE Office of Science Early Career Research Program through the Office of Basic Energy Sciences. Computational resources were provided at W. R. Wiley Environmental Molecular Science Laboratory (EMSL), a national scientific user facility sponsored by the Department of Energy’s Office of

  15. The functional investigation of the ldh-luc reporter strain of Streptococcus mutans%变异链球菌ldh-luc荧光报告株的生物学活性检测

    Institute of Scientific and Technical Information of China (English)

    霍丽珺; 凌均棨

    2014-01-01

    Objective To investigate the efficacy of ldh-luc reporter strain of Streptococcus mutans (S.mutans), which was constructed in our previous study, and to probe the metabolic status of the reporter strain of S.mutans growing as either in a planktonic culture or a in biofilm community . Methods Growth curve, scanning electron microscope (SEM) and colony-forming units (CFU) counts were used to investigate the metabolic status of the reporter strain and the wide type of S.mutans growing as either in a planktonic culture or in a biofilm community . The fluorescence activity of this reporter strain was also measured. Results Growth curves and CFU counts of the reporter strain and the wide type of S.mutans were similar. There was no significant difference between the 4, 12 and 24 h biofilm of the reporter strain and the wide type of S.mutans by SEM. The fluorescence expression of the report strain was steady from exponential phase to stationary phases . The RLU reflected the amount of live S.mutans. Conclusions The growth abilities of the reporter strain were similar with the wide type of S.mutans. And the fluorescence activity of this reporter strain was stable . All of these results suggested that the reporter strain could be used to probe the metabolic status of S.mutans and response of S.mutans to antimicrobial agents instead of the wild type .%目的:探讨本课题组前期所构建的变异链球菌(S.mutans)ldh-luc荧光报告株在浮游态及生物膜状态下的活性及代谢状态的有效性,为探讨抗菌剂对多菌种生物膜中S.mutans的作用提供研究基础。方法采用生长曲线、扫描电子显微镜、活菌菌落计数等方法研究浮游态及生物膜状态下S.mutans ldh-luc荧光报告株的生长活性及代谢状态,并测定荧光报告株的荧光活性。结果 S.mutans ldh-luc荧光报告株和野生株生长趋势一致,生物膜内可培养活菌数无差别(4h:F=2.13,P>0.05;12 h:F=1.45,P >0.05;24

  16. The expression of Ldh-c in the skeletal muscle of plateau pika (Ochotona curzoniae enhances adaptation to a hypoxic environment

    Directory of Open Access Journals (Sweden)

    Zhi F. An

    2017-09-01

    Full Text Available The plateau pika (Ochotona curzoniae is a species of sprint-running alpine animals in the Qinghai-Tibet Plateau, which is a harsh highland hypoxic environment. Ldh-c is expressed in the testis, sperm and somatic tissues of plateau pika. To reveal the role and physiological mechanisms of sperm-specific lactate dehydrogenase (LDH-C4, in plateau pika to adapt to hypoxic environment, an adenoviral line of pMultiRNAi-Ldhc was constructed and injected into the bilateral biceps femoris of the hind legs. The swimming times of the pikas, and the Ldh-c expression levels, total LDH activities and ATP levels in skeletal muscle, were measured after the pikas were raised in the trapped site for 5 days. Our results showed that after Ldh-c was silenced, the sprint-running ability (swimming time of the plateau pikas was significant decreased, and the total LDH activities and ATP levels were reduced by 28.21% and 27.88%, respectively. Our results indicated that expression of Ldh-c in the skeletal muscle of plateau pika increased anaerobic glycolysis and enhanced adaptation to highland hypoxic environments.

  17. A novel platform designed by Au core/inorganic shell structure conjugated onto MTX/LDH for chemo-photothermal therapy.

    Science.gov (United States)

    Tian, De-Ying; Wang, Wei-Yuan; Li, Shu-Ping; Li, Xiao-Dong; Sha, Zhao-Lin

    2016-05-30

    A novel platform making up of methotrexate intercalated layered double hydroxide (MTX/LDH) hybrid doped with gold nanoparticles (NPs) may have great potential both in chemo-photothermal therapy and the simultaneous drug delivery. In this paper, a promising platform of Au@PDDA-MTX/LDH was developed for anti-tumor drug delivery and synergistic therapy. Firstly, Au NPs were coated using Layer-by-Layer (LbL) technology by alternate deposition of poly (diallyldimethylammonium chloride) (PDDA) and MTX molecules, and then the resulting core-shell structures (named as Au@PDDA-MTX) were directly conjugated onto the surface of MTX/LDH hybrid by electrostatic attraction to afford Au@PDDA-MTX/LDH NPs. Here MTX was used as both the agent for surface modification and the anti-tumor drug for chemotherapy. The platform of Au@PDDA-MTX/LDH NPs not only had a high drug-loading capacity, but also showed excellent colloidal stability and interesting pH-responsive release profile. In vitro drug release studies demonstrated that MTX released from Au@PDDA-MTX/LDH was relatively slow under normal physiological pH, but it was enhanced significantly at a weak acidic pH value. Furthermore, the combined treatment of cancer cells by using Au@PDDA-MTX/LDH for synergistic hyperthermia ablation and chemotherapy was demonstrated to exhibit higher therapeutic efficacy than either single treatment alone, underscoring the great potential of the platform for cancer therapy.

  18. Intercalation chemistry in a LDH system: anion exchange process and staging phenomenon investigated by means of time-resolved, in situ X-ray diffraction.

    Science.gov (United States)

    Taviot-Guého, Christine; Feng, Yongjun; Faour, Azzam; Leroux, Fabrice

    2010-07-14

    Using time-resolved, in situ energy-dispersive X-ray diffraction (EDXRD), the formation of interstratified LDH structures, with alternate interlayer spaces occupied by different anions, have been demonstrated during anion exchange reactions. Novel hybrid LDH nanostructures can thus be prepared, combining the physicochemical properties of two intercalated anions plus those of the LDH host. A general trend is that inorganic-inorganic anion exchange reactions occur in a one-step process while inorganic-organic exchanges may proceed via a second-stage intermediate, suggesting that staging occurs partly as a result of organic-inorganic separation. Yet, other influencing parameters must be considered such as LDH host composition, LDH affinity for different anions and LDH particle size as well as extrinsic parameters like the reaction temperature. Hence, a correlation between the occurrence of staging phenomenon and the difficulty of the exchange of the initial anion is observed, suggesting that staging is needed to overcome the energy barrier in the case of the exchange by organic anions. Notwithstanding the LiAl(2) system, staging has mainly been observed with Zn(2)Cr LDH host so far, a peculiar LDH composition with a unique Zn/Cr ratio of two and a local order of the cations within the hydroxide layers. The formation of a higher order-staged intermediate than stage two, observed during the exchange reaction of CO(3)(2-) or SO(4)(2-) anions with Zn(2)Cr-tartrate, is in favour of a Daumas-Herold model although this model implies a bending of LDH layers. The analysis of the X-ray powder diffraction pattern of Zn(2)Cr-Cl/tartrate second-stage intermediate, isolated almost as a pure phase during the exchange of Cl(-) with tartrate anions in Zn(2)Cr LDH, indicates a disorder in the stacking sequence and a relative proportion of the two kinds of interlayers slightly different from 50/50. Besides, the microstructural analysis of the XRD pattern reveals a great reduction of the

  19. Raman Spectroscopy of Charge Transfer Interactions Between Single Wall Carbon Nanotubes and [FeFe] Hydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Blackburn, J. L. Svedruzic, D.; McDonald, T. J.; Kim, Y. H.; King, P. W.; Heben, M. J.

    2008-01-01

    We report a Raman spectroscopy study of charge transfer interactions in complexes formed by single-walled carbon nanotubes (SWNTs) and [FeFe] hydrogenase I (CaHydI) from Clostridium acetobutylicum. The choice of Raman excitation wavelength and sample preparation conditions allows differences to be observed for complexes involving metallic (m) and semiconducting (s) species. Adsorbed CaHydI can reversibly inject electronic charge into the LUMOs of s-SWNTs, while charge can be injected and removed from m-SWNTs at lower potentials just above the Fermi energy. Time-dependent enzymatic assays demonstrated that the reduced and oxidized forms of CaHydI are deactivated by oxygen, but at rates that varied by an order of magnitude. The time evolution of the oxidative decay of the CaHydI activity reveals different time constants when complexed with m-SWNTs and s-SWNTs. The correlation of enzymatic assays with time-dependent Raman spectroscopy provides a novel method by which the charge transfer interactions may be investigated in the various SWNT-CaHydI complexes. Surprisingly, an oxidized form of CaHydI is apparently more resistant to oxygen deactivation when complexed to m-SWNTs rather than s-SWNTs.

  20. Electrocatalytic proton reduction by a model for [NiFeSe] hydrogenases.

    Science.gov (United States)

    Gezer, Gamze; Durán Jiménez, Dinesh; Siegler, Maxime A; Bouwman, Elisabeth

    2017-06-13

    Two new heterodinuclear nickel-iron complexes [Ni(pbSmSe)FeCpCO]PF6 and [Ni(xbSmSe)FeCpCO]PF6 were synthesized as mimics of the [NiFeSe] hydrogenase active site (HCp = cyclopentadiene; H2pbSmSe = 1,9-diselenol-3,7-dithia-2,2,8,8-tetramethylnonane; H2xbSmSe = 1,2,-bis(2-thiabutyl-3,3-dimethyl-4-selenol)benzene). The compounds were characterized by single crystal X-ray diffraction and cyclic voltammetry. X-ray structure determinations showed that in both NiFe complexes the nickel(ii) center is in a square-planar S2Se2 environment; the two selenolate donors are bridging to the iron(ii) center that is further coordinated to an η(5)-cyclopentadienyl group and a carbon monoxide ligand. Electrochemical studies showed that the complex [Ni(pbSmSe)FeCpCO]PF6 is an electrocatalyst for the production of H2 in DMF in the presence of acetic acid at -2.1 V vs. Fc(+)/Fc; a foot-of-the-wave (FOWA) analysis of the catalytic currents yielded an estimation of kobs of 24 s(-1).

  1. Biomimetic peptide-based models of [FeFe]-hydrogenases: utilization of phosphine-containing peptides.

    Science.gov (United States)

    Roy, Souvik; Nguyen, Thuy-Ai D; Gan, Lu; Jones, Anne K

    2015-09-07

    Two synthetic strategies for incorporating diiron analogues of [FeFe]-hydrogenases into short peptides via phosphine functional groups are described. First, utilizing the amine side chain of lysine as an anchor, phosphine carboxylic acids can be coupled via amide formation to resin-bound peptides. Second, artificial, phosphine-containing amino acids can be directly incorporated into peptides via solution phase peptide synthesis. The second approach is demonstrated using three amino acids each with a different phosphine substituent (diphenyl, diisopropyl, and diethyl phosphine). In total, five distinct monophosphine-substituted, diiron model complexes were prepared by reaction of the phosphine-peptides with diiron hexacarbonyl precursors, either (μ-pdt)Fe2(CO)6 or (μ-bdt)Fe2(CO)6 (pdt = propane-1,3-dithiolate, bdt = benzene-1,2-dithiolate). Formation of the complexes was confirmed by UV/Vis, FTIR and (31)P NMR spectroscopy. Electrocatalysis by these complexes is reported in the presence of acetic acid in mixed aqueous-organic solutions. Addition of water results in enhancement of the catalytic rates.

  2. Hydrogen production by the naked active site of the di-iron hydrogenases in water.

    Science.gov (United States)

    Zipoli, Federico; Car, Roberto; Cohen, Morrel H; Selloni, Annabella

    2009-10-01

    We explored the reactivity of the active center of the [FeFe]-hydrogenases detached from the enzyme and immersed in acidified water by first-principles Car-Parrinello molecular-dynamics simulations. We focused on the identification of the structures that are stable and metastable in acidified water and on their activity for hydrogen production. Our calculations revealed that the naked active center could be an efficient catalyst provided that electrons are transferred to the cluster. We found that both bridging and terminal isomers are present at equilibrium and that the bridging configuration is essential for efficient hydrogen production. The formation of the hydrogen molecule occurs via sequential protonations of the distal iron and of the N-atom of the S-CH(2)-NH-CH(2)-S chelating group. H(2) desorption does not involve a significant energy barrier, making the process very efficient at room temperature. We established that the bottleneck in the reaction is the direct proton transfer from water to the vacant site of the distal iron. Moreover, we found that even if the terminal isomer is present at the equilibrium, its strong local hydrophobicity prevents poisoning of the cluster.

  3. Hydrogen-Activation Mechanism of [Fe] Hydrogenase Revealed by Multi-Scale Modeling

    CERN Document Server

    Finkelmann, Arndt Robert; Reiher, Markus

    2014-01-01

    When investigating the mode of hydrogen activation by [Fe] hydrogenases, not only the chemical reactivity at the active site is of importance but also the large-scale conformational change between the so-called open and closed conformations, which leads to a special spatial arrangement of substrate and iron cofactor. To study H2 activation, a complete model of the solvated and cofactor-bound enzyme in complex with the substrate methenyl-H4MPT+ was constructed. Both the closed and open conformations were simulated with classical molecular dynamics on the 100 ns time scale. Quantum-mechanics/molecular-mechanics calculations on snapshots then revealed the features of the active site that enable the facile H2 cleavage. The hydroxyl group of the pyridinol ligand can easily be deprotonated. With the deprotonated hydroxyl group and the structural arrangement in the closed conformation, H2 coordinated to the Fe center is subject to an ionic and orbital push-pull effect and can be rapidly cleaved with a concerted hydr...

  4. Functional Analysis by Site-Directed Mutagenesis of the NAD+-Reducing Hydrogenase from Ralstonia eutropha

    Science.gov (United States)

    Burgdorf, Tanja; De Lacey, Antonio L.; Friedrich, Bärbel

    2002-01-01

    The tetrameric cytoplasmic [NiFe] hydrogenase (SH) of Ralstonia eutropha couples the oxidation of hydrogen to the reduction of NAD+ under aerobic conditions. In the catalytic subunit HoxH, all six conserved motifs surrounding the [NiFe] site are present. Five of these motifs were altered by site-directed mutagenesis in order to dissect the molecular mechanism of hydrogen activation. Based on phenotypic characterizations, 27 mutants were grouped into four different classes. Mutants of the major class, class I, failed to grow on hydrogen and were devoid of H2-oxidizing activity. In one of these isolates (HoxH I64A), H2 binding was impaired. Class II mutants revealed a high D2/H+ exchange rate relative to a low H2-oxidizing activity. A representative (HoxH H16L) displayed D2/H+ exchange but had lost electron acceptor-reducing activity. Both activities were equally affected in class III mutants. Mutants forming class IV showed a particularly interesting phenotype. They displayed O2-sensitive growth on hydrogen due to an O2-sensitive SH protein. PMID:12399498

  5. Effect of surfactant alkyl chain length on the dispersion, and thermal and dynamic mechanical properties of LDPE/organo-LDH composites

    National Research Council Canada - National Science Library

    Muksing, N; Magaraphan, R; Coiai, S; Passaglia, E

    2011-01-01

    Low density polyethylene/layered double hydroxide (LDH) composites were prepared via melt compounding using different kinds of organo-LDHs and polyethylene-grafted maleic anhydride as the compatibilizer...

  6. [The expression of the sperm-specific lactate dehydrogenase gene Ldh-c in plateau pika (Ochotona curzoniae) cardiac muscle and its effect on the anaerobic glycolysis].

    Science.gov (United States)

    Li, Xiao; Wei, Lian; Wang, Yang; Xu, Li-Na; Wei, Lin-Na; Wei, Deng-Bang

    2015-06-25

    The plateau pika (Ochotona curzoniae) has a strong adaptability to hypoxic plateau environment. We found that the sperm-specific lactate dehydrogenase (LDH-C4) gene Ldh-c expressed in plateau pika cardiac muscle. In order to shed light on the effect of LDH-C4 on the anaerobic glycolysis in plateau pika cardiac muscle, 20 pikas were randomly divided into the inhibitor group and the control group, and the sample size of each group was 10. The pikas of inhibitor group were injected with 1 mL 1 mol/L N-isopropyl oxamate, a specific LDH-C4 inhibitor, in biceps femoris muscle of hind legs, each leg with 500 μL. The pikas of control group were injected with the same volume of normal saline (0.9% NaCl). The mRNA and protein expression levels of Ldh-c gene in plateau pika cardiac muscle were determined by real-time PCR and Western blot. The activities of LDH, and the contents of lactate (LD) and ATP in cardiac muscle were compared between the inhibitor group and the control group. The results showed that 1) the expression levels of Ldh-c mRNA and protein were 0.47 ± 0.06 and 0.68 ± 0.08, respectively; 2) 30 min after injection of 1 mL 1 mol/L N-isopropyl oxamate in biceps femoris muscle, the concentration of N-isopropyl oxamate in blood was 0.08 mmol/L; 3) in cardiac muscle of the inhibitor group and the control group, the LDH activities were (6.18 ± 0.48) U/mg and (9.08 ± 0.58) U/mg, the contents of LD were (0.21 ± 0.03) mmol/g and (0.26 ± 0.04) mmol/g, and the contents of ATP were (4.40 ± 0.69) nmol/mg and (6.18 ± 0.73) nmol/mg (P < 0.01); 5) the inhibition rates of N-isopropyl oxamate to LDH, LD and ATP were 31.98%, 20.90% and 28.70%, respectively. The results suggest that Ldh-c expresses in cardiac muscle of plateau pika, and the pika cardiac muscle may get at least 28% ATP for its activities by LDH-C4 catalyzed anaerobic glycolysis, which reduces the dependence on oxygen and enhances the adaptation to the hypoxic environments.

  7. Gas exchange in the filamentous cyanobacterium Nostoc punctiforme strain ATCC 29133 and Its hydrogenase-deficient mutant strain NHM5.

    Science.gov (United States)

    Lindberg, Pia; Lindblad, Peter; Cournac, Laurent

    2004-04-01

    Nostoc punctiforme ATCC 29133 is a nitrogen-fixing, heterocystous cyanobacterium of symbiotic origin. During nitrogen fixation, it produces molecular hydrogen (H(2)), which is recaptured by an uptake hydrogenase. Gas exchange in cultures of N. punctiforme ATCC 29133 and its hydrogenase-free mutant strain NHM5 was studied. Exchange of O(2), CO(2), N(2), and H(2) was followed simultaneously with a mass spectrometer in cultures grown under nitrogen-fixing conditions. Isotopic tracing was used to separate evolution and uptake of CO(2) and O(2). The amount of H(2) produced per molecule of N(2) fixed was found to vary with light conditions, high light giving a greater increase in H(2) production than N(2) fixation. The ratio under low light and high light was approximately 1.4 and 6.1 molecules of H(2) produced per molecule of N(2) fixed, respectively. Incubation under high light for a longer time, until the culture was depleted of CO(2), caused a decrease in the nitrogen fixation rate. At the same time, hydrogen production in the hydrogenase-deficient strain was increased from an initial rate of approximately 6 micro mol (mg of chlorophyll a)(-1) h(-1) to 9 micro mol (mg of chlorophyll a)(-1) h(-1) after about 50 min. A light-stimulated hydrogen-deuterium exchange activity stemming from the nitrogenase was observed in the two strains. The present findings are important for understanding this nitrogenase-based system, aiming at photobiological hydrogen production, as we have identified the conditions under which the energy flow through the nitrogenase can be directed towards hydrogen production rather than nitrogen fixation.

  8. Overproduction of the membrane-bound [NiFe]-hydrogenase in Thermococcus kodakarensis and its effect on hydrogen production

    Directory of Open Access Journals (Sweden)

    Tamotsu eKanai

    2015-08-01

    Full Text Available The hyperthermophilic archaeon Thermococcus kodakarensis can utilize sugars or pyruvate for growth. In the absence of elemental sulfur, the electrons via oxidation of these substrates are accepted by protons, generating molecular hydrogen (H2. The hydrogenase responsible for this reaction is a membrane-bound [NiFe]-hydrogenase (Mbh. In this study, we have examined several possibilities to increase the protein levels of Mbh in T. kodakarensis by genetic engineering. Highest levels of intracellular Mbh levels were achieved when the promoter of the entire mbh operon (TK2080-TK2093 was exchanged to a strong constitutive promoter from the glutamate dehydrogenase gene (TK1431 (strain MHG1. When MHG1 was cultivated under continuous culture using pyruvate-based medium, a nearly 25 % higher specific hydrogen production rate (SHPR of 35.3 mmol H2 g-dcw-1 h-1 was observed at a dilution rate of 0.31 h-1. We also combined mbh overexpression using an even stronger constitutive promoter from the cell surface glycoprotein gene (TK0895 with disruption of the genes encoding the cytosolic hydrogenase (Hyh and an alanine aminotransferase (AlaAT, both of which are involved in hydrogen consumption (strain MAH1. At a dilution rate of 0.30 h-1, the SHPR was 36.2 mmol H2 g-dcw-1 h-1, corresponding to a 28 % increase compared to that of the host T. kodakarensis strain. Increasing the dilution rate to 0.83 h-1 resulted in a SHPR of 120 mmol H2 g-dcw-1 h-1, which is one of the highest production rates observed in microbial fermentation.

  9. Overproduction of the membrane-bound [NiFe]-hydrogenase in Thermococcus kodakarensis and its effect on hydrogen production.

    Science.gov (United States)

    Kanai, Tamotsu; Simons, Jan-Robert; Tsukamoto, Ryohei; Nakajima, Akihito; Omori, Yoshiyuki; Matsuoka, Ryoji; Beppu, Haruki; Imanaka, Tadayuki; Atomi, Haruyuki

    2015-01-01

    The hyperthermophilic archaeon Thermococcus kodakarensis can utilize sugars or pyruvate for growth. In the absence of elemental sulfur, the electrons via oxidation of these substrates are accepted by protons, generating molecular hydrogen (H2). The hydrogenase responsible for this reaction is a membrane-bound [NiFe]-hydrogenase (Mbh). In this study, we have examined several possibilities to increase the protein levels of Mbh in T. kodakarensis by genetic engineering. Highest levels of intracellular Mbh levels were achieved when the promoter of the entire mbh operon (TK2080-TK2093) was exchanged to a strong constitutive promoter from the glutamate dehydrogenase gene (TK1431) (strain MHG1). When MHG1 was cultivated under continuous culture conditions using pyruvate-based medium, a nearly 25% higher specific hydrogen production rate (SHPR) of 35.3 mmol H2 g-dcw(-1) h(-1) was observed at a dilution rate of 0.31 h(-1). We also combined mbh overexpression using an even stronger constitutive promoter from the cell surface glycoprotein gene (TK0895) with disruption of the genes encoding the cytosolic hydrogenase (Hyh) and an alanine aminotransferase (AlaAT), both of which are involved in hydrogen consumption (strain MAH1). At a dilution rate of 0.30 h(-1), the SHPR was 36.2 mmol H2 g-dcw(-1) h(-1), corresponding to a 28% increase compared to that of the host T. kodakarensis strain. Increasing the dilution rate to 0.83 h(-1) or 1.07 h(-1) resulted in a SHPR of 120 mmol H2 g-dcw(-1) h(-1), which is one of the highest production rates observed in microbial fermentation.

  10. Highly efficient and selective adsorption of In3+ on pristine Zn/Al layered double hydroxide (Zn/Al-LDH) from aqueous solutions

    Science.gov (United States)

    Barnabas, Mary Jenisha; Parambadath, Surendran; Mathew, Aneesh; Park, Sung Soo; Vinu, Ajayan; Ha, Chang-Sik

    2016-01-01

    A pristine Zn/Al-layered double hydroxide (Zn/Al-LDH) showed excellent adsorption ability and selectivity towards In3+ ions from aqueous solutions. The adsorption behaviour as a function of the contact time, solution pH, ionic strength, and amount of adsorbent under ambient conditions revealed a strong dependency on the pH and ionic strength over In3+ intake. The structure and properties of Zn/Al-LDH and In3+ adsorbed Zn/Al-LDH (In-Zn/Al-LDH) were examined carefully by X-ray diffraction, Fourier transform infrared spectroscopy, N2-sorption/desorption, UV-vis spectroscopy, and X-ray photoelectron spectroscopy. The adsorbent had a sufficient number of active sites that were responsible for the In3+ adsorption and quite stable even after the adsorption process. The selective adsorption of In3+ on Zn/Al-LDH was also observed even from a mixture containing competing ions, such as Mn2+, Co2+, Ni2+, Cd2+, Pb2+, and Cu2+. The adsorption experiments showed that Zn/Al-LDH is a promising material for the pre-concentration and selective removal of In3+ from large volumes of aqueous solutions.

  11. Effect of Hypertension on Hearing Function, LDH and ChE of the Cochlea in Older Rats

    Institute of Scientific and Technical Information of China (English)

    李穗; 龚树生; 杨燕珍; 余青松

    2003-01-01

    The relationship between the hypertension and the aging process of hearing organ was in-vestigated. Twenty Wistar 3-month old rats and 20 Wistar 12-month old rats, 20 spontaneously hy-pertensive rat stroke-prone (SHRSP) 3-month old rats and 20 SHRSP 12-month old rats free ofmiddle ear infections as observed under otomicroscopy, with normal tympanic membrane and auriclereflex, were selected to be divided into two experimental groups and two control groups respective-ly. The tail artery blood pressure was measured non-invasively. The threshold of auditory brain-stem response (ABR) was measured by SpiritTM evoked potential meter. The LDH and ChE stai-ning in the inner ear was performed and the optical density was analyzed by the HPIAS analysis sys-tem. The results showed that there was no difference in the ABR thresholds, the activities of LDHand ChE between Wistar 3-month old group and SHRSP 3-month old group (P>0. 05). The meanvalue of ABR threshold and the activities of LDH and ChE in the Wistar 12-month old group at rel-evant sections were significantly greater than those in the two 3-month old groups (P<0.05),whereas the mean value of ABR threshold and the activities of LDH and ChE in the SHRSP 12-month old group at relevant sections were significantly higher than those in the 3-month old controlgroup (P<0. 01). It was concluded that presbycusis existed in the Wistar 12-month old group rats.The glycogenosis and the abnormal secretion of neural transmitter were discerned after hyperten-sion. All the above factors may worsen the aging of the hearing system.

  12. Role of the Azadithiolate Cofactor in Models for the [FeFe]-Hydrogenase: Novel Structures and Catalytic Implications

    OpenAIRE

    Olsen, Matthew T.; Thomas B. Rauchfuss; Wilson, Scott R.

    2010-01-01

    The report summarizes studies on the redox behavior of synthetic models for the [FeFe]-hydrogenases, consisting of diiron dithiolato carbonyl complexes bearing the amine cofactor and its N-benzyl derivative. Of specific interest are the causes of the low reactivity of oxidized models toward H2, which contrasts with the high activity of these enzymes for H2 oxidation. The redox and acid-base properties of the model complexes [Fe2[(SCH2)2NR](CO)3(dppv)(PMe3)]+ ([2]+ for R = H and [2′]+ for R = ...

  13. Synthetic Active Site Model of the [NiFeSe] Hydrogenase.

    Science.gov (United States)

    Wombwell, Claire; Reisner, Erwin

    2015-05-26

    A dinuclear synthetic model of the [NiFeSe] hydrogenase active site and a structural, spectroscopic and electrochemical analysis of this complex is reported. [NiFe('S2Se2')(CO)3] (H2'S2Se2' = 1,2-bis(2-thiabutyl-3,3-dimethyl-4-selenol)benzene) has been synthesized by reacting the nickel selenolate complex [Ni('S2Se2')] with [Fe(CO)3bda] (bda = benzylideneacetone). X-ray crystal structure analysis confirms that [NiFe('S2Se2')(CO)3] mimics the key structural features of the enzyme active site, including a doubly bridged heterobimetallic nickel and iron center with a selenolate terminally coordinated to the nickel center. Comparison of [NiFe('S2Se2')(CO)3] with the previously reported thiolate analogue [NiFe('S4')(CO)3] (H2'S4' = H2xbsms = 1,2-bis(4-mercapto-3,3-dimethyl-2-thiabutyl)benzene) showed that the selenolate groups in [NiFe('S2Se2')(CO)3] give lower carbonyl stretching frequencies in the IR spectrum. Electrochemical studies of [NiFe('S2Se2')(CO)3] and [NiFe('S4')(CO)3] demonstrated that both complexes do not operate as homogenous H2 evolution catalysts, but are precursors to a solid deposit on an electrode surface for H2 evolution catalysis in organic and aqueous solution. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

  14. Redox reactions of [FeFe]-hydrogenase models containing an internal amine and a pendant phosphine.

    Science.gov (United States)

    Zheng, Dehua; Wang, Mei; Chen, Lin; Wang, Ning; Sun, Licheng

    2014-02-03

    A diiron dithiolate complex with a pendant phosphine coordinated to one of the iron centers, [(μ-SCH2)2N(CH2C6H4-o-PPh2){Fe2(CO)5}] (1), was prepared and structurally characterized. The pendant phosphine is dissociated together with a CO ligand in the presence of excess PMe3, to afford [(μ-SCH2)2N(CH2C6H4-o-PPh2){Fe(CO)2(PMe3)}2] (2). Redox reactions of 2 and related complexes were studied in detail by in situ IR spectroscopy. A series of new Fe(II)Fe(I) ([3](+) and [6](+)), Fe(II)Fe(II) ([4](2+)), and Fe(I)Fe(I) (5) complexes relevant to Hox, Hox(CO), and Hred states of the [FeFe]-hydrogenase active site were detected. Among these complexes, the molecular structures of the diferrous complex [4](2+) with the internal amine and the pendant phosphine co-coordinated to the same iron center and the triphosphine diiron complex 5 were determined by X-ray crystallography. To make a comparison, the redox reactions of an analogous complex, [(μ-SCH2)2N(CH2C6H5){Fe(CO)2(PMe3)}2] (7), were also investigated by in situ IR spectroscopy in the absence or presence of extrinsic PPh3, which has no influence on the oxidation reaction of 7. The pendant phosphine in the second coordination sphere makes the redox reaction of 2 different from that of its analogue 7.

  15. LDH, proliferation curves and cell cycle analysis are the most suitable assays to identify and characterize new phytotherapeutic compounds.

    Science.gov (United States)

    Specian, Ana Flávia L; Serpeloni, Juliana M; Tuttis, Katiuska; Ribeiro, Diego L; Cilião, Heloísa L; Varanda, Eliana A; Sannomiya, Miriam; Martinez-Lopez, Wilner; Vilegas, Wagner; Cólus, Ilce M S

    2016-12-01

    Brazilian flora biodiversity has been widely investigated to identify effective and safe phytotherapeutic compounds. Among the investigated plant species, the Byrsonima genus exhibits promising biological activities. This study aimed at evaluating the cytotoxicity of B. correifolia, B. verbascifolia, B. fagifolia and B. intermedia extracts using different assays in two cell lines (primary gastric and HepG2 cells). The different extract concentrations effects on cell viability were assayed using the MTT, aquabluer, neutral red and LDH assays. Non-cytotoxic concentrations were selected to generate cell proliferation curves and to assess cell cycle kinetics by flow cytometry. Byrsonima extracts differentially affected cell viability depending on the metabolic cellular state and the biological parameter evaluated. B. fagifolia and B. intermedia extracts exhibited lower cytotoxic effects than B. correifolia and B. verbascifolia in all assays. The results obtained with LDH and flow cytometry assays were more reliable, suggesting that they can be useful in the screening for herbal medicine and to further characterize these extracts as phytotherapeutic compounds.

  16. Towards novel adsorptive nanomaterials: Synthesis of Co2+Mo6+ LDH for sulfur and aromatic removal from crude petrolatum

    Directory of Open Access Journals (Sweden)

    Mohsen S. Mostafa

    2016-06-01

    Full Text Available In the present work Co/Mo(CO32−-LDH material of highly energetic surface was prepared by controlled titration of ammonium carbonate and ammonium hydroxide against Co and Mo cations at elevated temperatures, while different analytical techniques were applied to proof the chemical constitution and surface features of the material as XPS (X-ray Photoelectric Spectroscopy in addition to XRF metal analysis, FT-IR, SEM, XRD, DSC-TGA and N2 adsorption–desorption isotherm. The highly energetic surface due to formation of 4+ surface charge in the brucite layer between Co and Mo as confirmed by XPS was practically ensured when the freshly prepared Co2+Mo6+-LDH dried at 60 °C overnight without any activation has been applied as a novel adsorbent for the removal of the undesirable compounds (sulfur and aromatics compounds from crude waxes. Suez crude petrolatum has been used during this study. Depending on the experimental data, we were successful to prepare a new type of LDHs showed high ability for removing the undesirable compounds (sulfur and aromatics from Suez crude petrolatum. Also, in the same trend it removed low melting waxes. This leads to isolation of microcrystalline waxes from petroleum wastes which used as a lubricant, rust preventive, in the manufacture of cosmetics, and in medicine as a protective dressing, emollient, and in a lot of industrial applications.

  17. LDH is an adverse prognostic factor independent of ISS in transplant-eligible myeloma patients receiving bortezomib-based induction regimens.

    Science.gov (United States)

    Chim, Chor Sang; Sim, Joycelyn; Tam, Sidney; Tse, Eric; Lie, Albert Kwok Wai; Kwong, Yok Lam

    2015-04-01

    Serum lactate dehydrogenase (LDH) has been an adverse prognostic factor for myeloma but does not feature in the International Staging System (ISS). We examined whether elevated serum LDH at diagnosis remains an adverse risk factor independent of ISS for survivals transplant-eligible myeloma patients receiving early/frontline bortezomib-based induction, followed by autologous stem cell transplantation (ASCT). Seventy-seven transplant-eligible Chinese patients received three induction regimens [staged approach (N = 25), PAD (N = 19), VTD (N = 33)], followed by ASCT and thalidomide maintenance. Five-year overall (OS) and event-free (EFS) survivals were 66.4% and 36.2%. There was no difference in demographics, complete remission/near complete remission (CR/nCR rates postinduction or ASCT, and survivals among patients induced by the three induction regimens. Elevated LDH was associated with male gender (P = 0.006), ISS III (P = 0.042) and serum β2-microglobulin (P = 0.040). Univariate analysis showed that elevated LDH, ISS III, high β2-microglobulin, and failure to attain CR/nCR post-ACST were risk factors adversely impacting both OS and EFS. Multivariate analysis showed that elevated LDH was the only factor impacting both OS (P = 0.007) and EFS (P = 0.008). In this uniformly treated cohort of transplant-eligible myeloma patients, elevated serum LDH is an adverse risk factor independent of ISS for both OS and EFS. Bortezomib-based induction/ASCT regimen had not abolished the adverse impact of elevated LDH. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. Water decontamination via the removal of Pb (II) using a new generation of highly energetic surface nano-material: Co(+2)Mo(+6) LDH.

    Science.gov (United States)

    Mostafa, Mohsen S; Bakr, Al-Sayed A; El Naggar, Ahmed M A; Sultan, El-Sayed A

    2016-01-01

    CoMo(CO3(2-)) layered double hydroxide of a highly energetic surface, as a new LDH consisting of divalent and hexavalent cations (M(+2)/M(+6)-LDH), was prepared by a homogeneous co-precipitation method. The structure and morphology of the prepared material was confirmed by several analytical techniques namely; X-ray diffraction analysis (XRD), X-ray fluorescence (XRF), Fourier transform infra-red (FT-IR) spectroscopy, differential scanning calorimetry and thermal gravimetric analysis (DSC-TGA), N2 adsorption-desorption isotherm and scanning electron microscope (SEM). The highly energetic surface of the prepared LDH was demonstrated via the X-ray photoelectron spectroscopy (XPS). The surface energy is due to the formation of +4 surface charges in the brucite layer between Co(+2) and Mo(+6). The prepared LDH was applied as a novel adsorbent for the removal of Pb (II) from its aqueous solution at different experimental conditions of time, temperature and initial Pb (II) concentrations. The change of the Pb (II) concentrations; due to adsorption, was monitored by atomic absorption spectrophotometer (AAS). The maximum uptake of Pb (II) by the Co Mo LDH was (73.4 mg/g) at 298 K. The Pb (II) adsorption was found to follow Langmuir isotherm and pseudo second order model. The adsorption process was spontaneous and endothermic. The interference of other cations on the removal of the Pb (II) was studied. Na(+) and K(+) were found to increase the adsorption capacity of the Co Mo LDH toward Pb (II) while it was slightly decreased by the presence of Mn(+2) and Cu(+2). The synthesized LDH showed a great degree of recoverability (7 times) while completely conserving its parental morphology and adsorption capacity. The mechanism of the lead ions removal had exhibited more reliability through a surface adsorption by the coordination between the Mo(+6) of the brucite layers and the oxygen atoms of the nitrates counter ions.

  19. Is Serum Total LDH Evaluation Able to Differentiate between Alimentary Lymphoma and Inflammatory Bowel Disease in a Real World Clinical Setting?

    Science.gov (United States)

    Terragni, Rossella; Morselli-Labate, Antonio M.; Vignoli, Massimo; Bottero, Enrico; Brunetti, Barbara; Saunders, Jimmy H.

    2016-01-01

    Context An increase in enzyme lactate dehydrogenase (LDH) in serum is a negative prognostic factor for survival in cats affected by lymphoma. Measuring LDH at the time of diagnosis has been studied for differentiating neoplastic disease from non-neoplastic disease in dogs. Inflammatory bowel disease (IBD) and alimentary lymphoma are common diseases in cats. Objective The aim of this study was to determine whether elevation of total LDH occurred in cats with alimentary lymphoma and non-neoplastic gastrointestinal disease, such as IBD, and to evaluate whether this enzyme is useful in supporting the differential diagnosis of these specific diseases. Materials and Methods A prospective non-randomized controlled study was carried-out in a real world setting of three Italian private veterinary clinics. Seventy-one client-owned cats with a history of chronic gastrointestinal symptoms were enrolled; 33 cats were histologically diagnosed as having alimentary lymphoma and 38 cats as having IBD. Serum samples of total LDH analysis were measured. Results Gender (P = 0.016) and age (P = 0.046) were found to be significant factors influencing the differentiation of serum total LDH between cats with alimentary lymphoma and those with IBD. Despite low diagnostic accuracy in the overall population (63%), a cut-off value of serum total LDH ranging from 0.85- to 1.04-times the upper reference limit showed good capability (accuracy >80%) of differentiating these two conditions in neutered males and cats younger than 8 years of age (AUC: 0.805, 0.833; sensitivities: 76.9%, 83.3%; specificities: 80.0%, 76.5%; PPV: 76.9%, 55.6%; NPV: 80.0%, 92.9%; respectively). Conclusions Although our study showed that gender and age are significant factors in differentiating serum total LDH between cats with alimentary lymphoma and those with IBD, this test had poor diagnostic accuracy in differentiating between these two conditions in the overall population. PMID:26986208

  20. Is Serum Total LDH Evaluation Able to Differentiate between Alimentary Lymphoma and Inflammatory Bowel Disease in a Real World Clinical Setting?

    Directory of Open Access Journals (Sweden)

    Rossella Terragni

    Full Text Available An increase in enzyme lactate dehydrogenase (LDH in serum is a negative prognostic factor for survival in cats affected by lymphoma. Measuring LDH at the time of diagnosis has been studied for differentiating neoplastic disease from non-neoplastic disease in dogs. Inflammatory bowel disease (IBD and alimentary lymphoma are common diseases in cats.The aim of this study was to determine whether elevation of total LDH occurred in cats with alimentary lymphoma and non-neoplastic gastrointestinal disease, such as IBD, and to evaluate whether this enzyme is useful in supporting the differential diagnosis of these specific diseases.A prospective non-randomized controlled study was carried-out in a real world setting of three Italian private veterinary clinics. Seventy-one client-owned cats with a history of chronic gastrointestinal symptoms were enrolled; 33 cats were histologically diagnosed as having alimentary lymphoma and 38 cats as having IBD. Serum samples of total LDH analysis were measured.Gender (P = 0.016 and age (P = 0.046 were found to be significant factors influencing the differentiation of serum total LDH between cats with alimentary lymphoma and those with IBD. Despite low diagnostic accuracy in the overall population (63%, a cut-off value of serum total LDH ranging from 0.85- to 1.04-times the upper reference limit showed good capability (accuracy >80% of differentiating these two conditions in neutered males and cats younger than 8 years of age (AUC: 0.805, 0.833; sensitivities: 76.9%, 83.3%; specificities: 80.0%, 76.5%; PPV: 76.9%, 55.6%; NPV: 80.0%, 92.9%; respectively.Although our study showed that gender and age are significant factors in differentiating serum total LDH between cats with alimentary lymphoma and those with IBD, this test had poor diagnostic accuracy in differentiating between these two conditions in the overall population.

  1. Improved hydrogen production by coupled systems of hydrogenase negative photosynthetic bacteria and fermentative bacteria in reverse micelles

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Anita [Centre for Biotechnology, University of Allahabad, Allahabad 211002 (India); Misra, Krishna [Indo-Russian Center for Bioinformatics, Indian Institute of Information Technology, Allahabad 211011 (India)

    2008-11-15

    Significant improvement in biological hydrogen production is achieved by the use of coupled bacterial cells in reverse micellar systems. Two coupled systems (a) Rhodopseudomonas palustris CGA009/Citrobacter Y19, and (b) Rhodobacter sphaeroides 2.4.1/Citrobacter Y19 bacteria have been immobilized separately in aqueous pool of the reverse micelles fabricated by various surfactants (AOT, CBAC and SDS) and apolar organic solvents (benzene and isooctane). The gene for uptake hydrogenase enzyme has been manipulated further for hydrogen generation. Mutants deficient in uptake hydrogenase (Hup{sup -}) were obtained from R. palustris CGA009 and R. sphaeroides 2.4.1, and entrapped with Citrobacter Y19 in the reverse micellar systems. More than two fold increase in hydrogen production was obtained by the use of Hup{sup -} mutants instead of wild-type photosynthetic bacteria together with Citrobacter Y19. Addition of sodium dithionite, a reducing agent to AOT/H{sub 2}O/isooctane reverse micellar system with the coupled systems of wild-type photosynthetic bacteria and fermentative bacterium Y19 effected similar increase in hydrogen production rate as it is obtained by the use of mutants. CBAC/H{sub 2}O/isooctane reverse micellar system is used for the first time for hydrogen production and is as promising as AOT/H{sub 2}O/isooctane reverse micellar system. All reverse micellar systems of coupled bacterial cultures gave encouraging hydrogen production (rate as well as yield) compared to uncoupled bacterial culture. (author)

  2. Proton Inventory and Dynamics in the Nia-S to Nia-C Transition of a [NiFe] Hydrogenase.

    Science.gov (United States)

    Greene, Brandon L; Wu, Chang-Hao; Vansuch, Gregory E; Adams, Michael W W; Dyer, R Brian

    2016-03-29

    Hydrogenases (H2ases) represent one of the most striking examples of biological proton-coupled electron transfer (PCET) chemistry, functioning in facile proton reduction and H2 oxidation involving long-range proton and electron transport. Spectroscopic and electrochemical studies of the [NiFe] H2ases have identified several catalytic intermediates, but the details of their interconversion are still a matter of debate. Here we use steady state and time-resolved infrared spectroscopy, sensitive to the CO ligand of the active site iron, as a probe of the proton inventory as well as electron and proton transfer dynamics in the soluble hydrogenase I from Pyrococcus furiosus. Subtle shifts in infrared signatures associated with the Nia-C and Nia-S states as a function of pH revealed an acid-base equilibrium associated with an ionizable amino acid near the active site. Protonation of this residue was found to correlate with the photoproduct distribution that results from hydride photolysis of the Nia-C state, in which one of the two photoproduct states becomes inaccessible at low pH. Additionally, the ability to generate Nia-S via PCET from Nia-C was weakened at low pH, suggesting prior protonation of the proton acceptor. Kinetic and thermodynamic analysis of electron and proton transfer with respect to the various proton inventories was utilized to develop a chemical model for reversible hydride oxidation involving two intermediates differing in their hydrogen bonding character.

  3. Disruption of the Operon Encoding Ehb Hydrogenase Limits AnabolicCO2 Assimilation in the Archaeon Methanococcus maripaludis

    Energy Technology Data Exchange (ETDEWEB)

    Porat, Iris; Kim, Wonduck; Hendrickson, Erik L.; Xia, Qiangwei; Zhang, Yi; Wang, Tiansong; Taub, Fred; Moore, Brian C.; Anderson, IainJ.; Hackett, Murray; Leigh, John A.; Whitman, William B.

    2006-02-01

    Methanococcus maripaludis is a mesophilic archaeon thatreduces CO2 to methane with H2 or formate as an energy source. Itcontains two membrane-bound energy-conserving hydrogenases, Eha and Ehb.To determine therole of Ehb, a deletion in the ehb operon wasconstructed to yield the mutant, strain S40. Growth of S40 was severelyimpaired in minimal medium. Both acetate and yeast extract were necessaryto restore growth to nearly wild-type levels, suggesting that Ehb wasinvolved in multiple steps in carbon assimilation. However, nodifferences in the total hydrogenase specific activities were foundbetween the wild type and mutant in either cell extracts ormembrane-purified fractions. Methanogenesis by resting cells withpyruvate as the electron donor was also reduced by 30 percent in S40,suggesting a defect in pyruvate oxidation. CO dehydrogenase/acetylcoenzyme A (CoA) synthase and pyruvate oxidoreductase had higher specificactivities in the mutant, and genes encoding these enzymes, as well asAMP-forming acetyl-CoA synthetase, were expressed at increased levels.These observations support a role for Ehb in anabolic CO2 assimilation inmethanococci.

  4. Construction and use of a Cupriavidus necator H16 soluble hydrogenase promoter (PSH) fusion to gfp (green fluorescent protein).

    Science.gov (United States)

    Jugder, Bat-Erdene; Welch, Jeffrey; Braidy, Nady; Marquis, Christopher P

    2016-01-01

    Hydrogenases are metalloenzymes that reversibly catalyse the oxidation or production of molecular hydrogen (H2). Amongst a number of promising candidates for application in the oxidation of H2 is a soluble [Ni-Fe] uptake hydrogenase (SH) produced by Cupriavidus necator H16. In the present study, molecular characterisation of the SH operon, responsible for functional SH synthesis, was investigated by developing a green fluorescent protein (GFP) reporter system to characterise PSH promoter activity using several gene cloning approaches. A PSH promoter-gfp fusion was successfully constructed and inducible GFP expression driven by the PSH promoter under de-repressing conditions in heterotrophic growth media was demonstrated in the recombinant C. necator H16 cells. Here we report the first successful fluorescent reporter system to study PSH promoter activity in C. necator H16. The fusion construct allowed for the design of a simple screening assay to evaluate PSH activity. Furthermore, the constructed reporter system can serve as a model to develop a rapid fluorescent based reporter for subsequent small-scale process optimisation experiments for SH expression.

  5. Improved purification, crystallization and crystallographic study of Hyd-2-type [NiFe]-hydrogenase from Citrobacter sp. S-77.

    Science.gov (United States)

    Muhd Noor, Noor Dina; Nishikawa, Koji; Nishihara, Hirofumi; Yoon, Ki Seok; Ogo, Seiji; Higuchi, Yoshiki

    2016-01-01

    The purification procedure of Hyd-2-type [NiFe]-hydrogenase from Citrobacter sp. S-77 was improved by applying treatment with trypsin before chromatography. Purified protein samples both with and without trypsin treatment were successfully crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol as a precipitant. Both crystals belonged to space group P21, with unit-cell parameters a = 63.90, b = 118.89, c = 96.70 Å, β = 100.61° for the protein subjected to trypsin treatment and a = 65.38, b = 121.45, c = 98.63 Å, β = 102.29° for the sample not treated with trypsin. The crystal obtained from the trypsin-treated protein diffracted to 1.60 Å resolution, which is considerably better than the 2.00 Å resolution obtained without trypsin treatment. The [NiFe]-hydrogenase from Citrobacter sp. S-77 retained catalytic activity with some amount of O2, indicating that it has clear O2 tolerance.

  6. Construction and use of a Cupriavidus necator H16 soluble hydrogenase promoter (PSH fusion to gfp (green fluorescent protein

    Directory of Open Access Journals (Sweden)

    Bat-Erdene Jugder

    2016-07-01

    Full Text Available Hydrogenases are metalloenzymes that reversibly catalyse the oxidation or production of molecular hydrogen (H2. Amongst a number of promising candidates for application in the oxidation of H2 is a soluble [Ni–Fe] uptake hydrogenase (SH produced by Cupriavidus necator H16. In the present study, molecular characterisation of the SH operon, responsible for functional SH synthesis, was investigated by developing a green fluorescent protein (GFP reporter system to characterise PSH promoter activity using several gene cloning approaches. A PSH promoter-gfp fusion was successfully constructed and inducible GFP expression driven by the PSH promoter under de-repressing conditions in heterotrophic growth media was demonstrated in the recombinant C. necator H16 cells. Here we report the first successful fluorescent reporter system to study PSH promoter activity in C. necator H16. The fusion construct allowed for the design of a simple screening assay to evaluate PSH activity. Furthermore, the constructed reporter system can serve as a model to develop a rapid fluorescent based reporter for subsequent small-scale process optimisation experiments for SH expression.

  7. Reactions of H2, CO, and O2 with active [NiFe]-hydrogenase from Allochromatium vinosum. A stopped-flow infrared study.

    NARCIS (Netherlands)

    George, S.J.; Kurkin, S.; Thorneley, R.N.; Albracht, S.P.J.

    2004-01-01

    The Ni-Fe site in the active membrane-bound [NiFe]-hydrogenase from Allochromatium Vinosum can exist in three different redox states. In the most oxidized state (Nia-S) the nickel is divalent. The most reduced state (Nia-SR) likewise has Ni2+, while the intermediate state (Nia-C*) has Ni3+. The tran

  8. Hydrogen-induced activation of the [NiFe]-hydrogenase from Allochromatium vinosum as studied by stopped-flow infrared spectroscopy.

    NARCIS (Netherlands)

    Kurkin, S.; George, S.J.; Thorneley, R.N.; Albracht, S.P.J.

    2004-01-01

    The reaction between hydrogen and the [NiFe]-hydrogenase from Allochromatium Vinosum in its inactive form has been studied by stopped-flow infrared spectroscopy. The data, for the first time, clearly show that at room temperature enzyme in the unready state, either oxidized or reduced, does not

  9. Influence of pH and ionic strength on electrostatic properties of ferredoxin, FNR, and hydrogenase and the rate constants of their interaction

    Science.gov (United States)

    Diakonova, A. N.; Khrushchev, S. S.; Kovalenko, I. B.; Riznichenko, G. Yu; Rubin, A. B.

    2016-10-01

    Ferredoxin (Fd) protein transfers electrons from photosystem I (PSI) to ferredoxin:NADP+-reductase (FNR) in the photosynthetic electron transport chain, as well as other metabolic pathways. In some photosynthetic organisms including cyanobacteria and green unicellular algae under anaerobic conditions Fd transfers electrons not only to FNR but also to hydrogenase—an enzyme which catalyzes reduction of atomic hydrogen to H2. One of the questions posed by this competitive relationship between proteins is which characteristics of thylakoid stroma media allow switching of the electron flow between the linear path PSI-Fd-FNR-NADP+ and the path PSI-Fd-hydrogenase-H2. The study was conducted using direct multiparticle simulation approach. In this method protein molecules are considered as individual objects that experience Brownian motion and electrostatic interaction with the surrounding media and each other. Using the model we studied the effects of pH and ionic strength (I) upon complex formation between ferredoxin and FNR and ferredoxin and hydrogenase. We showed that the rate constant of Fd-FNR complex formation is constant in a wide range of physiologically significant pH values. Therefore it can be argued that regulation of FNR activity doesn’t involve pH changes in stroma. On the other hand, in the model rate constant of Fd-hydrogenase interaction dramatically depends upon pH: in the range 7-9 it increases threefold. It may seem that because hydrogenase reduces protons it should be more active when pH is acidic. Apparently, regulation of hydrogenase’s affinity to both her reaction partners (H+ and Fd) is carried out by changes in its electrostatic properties. In the dark, the protein is inactive and in the light it is activated and starts to interact with both Fd and H+. Therefore, we can conclude that in chloroplasts the rate of hydrogen production is regulated by pH through the changes in the affinity between hydrogenase and ferredoxin.

  10. Nickel binding and [NiFe]-hydrogenase maturation by the metallochaperone SlyD with a single metal-binding site in Escherichia coli.

    Science.gov (United States)

    Kaluarachchi, Harini; Altenstein, Matthias; Sugumar, Sonia R; Balbach, Jochen; Zamble, Deborah B; Haupt, Caroline

    2012-03-16

    SlyD (sensitive to lysis D) is a nickel metallochaperone involved in the maturation of [NiFe]-hydrogenases in Escherichia coli (E. coli) and specifically contributes to the nickel delivery step during enzyme biosynthesis. This protein contains a C-terminal metal-binding domain that is rich in potential metal-binding residues that enable SlyD to bind multiple nickel ions with high affinity. The SlyD homolog from Thermus thermophilus does not contain the extended cysteine- and histidine-rich C-terminal tail of the E. coli protein, yet it binds a single Ni(II) ion tightly. To investigate whether a single metal-binding motif can functionally replace the full-length domain, we generated a truncation of E. coli SlyD, SlyD155. Ni(II) binding to SlyD155 was investigated by using isothermal titration calorimetry, NMR and electrospray ionization mass spectrometry measurements. This in vitro characterization revealed that SlyD155 contains a single metal-binding motif with high affinity for nickel. Structural characterization by X-ray absorption spectroscopy and NMR indicated that nickel was coordinated in an octahedral geometry with at least two histidines as ligands. Heterodimerization between SlyD and another hydrogenase accessory protein, HypB, is essential for optimal hydrogenase maturation and was confirmed for SlyD155 via cross-linking experiments and NMR titrations, as were conserved chaperone and peptidyl-prolyl isomerase activities. Although these properties of SlyD are preserved in the truncated version, it does not modulate nickel binding to HypB in vitro or contribute to the maturation of [NiFe]-hydrogenases in vivo, unlike the full-length protein. This study highlights the importance of the unusual metal-binding domain of E. coli SlyD in hydrogenase biogenesis.

  11. An analysis of the changes in soluble hydrogenase and global gene expression in Cupriavidus necator (Ralstonia eutropha) H16 grown in heterotrophic diauxic batch culture.

    Science.gov (United States)

    Jugder, Bat-Erdene; Chen, Zhiliang; Ping, Darren Tan Tek; Lebhar, Helene; Welch, Jeffrey; Marquis, Christopher P

    2015-03-25

    Soluble hydrogenases (SH) are enzymes that catalyse the oxidation of molecular hydrogen. The SH enzyme from Cupriavidus necator H16 is relatively oxygen tolerant and makes an attractive target for potential application in biochemical hydrogen fuel cells. Expression of the enzyme can be mediated by derepression of the hox promoter system under heterotrophic conditions. However, the overall impact of hox derepression, from a transcriptomic perspective, has never been previously reported. Derepression of hydrogenase gene expression upon fructose depletion was confirmed in replicate experiments. Using qRT-PCR, hoxF was 4.6-fold up-regulated, hypF2 was up-regulated in the cells grown 2.2-fold and the regulatory gene hoxA was up-regulated by a mean factor of 4.5. A full transcriptomic evaluation revealed a substantial shift in the global pattern of gene expression. In addition to up-regulation of genes associated with hydrogenase expression, significant changes were observed in genes associated with energy transduction, amino acid metabolism, transcription and translation (and regulation thereof), genes associated with cell stress, lipid and cell wall biogenesis and other functions, including cell motility. We report the first full transcriptome analysis of C. necator H16 grown heterotrophically on fructose and glycerol in diauxic batch culture, which permits expression of soluble hydrogenase under heterotrophic conditions. The data presented deepens our understanding of the changes in global gene expression patterns that occur during the switch to growth on glycerol and suggests that energy deficit is a key driver for induction of hydrogenase expression in this organism.

  12. Site saturation mutagenesis demonstrates a central role for cysteine 298 as proton donor to the catalytic site in CaHydA [FeFe]-hydrogenase.

    Directory of Open Access Journals (Sweden)

    Simone Morra

    Full Text Available [FeFe]-hydrogenases reversibly catalyse molecular hydrogen evolution by reduction of two protons. Proton supply to the catalytic site (H-cluster is essential for enzymatic activity. Cysteine 298 is a highly conserved residue in all [FeFe]-hydrogenases; moreover C298 is structurally very close to the H-cluster and it is important for hydrogenase activity. Here, the function of C298 in catalysis was investigated in detail by means of site saturation mutagenesis, simultaneously studying the effect of C298 replacement with all other 19 amino acids and selecting for mutants with high retained activity. We demonstrated that efficient enzymatic turnover was maintained only when C298 was replaced by aspartic acid, despite the structural diversity between the two residues. Purified CaHydA C298D does not show any significant structural difference in terms of secondary structure and iron incorporation, demonstrating that the mutation does not affect the overall protein fold. C298D retains the hydrogen evolution activity with a decrease of k(cat only by 2-fold at pH 8.0 and it caused a shift of the optimum pH from 8.0 to 7.0. Moreover, the oxygen inactivation rate was not affected demonstrating that the mutation does not influence O(2 diffusion to the active site or its reactivity with the H-cluster. Our results clearly demonstrate that, in order to maintain the catalytic efficiency and the high turnover number typical of [FeFe] hydrogenases, the highly conserved C298 can be replaced only by another ionisable residue with similar steric hindrance, giving evidence of its involvement in the catalytic function of [FeFe]-hydrogenases in agreement with an essential role in proton transfer to the active site.

  13. Expressão do Mg+2, CK, AST e LDH em equinos finalistas de provas de enduro Endurance horses finalists: expression of Mg+2, CK, AST and LDH in horse finalists of endurance race

    Directory of Open Access Journals (Sweden)

    Juliana V.F. Sales

    2013-01-01

    Full Text Available Nos últimos anos, o equino atleta vem sendo cada vez mais requerido. Dessa forma, as exigências por alto desempenho têm fomentado o interesse pelo estudo das afecções relacionadas com a fisiopatologia de diversas enfermidades dos equinos. A relação entre o íon magnésio e o exercício físico tem recebido atenção significativa visto que este íon está intimamente relacionado ao tecido muscular estriado esquelético. Além disso, dentre as principais estratégias para a detecção e acompanhamento clínico de lesões musculares, destacam-se a avaliação das atividades das enzimas creatino quinase (CK, lactato desidrogenase (LDH e aspartato aminotransferase (AST. A busca pelo estabelecimento de parâmetros que se relacionam entre si é um fator determinante na compreensão de alterações fisiológicas encontradas diante do esforço em equinos atletas. Desta forma, o presente trabalho teve como objetivo determinar como as concentrações sanguíneas do íon magnésio e as atividades enzimáticas das enzimas CK, LDH e AST comportaram-se em equinos Puro Sangue Árabe finalistas de provas de enduro de 90km e relacionar as possíveis alterações com o tipo de esforço físico desempenhado pelos animais. Foram avaliadas a atividade enzimática das enzimas CK, LDH, AST e a concentração do íon magnésio no exercício em relação ao repouso de 14 equinos clinicamente hígidos da raça Puro Sangue Árabe, sendo 9 machos e 5 fêmeas, com idades variando entre 6 a 12 anos, submetidos a treinamento para enduro e participantes de provas de 90 km. Pode-se observar que as variáveis acima mencionadas sofreram aumento com diferença estatística em relação ao repouso. O exercício físico de enduro determinou a ocorrência de alterações nas atividades enzimáticas das enzimas CK (p≤0,001, LDH (p=0,0001, AST (p=0,0007 e na concentração do íon magnésio (p=0,0004, no exercício em relação ao repouso (p≤0,05. Fato que determinou altera

  14. Purification and Characterization of [NiFe]-Hydrogenase of Shewanella oneidensis MR-1

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Liang; Belchik, Sara M.; Plymale, Andrew E.; Heald, Steve M.; Dohnalkova, Alice; Sybirna, Kateryna; Bottin, Herve; Squier, Thomas C.; Zachara, John M.; Fredrickson, Jim K.

    2011-08-02

    The γ-proteobacterium Shewanella oneidensis MR-1 possesses a periplasmic [NiFe]-hydrogenase (MR-1 [NiFe]-H2ase) that was implicated in both H2 production and oxidation as well as technetium [Tc(VII)] reduction. To characterize the roles of MR-1 [NiFe]-H2ase in these proposed reactions, the genes encoding both subunits of MR-1 [NiFe]-H2ase were cloned into a protein expression vector. The resulting plasmid was transformed into a MR-1 mutant deficient in H2 formation. Expression of MR-1 [NiFe]-H2ase in trans restored the mutant’s ability to produce H2 at 37% of that for wild type. Following expression, MR-1 [NiFe]-H2ase was purified to near homogeneity. The purified MR-1 [NiFe]-H2ase could couple H2 oxidation to reduction of Tc(VII) and methyl viologen directly. Change of the buffers used affected MR-1 [NiFe]-H2ase-mediated Tc(VII) but not methyl viologen reductions. Under the conditions tested, Tc(VII) reduction was complete in Tris buffer but not in HEPES buffer. The reduced Tc(IV) was soluble in Tris buffer but insoluble in HEPES buffer. Transmission electron microscopy analysis revealed that Tc(IV) precipitates formed in HEPES buffer were packed with crystallites. Although X-ray absorption near-edge spectroscopy measurements confirmed that the reduction products found in both buffers were Tc(IV), extended X-ray adsorption fine-structure measurements revealed that these products were very different. While the product in Tris buffer could not be determined, the Tc(IV) product in HEPES buffer was very similar to Tc(IV)O2•nH2O. These results shows for the first time that MR-1 [NiFe]-H2ase is a bidirectional enzyme that catalyzes both H2 formation and oxidation as well as Tc(VII) reduction directly by coupling H2 oxidation.

  15. 胶原酶治疗LDH 5 1例近期疗效观察

    Institute of Scientific and Technical Information of China (English)

    杨希; 陈龙尧

    2001-01-01

    @@ 腰椎间盘突出症(Lumbar dise hemiation,LDH)的发生率占所有门诊腰腿痛患者的15%,本病除少数需要手术治疗外,大部分采用非手术治疗,其方法较多,但各有利弊,而应用胶原酶治疗在我省应用不多,报道较少.现结合本组51例近期观察及有关文献,就胶原酶溶解术(简称溶盘术)的有关问题进行探讨.1资料与方法1.1一般资料自2000年元月至2000年7月,共治疗LDH 51例,其中男39例,女12例;年龄19~63岁,平均46岁.病程10天至12年,平均14个月.全部病例为住院病人,均有腰痛及下肢放射痛症状,经CT扫描确诊,其中腰3-4椎间盘突出12例,腰4-5椎间盘突出25例,腰5骶1椎间盘突出14例.

  16. Minimal Influence of [NiFe] Hydrogenase on Hydrogen Isotope Fractionation in H2-Oxidizing Cupriavidus necator

    Directory of Open Access Journals (Sweden)

    Brian J. Campbell

    2017-10-01

    Full Text Available Fatty acids produced by H2-metabolizing bacteria are sometimes observed to be more D-depleted than those of photoautotrophic organisms, a trait that has been suggested as diagnostic for chemoautotrophic bacteria. The biochemical reasons for such a depletion are not known, but are often assumed to involve the strong D-depletion of H2. Here, we cultivated the bacterium Cupriavidus necator H16 (formerly Ralstonia eutropha H16 under aerobic, H2-consuming, chemoautotrophic conditions and measured the isotopic compositions of its fatty acids. In parallel with the wild type, two mutants of this strain, each lacking one of two key hydrogenase enzymes, were also grown and measured. In all three strains, fractionations between fatty acids and water ranged from -173‰ to -235‰, and averaged -217‰, -196‰, and -226‰, respectively, for the wild type, SH- mutant, and MBH- mutant. There was a modest increase in δD as a result of loss of the soluble hydrogenase enzyme. Fractionation curves for all three strains were constructed by growing parallel cultures in waters with δDwater values of approximately -25‰, 520‰, and 1100‰. These curves indicate that at least 90% of the hydrogen in fatty acids is derived from water, not H2. Published details of the biochemistry of the soluble and membrane-bound hydrogenases confirm that these enzymes transfer electrons rather than intact hydride (H- ions, providing no direct mechanism to connect the isotopic composition of H2 to that of lipids. Multiple lines of evidence thus agree that in this organism, and presumably others like it, environmental H2 plays little or no direct role in controlling lipid δD values. The observed fractionations must instead result from isotope effects in the reduction of NAD(PH by reductases with flavin prosthetic groups, which transfer two electrons and acquire H+ (or D+ from solution. Parallels to NADPH reduction in photosynthesis may explain why D/H fractionations in C. necator

  17. Highly efficient and selective adsorption of In{sup 3+} on pristine Zn/Al layered double hydroxide (Zn/Al-LDH) from aqueous solutions

    Energy Technology Data Exchange (ETDEWEB)

    Barnabas, Mary Jenisha; Parambadath, Surendran; Mathew, Aneesh; Park, Sung Soo [Department of Polymer Science and Engineering, Pusan National University, Geumjeong-gu, Busan 46241 (Korea, Republic of); Vinu, Ajayan [Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, #75 Corner College and Cooper Road, Brisbane 4072, QLD (Australia); Ha, Chang-Sik, E-mail: csha@pnu.edu [Department of Polymer Science and Engineering, Pusan National University, Geumjeong-gu, Busan 46241 (Korea, Republic of)

    2016-01-15

    A pristine Zn/Al-layered double hydroxide (Zn/Al-LDH) showed excellent adsorption ability and selectivity towards In{sup 3+} ions from aqueous solutions. The adsorption behaviour as a function of the contact time, solution pH, ionic strength, and amount of adsorbent under ambient conditions revealed a strong dependency on the pH and ionic strength over In{sup 3+} intake. The structure and properties of Zn/Al-LDH and In{sup 3+} adsorbed Zn/Al-LDH (In–Zn/Al-LDH) were examined carefully by X-ray diffraction, Fourier transform infrared spectroscopy, N{sub 2}-sorption/desorption, UV–vis spectroscopy, and X-ray photoelectron spectroscopy. The adsorbent had a sufficient number of active sites that were responsible for the In{sup 3+} adsorption and quite stable even after the adsorption process. The selective adsorption of In{sup 3+} on Zn/Al-LDH was also observed even from a mixture containing competing ions, such as Mn{sup 2+}, Co{sup 2+}, Ni{sup 2+}, Cd{sup 2+}, Pb{sup 2+}, and Cu{sup 2+}. The adsorption experiments showed that Zn/Al-LDH is a promising material for the pre-concentration and selective removal of In{sup 3+} from large volumes of aqueous solutions. - Highlights: • A pristine Zn/Al-layered double hydroxide showed good selectivity for In{sup 3+} ions. • The material exhibited a maximum In{sup 3+} intake of 205 mg g{sup −1} at pH 6. • The materials showed good affinity of In{sup 3+} over Cu{sup 2+} and Pb{sup 2+} from ion mixtures.

  18. Characteristic LDH isozyme electrophoretic patterns in six flatfish species in the Trondheimsfjord, Norway and their utility for the detection of natural species hybrids

    KAUST Repository

    He, Song

    2014-11-19

    Abstract: LDH isozyme electrophoretic patterns among 621 specimens of six different flatfish species collected in the Trondheimsfjord, Norway, were characterized by using the isoelectric focusing in polyacrylamide gel (IFPAG) technique. The LDH locus appears to be a reliable tool for species identification in the Trondheimsfjord flatfishes. Hence, these patterns were used to detect and identify potential hybrids, together with morphological traits. Among all the specimens collected during this study no hybrids were detected. From the actual numbers analysed, the natural hybridization rate between European plaice and European flounder in the Trondheimsfjord can be roughly estimated to be less than 1%. This is substantially lower than corresponding values reported from Baltic and Danish waters.

  19. [A Fluorescent Chemical Sensor Based on MgAl-8-HQ LDH Composite Particle for the Selective Detection of Fe3+].

    Science.gov (United States)

    Yang, Lei; Yao, Qi; Yuan, Xue-hua; Yang, Yan-ling

    2015-03-01

    In order to achieve the highly selective and Simple detection for ferric ion, strong-fluorescent 8-hydroxyquinoline (8-HQ) Mg-Al layered double hydroxide(Mg(χ)Al-8-HQ LDH) was designed and prepared by 8-HQ's intercalation and ready coordination based on adjustment of Al3+ on Mg-Al layered double hydroxides (MgAl LDH) laminates. Meanwhile its structure and property were characterized by IR, XRD, UV-Vis and fluorescent spectrometer. IR analysis showed coordinate bonds of C-O-Al and C-N-Al between 8-HQ and Al3+ were generated. XRD revealed that 8-HQ had already inserted in MgAl LDH laminates, and it made (003) diffraction peaks move to low 2θ angle direction, and the diffraction peak intensity was enhanced with the molar ratio of Mg and Al increasing. Because the coordination reaction between 8-HQ and Al3+ in MgAl LDH laminates took place, it induced the absorption peak of 8-HQ at 314 nm disappeared, at the same time the transition absorption peak at 376 nm between metal ions and ligands appeared. As demonstrated by fluorescence spectroscopic analysis, fluorescence intensity of Mg(χ)Al-8-HQ LDH increased with the content of Al3+ reducing, when the molar ratio of Magnesium and Aluminium ion is 4 : 1, its fluorescence intensity enhanced more significantly than 8-hydroxyquinoline aluminum. Through the research on the influence of metal ions on the fluorescence spectra of Mg4 Al-8-HQ LDH particle, it was found that the particle to metal ions exhibited significant selection and difference, especially with high selectivity for Fe3+ ion. The effect of [Fe3+] on the color and fluorescence intensity of Mg4Al-8-HQ LDH particle solution was further studied, and the results showed that the solution varied from light yellow to dark green with the content of Fe3+ in 10(-6) to 10(-2) mol x L(-1) increasing, so it can implement colorimetric sensing for Fe3+ in the above range. And at the same time its fluorescence intensity significantly decreased, and its fluorescence could be

  20. The effects of the dimethylether bridging moiety in the H-cluster of the Clostridium pasteurianum hydrogenase on the mechanism of H{sub 2} production: A quantum mechanics/molecular mechanics study

    Energy Technology Data Exchange (ETDEWEB)

    Trohalaki, Steven [Air Force Research Laboratory, Materials and Manufacturing Directorate, Wright-Patterson Air Force Base, OH 45433 (United States); General Dynamics Information Technology, Inc. (United States); Pachter, Ruth [Air Force Research Laboratory, Materials and Manufacturing Directorate, Wright-Patterson Air Force Base, OH 45433 (United States)

    2010-12-15

    [Fe-Fe]-hydrogenases are naturally occurring metalloenzymes that catalyze the reversible production of H{sub 2} from two protons and two electrons. [Fe-Fe]-hydrogenases found in two species -Clostridium pasteurianum (CpI) and Desulfovibrio desulfuricans (DdH) - were shown with x-ray crystallography to have active sites that are very similar, although several atoms that bridge the dithiolate ligand were unresolved. In earlier work, we employed density functional theory (DFT) within a QM/MM method to investigate two previously proposed mechanisms of hydrogen production by DdH and CpI hydrogenases. In one mechanism (I), a CO ligand bridging two Fe atoms in the active site rotates to a terminal position while in the other (II) the CO bridge remains intact throughout the catalytic cycle. We previously assumed that the active sites for the two hydrogenases were identical; each had a dimethylamine bridging moiety, whose basicity is important for Mechanism II. Our overall conclusion, taking into consideration an energy comparison for the two mechanisms and activation energies for the CO-unbridging step in Mechanism I, was that Mechanism II was favored for both hydrogenases. In this paper, we extend our previous work to show that Mechanism II is favored over Mechanism I even if the bridging moiety in CpI hydrogenase is dimethylether, a significantly weaker base than dimethylamine, providing further support for Mechanism II even though experimental verification of the bridging moiety for the CpI H-cluster is lacking. (author)

  1. Multiscale modeling of the active site of [Fe] hydrogenase: the H₂ binding site in open and closed protein conformations.

    Science.gov (United States)

    Hedegård, Erik Donovan; Kongsted, Jacob; Ryde, Ulf

    2015-05-18

    A series of QM/MM optimizations of the full protein of [Fe] hydrogenase were performed. The FeGP cofactor has been optimized in the water-bound resting state (1), with a side-on bound dihydrogen (2), or as a hydride intermediate (3). For inclusion of H4MPT in the closed structure, advanced multiscale modeling appears to be necessary, especially to obtain reliable distances between CH-H4MPT(+) and the dihydrogen (H2) or hydride (H(-)) ligand in the FeGP cofactor. Inclusion of the full protein is further important for the relative energies of the two intermediates 2 and 3. We finally find that hydride transfer from 3 has a significantly higher barrier than found in previous studies neglecting the full protein environment.

  2. The weak, fluctuating, dipole moment of membrane-bound hydrogenase from Aquifex aeolicus accounts for its adaptability to charged electrodes.

    Science.gov (United States)

    Oteri, Francesco; Ciaccafava, Alexandre; de Poulpiquet, Anne; Baaden, Marc; Lojou, Elisabeth; Sacquin-Mora, Sophie

    2014-06-21

    [NiFe] hydrogenases from Aquifex aeolicus (AaHase) and Desulfovibrio fructosovorans (DfHase) have been mainly studied to characterize physiological electron transfer processes, or to develop biotechnological devices such as biofuel cells. In this context, it remains difficult to control the orientation of AaHases on electrodes to achieve a fast interfacial electron transfer. Here, we study the electrostatic properties of these two proteins based on microsecond-long molecular dynamics simulations that we compare to voltammetry experiments. Our calculations show weak values and large fluctuations of the dipole direction in AaHase compared to DfHase, enabling the AaHase to absorb on both negatively and positively charged electrodes, with an orientation distribution that induces a spread in electron transfer rates. Moreover, we discuss the role of the transmembrane helix of AaHase and show that it does not substantially impact the general features of the dipole moment.

  3. Photoinduced reduction of the medial FeS center in the hydrogenase small subunit HupS from Nostoc punctiforme.

    Science.gov (United States)

    Raleiras, Patrícia; Hammarström, Leif; Lindblad, Peter; Styring, Stenbjörn; Magnuson, Ann

    2015-07-01

    The small subunit from the NiFe uptake hydrogenase, HupSL, in the cyanobacterium Nostoc punctiforme ATCC 29133, has been isolated in the absence of the large subunit (P. Raleiras, P. Kellers, P. Lindblad, S. Styring, A. Magnuson, J. Biol. Chem. 288 (2013) 18,345-18,352). Here, we have used flash photolysis to reduce the iron-sulfur clusters in the isolated small subunit, HupS. We used ascorbate as electron donor to the photogenerated excited state of Ru(II)-trisbipyridine (Ru(bpy)3), to generate Ru(I)(bpy)3 as reducing agent. Our results show that the isolated small subunit can be reduced by the Ru(I)(bpy)3 generated through flash photolysis. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Modulation of Active Site Electronic Structure by the Protein Matrix to Control [NiFe] Hydrogenase Reactivity

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Dayle MA; Raugei, Simone; Squier, Thomas C.

    2014-09-30

    Control of the reactivity of the nickel center of the [NiFe] hydrogenase and other metalloproteins commonly involves outer coordination sphere ligands that act to modify the geometry and physical properties of the active site metal centers. We carried out a combined set of classical molecular dynamics and quantum/classical mechanics calculations to provide quantitative estimates of how dynamic fluctuations of the active site within the protein matrix modulate the electronic structure at the catalytic center. Specifically we focused on the dynamics of the inner and outer coordination spheres of the cysteinate-bound Ni–Fe cluster in the catalytically active Ni-C state. There are correlated movements of the cysteinate ligands and the surrounding hydrogen-bonding network, which modulate the electron affinity at the active site and the proton affinity of a terminal cysteinate. On the basis of these findings, we hypothesize a coupling between protein dynamics and electron and proton transfer reactions critical to dihydrogen production.

  5. [NiFe]Hydrogenase from Citrobacter sp. S-77 surpasses platinum as an electrode for H2 oxidation reaction.

    Science.gov (United States)

    Matsumoto, Takahiro; Eguchi, Shigenobu; Nakai, Hidetaka; Hibino, Takashi; Yoon, Ki-Seok; Ogo, Seiji

    2014-08-18

    Reported herein is an electrode for dihydrogen (H2) oxidation, and it is based on [NiFe]Hydrogenase from Citrobacter sp. S-77 ([NiFe]S77). It has a 637 times higher mass activity than Pt (calculated based on 1 mg of [NiFe]S77 or Pt) at 50 mV in a hydrogen half-cell. The [NiFe]S77 electrode is also stable in air and, unlike Pt, can be recovered 100 % after poisoning by carbon monoxide. Following characterization of the [NiFe]S77 electrode, a fuel cell comprising a [NiFe]S77 anode and Pt cathode was constructed and shown to have a a higher power density than that achievable by Pt.

  6. Occurrence of H2-Uptake Hydrogenases in Bradyrhizobium sp. (Lupinus) and Their Expression in Nodules of Lupinus spp. and Ornithopus compressus1

    Science.gov (United States)

    Murillo, Jesús; Villa, Ana; Chamber, Manuel; Ruiz-Argüeso, Tomás

    1989-01-01

    Fifty-four strains of Bradyrhizobium sp. (Lupinus) from worldwide collections were screened by a colony hybridization method for the presence of DNA sequences homologous to the structural genes of the Bradyrhizobium japonicum hydrogenase. Twelve strains exhibited strong colony hybridization signals, and subsequent Southern blot hybridization experiments showed that they fell into two different groups on the basis of the pattern of EcoRI fragments containing the homology to the hup probe. All strains in the first group (UPM860, UPM861, and 750) expressed uptake hydrogenase activity in symbiosis with Lupinus albus, Lupinus angustifolius, Lupinus luteus, and Ornithopus compressus, but both the rate of H2 uptake by bacteroids and the relative efficiency of N2 fixation (RE = 1 - [H2 evolved in air/acetylene reduced]) by nodules were markedly affected by the legume host. L. angustifolius was the less permissive host for hydrogenase expression in symbiosis with the three strains (average RE = 0.76), and O. compressus was the more permissive (average RE = 1.0). None of the strains in the second group expressed hydrogenase activity in lupine nodules, and only one exhibited low H2-uptake activity in symbiosis with O. compressus. The inability of these putative Hup+ strains to induce hydrogenase activity in lupine nodules is discussed on the basis of the legume host effect. Among the 42 strains showing no homology to the B. japonicum hup-specific probe in the colony hybridization assay, 10 were examined in symbiosis with L. angustifolius. The average RE for these strains was 0.51. However, one strain, IM43B, exhibited high RE values (higher than 0.80) and high levels of hydrogenase activity in symbiosis with L. angustifolius, L. albus, and L. luteus. In Southern blot hybridization experiments, no homology was detected between the B. japonicum hup-specific DNA probe and total DNA from vegetative cells or bacteroids from strain IM43B even under low stringency hybridization

  7. Molecular study of the chlorella algae and determining its functional features with the approach of hydrogenase gene expression

    Directory of Open Access Journals (Sweden)

    Seyedeh Tayebeh Mousavi

    2016-09-01

    Full Text Available Biohydrogen production by biological processes are known as a renewable energy source. The aim of the investigation was to find and optimize the most appropriate medium for the algae growth to produce the maximum amount of hydrogen. First of all, the bioinformatics and biosystematics studies were taken for identifying the collected microalgae which was detected as Chlorella with the following features: spherical appearance, spent protozoan, Cup-shaped chloroplast with no flagella. On the other hand, the molecular analysis by PCR and 18S sequence typing of interested microalgae demonstrated 100% similarity to that well known sequences for Chlorella vulgaris. Second, we assessed some culture media including BBM, Chu10, TAP, and Sorokin and Krauss for optimum growth conditions for Chlorella vulgaris. In general, our results showed that BBM medium had the highest efficiency for producing microalgae biomass under following conditions: pH=8, temperature of 30 ° with 16 to 8 h light to darkness periods ratio.Third, we designed a more efficient photo-bioreactor apparatus toward inducing more powerful bio-hydrogen production by hydrogenase enzyme activity of our given microalgae. Then, the performance of the apparatus as well as the gene expression was scrutinized under different conditions (light, pH, sulphorous, etc.. For this, after extracting RNA and constructing cDNA, hydrogenase gene was amplified with PCR and the product was evaluated by agarose gel. However, the relative expression of the gene measured by Real Time PCR showed the influence of light, pH and sulphourous on the expression as compared with control.

  8. In search of metal hydrides: an X-ray absorption and emission study of [NiFe] hydrogenase model complexes.

    Science.gov (United States)

    Hugenbruch, Stefan; Shafaat, Hannah S; Krämer, Tobias; Delgado-Jaime, Mario Ulises; Weber, Katharina; Neese, Frank; Lubitz, Wolfgang; DeBeer, Serena

    2016-04-28

    Metal hydrides are invoked as important intermediates in both chemical and biological H2 production. In the [NiFe] hydrogenase enzymes, pulsed EPR and high-resolution crystallography have argued that the hydride interacts primarily at the Ni site. In contrast, in [NiFe] hydrogenase model complexes, it is observed that the bridging hydride interacts primarily with the Fe. Herein, we utilize a combination of Ni and Fe X-ray absorption (XAS) and emission (XES) spectroscopies to examine the contribution of the bridging hydride to the observed spectral features in [(dppe)Ni(μ-pdt)(μ-H)Fe(CO)3](+). The corresponding data on (dppe)Ni(μ-pdt)Fe(CO)3 are used as a reference for the changes that occur in the absence of a hydride bridge. For further interpretation of the observed spectral features, all experimental spectra were calculated using a density functional theory (DFT) approach, with excellent agreement between theory and experiment. It is found that the iron valence-to-core (VtC) XES spectra reveal clear signatures for the presence of a Fe-H interaction in the hydride bridged model complex. In contrast, the Ni VtC XES spectrum largely reflects changes in the local Ni geometry and shows little contribution from a Ni-H interaction. A stepwise theoretical analysis of the hydride contribution and the Ni site symmetry provides insights into the factors, which govern the different metal-hydride interactions in both the model complexes and the enzyme. Furthermore, these results establish the utility of two-color XES to reveal important insights into the electronic structure of various metal-hydride species.

  9. Powerful Fermentative Hydrogen Evolution of Photosynthate in the Cyanobacterium Lyngbya aestuarii BL J Mediated by a Bidirectional Hydrogenase

    Directory of Open Access Journals (Sweden)

    Ankita eKothari

    2014-12-01

    Full Text Available Cyanobacteria are considered good models for biohydrogen production because they are relatively simple organisms with a demonstrable ability to generate H2 under certain physiological conditions. However, most produce only little H2, revert readily to H2 consumption, and suffer from hydrogenase sensitivity to O2. Strains of the cyanobacteria Lyngbya aestuarii and Microcoleus chthonoplastes obtained from marine intertidal cyanobacterial mats were recently found to display much better H2 production potential. Because of their ecological origin in environments that become quickly anoxic in the dark, we hypothesized that this differential ability may have evolved to serve a role in the fermentation of the photosynthate. Here we show that, when forced to ferment internal substrate, these cyanobacteria display desirable characteristics of physiological H2 production. Among them, the strain L. aestuarii BL J had the fastest specific rates and attained the highest H2 concentrations during fermentation of photosynthate, which proceeded via a mixed-acid fermentation pathway to yield acetate, ethanol, lactate, H2, CO2 and pyruvate. Contrary to expectations, the H2 yield per mole of glucose was only average compared to that of other cyanobacteria. Thermodynamic analyses point to the use of electron donors more electronegative than NAD(PH in Lyngbya hydrogenases as the basis for its strong H2 production ability. In any event, the high specific rates and H2 concentrations coupled with the lack of reversibility of the enzyme, at the expense of internal, photosynthetically generated reductants, makes L. aestuarii BL J and/or its enzymes, a potentially feasible platform for large-scale H2 production.

  10. Facile preparation of free-standing rGO paper-based Ni-Mn LDH/graphene superlattice composites as a pseudocapacitive electrode.

    Science.gov (United States)

    Quan, W; Tang, Z L; Wang, S T; Hong, Y; Zhang, Z T

    2016-03-04

    A novel film electrode was assembled via a simple filtration process, with an rGO paper as the substrate and Ni-Mn LDH/graphene superlattice composites as the functional layer. The electrode presented typical pseudocapacitive behaviours with excellent rate property and cycle stability.

  11. Effect of surfactant alkyl chain length on the dispersion, and thermal and dynamic mechanical properties of LDPE/organo-LDH composites

    Directory of Open Access Journals (Sweden)

    2011-05-01

    Full Text Available Low density polyethylene/layered double hydroxide (LDH composites were prepared via melt compounding using different kinds of organo-LDHs and polyethylene-grafted maleic anhydride as the compatibilizer. The organo-LDHs were successfully prepared by converting a commercial MgAl-carbonate LDH into a MgAl-nitrate LDH, which was later modified by anion exchange with linear and branched sodium alkyl sulfates having different alkyl chain lengths (nc = 6, 12 and 20. It was observed that, depending on the size of the surfactant alkyl chain, different degrees of polymer chain intercalation were achieved, which is a function of the interlayer distance of the organo-LDHs, of the packing level of the alkyl chains, and of the different interaction levels between the surfactant and the polymer chains. In particular, when the number of carbon atoms of the surfactant alkyl chain is larger than 12, the intercalation of polymer chains in the interlayer space and depression of the formation of large aggregates of organo-LDH platelets are favored. A remarkable improvement of the thermal-oxidative degradation was evidenced for all of the composites; whereas only a slight increase of the crystallization temperature and no significant changes of both melting temperature and degree of crystallinity were achieved. By thermodynamic mechanical analysis, it was evidenced that a softening of the matrix is may be due to the plasticizing effect of the surfactant.

  12. Anti‐Metastatic and Anti‐Angiogenic Activities of Core–Shell SiO2@LDH Loaded with Etoposide in Non‐Small Cell Lung Cancer

    Science.gov (United States)

    Zhu, Yanjing; Wang, Mei; Wu, Bin; He, Xiaolie

    2016-01-01

    Currently, nanoparticles have gained a great attention in the anti‐tumor research area. However, to date, studies on the anti‐metastasis action of core–shell SiO2@LDH (LDH: layered double hydroxide) nanoparticles remain untouched. Two emerging aspects considered are establishing research on the controlling delivery effect of SiO2@LDH combined with anti‐cancer medicine from a new perspective. The fine properties synthetic SiO2@LDH‐VP16 (VP16: etoposide) are practiced to exhibit the nanoparticle's suppression on migration and invasion of non‐small cell lung cancer (NSCLC). Both in vitro and in vivo inspection shows that SiO2@LDH can help VP16 better function as an anti‐metastasis agent. On the other hand, anti‐angiogenic efficiency, co‐localization, as well as western blot are investigated to explain the possible mechanism. A clear mergence of SiO2@LDH‐VP16 and cytomembrane/microtubule may be observed from co‐location images. Results offer evidence that SiO2@LDH‐VP16 plays positions on cytomembrane and microtubules. It efficiently inhibits metastasis on NSCLC by reducing vascularization, and eliciting depression of the PI3K‐AKT and FAK‐Paxillin signaling pathways. SiO2@LDH‐VP16, the overall particle morphology, and function on anti‐metastasis and anti‐angiogenic may be tuned to give new opportunities for novel strategies for cancer therapy. PMID:27980999

  13. Initial LDH level can predict the survival benefit from bevacizumab in the first-line setting in Chinese patients with metastatic colorectal cancer

    Directory of Open Access Journals (Sweden)

    Yin CX

    2014-08-01

    Full Text Available Chenxi Yin,1,2,* Chang Jiang,1,2,* Fangxin Liao,1,2 Yuming Rong,1,2 Xiuyu Cai,1,2 Guifang Guo,1,2 Huijuan Qiu,1,2 Xuxian Chen,1,2 Bei Zhang,1,2 Wenzhuo He,1,2 Liangping Xia1,2 1State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Collaborative Innovation Center for Cancer, Medicine, 2VIP Region, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, People’s Republic of China *These authors contributed equally to this paper Background: Markers to predict the efficacy of bevacizumab treatment have been not fully validated in most cancers, including metastatic colorectal cancer (mCRC. The aim of this study was to investigate the potential role of lactate dehydrogenase (LDH in predicting the survival benefit from first-line bevacizumab treatment, in Chinese patients with mCRC. Methods: All the patients were diagnosed with mCRC at the Sun Yat-sen University Cancer Center from 2003 to 2013. The study group and the control group were classified by receiving bevacizumab or not. The serum LDH value of all the patients had been detected before the first-line treatment. The primary end point was progression-free survival (PFS. Results: The median PFS of the study and the control group (patients who received bevacizumab or not was 11.3 and 9.1 months, respectively (P=0.004. In the control group, the median PFS of the high LDH level and the low LDH level groups was 6.9 and 10.2 months, respectively (P<0.001. However, in the study group, the corresponding median PFS was 9.9 and 11.9 months, respectively (P=0.145. In addition, for the low LDH level group, the median PFS was 11.9 and 10.2 months for patients who received bevacizumab or not, respectively (P=0.066; however, the median PFS of patients receiving bevacizumab or not was significantly different in the high LDH level group (9.9 and 6.9 months, respectively (P=0.012. Conclusion: The addition of bevacizumab in the first-line treatment setting could improve the

  14. [Effect of Guilingji Capsule on the fertility, liver functions, and serum LDH of male SD rats exposed by 900 mhz cell phone].

    Science.gov (United States)

    Ma, Hui-Rong; Li, Yuan-Yuan; Luo, Ya-Ping; Ma, Xue-Lian; Gong, Zhi-Qiang

    2014-04-01

    To observe the effect of Guilingji Capsule (GC) on the fertility, liver functions, and serum lactate dehydrogenase (LDH) of adult male SD rats exposed by 900 MHz cell phone. Totally 18 adult male SD rats and 36 adult female rats in child-bearing period were selected and randomly divided into three groups according to weight equilibrium principle, i.e., the normal group, the radiated group, and the GC group, 6 males and 12 females in each group. Male rats in the normal group and all female rats were not radiated. Male rats in the radiated group and the GC group received radiation for 4 h per day, lasting for 18 successive days. Rats in the GC group received GC suspension at the daily dose of 0. 15 g/kg by gastrogavage at the same time. Equal volume of normal saline was administrated to other male rats. Then male rats were mated with corresponding female rats from the 14th radiation night to the 18th radiation night in the ratio of 1:2. Male rats were killed following on the next morning of ending the radiation. Female rats were normally fed and then killed before delivery. The pregnant outcomes of female rats in responding groups (the rates of pregnancy and the number of death fetus, birth weight, body length, and tail length) were observed and compared. Serum alanine aminotransferase (ALT), aspartate transferase (AST), AST/ALT, and LDH levels of the male rats were detected by colorimetry. Histological and morphological changes of liver were observed by HE staining. Compared with the normal group, the pregnancy rates of female rats decreased and the number of death fetus increased, the serum LDH level obviously increased in the radiated group (P LDH decreased in the GC group (P LDH level increased by exposure toy GSM 900 MHz cell phone. GC could prevent and treat the aforesaid lesion. But there was no statistical difference in serum ALT or AST levels.

  15. PfHRP2 and PfLDH antigen detection for monitoring the efficacy of artemisinin-based combination therapy (ACT in the treatment of uncomplicated falciparum malaria

    Directory of Open Access Journals (Sweden)

    Deloron Philippe

    2009-09-01

    Full Text Available Abstract Background An assessment of the accuracy of two malaria rapid diagnostic tests (RDT for the detection of Plasmodium falciparum histidine-rich protein 2 (PfHRP2 or Pf lactate dehydrogenase (PfLDH was undertaken in children aged between six and 59 months included in an anti-malarial efficacy study in Benin. Methods In Allada (Benin, 205 children aged 6-59 months with falciparum malaria received either artesunate-amodiaquine (ASAQ, artemether-lumefantrine (AL, or sulphadoxine-pyrimethamine (SP. Children included in the study were simultaneously followed by both RDT and high-quality microscopy for up to 42 days. Results At the time of inclusion, PfHRP2-based tests were positive in 203 children (99% and PfLDH-based tests were positive in 204 (99.5%. During follow-up, independent of the treatment received, only 17.3% (28/162 of children effectively cured were negative with the PfHRP2 RDT at day 3, with a gradual increase in specificity until day 42. The specificity of antigen detection with the PfLDH test was 87% (141/162 on day 3, and between 92% and 100% on days 7 to 42. A statistical difference was observed between the persistence of PfHRP2 and PfLDH antigenaemia during follow-up in children treated with artemisinin-based combination therapy (ACT but not with SP. Conclusion Although both RDTs are as sensitive as microscopy in detecting true malaria cases, the PfHRP2 RDT had very low specificity during follow-up until day 28. On the other hand, the PfLDH test could be used to detect failures and, therefore, to assess anti-malarial efficacy.

  16. In situ platelets formation into aqueous polymer colloids: The topochemical transformation from single to double layered hydroxide (LSH-LDH) uncovered.

    Science.gov (United States)

    Stimpfling, Thomas; Langry, Arthur; Hintze-Bruening, Horst; Leroux, Fabrice

    2016-01-15

    Layered Single Hydroxide (LSH) of chemical composition Zn5(OH)8(acetate)2·nH2O is synthesized under in situ condition in an aqueous dispersion of an amphiphilic, carboxylate bearing polyester via a modified polyol route. The one-pot LSH generation yields agglomerates of well intercalated platelets, 9-10nm separated from each other. However the corresponding Layered Double Hydroxide (LDH) of formal composition Zn2Al(OH)6 (acetate)·nH2O is found to proceed via the formation of crystallized, similarly spaced LSH sheets in the neighborhood of amorphous Al rich domains as evidenced by X-ray diffraction and transmission electron micrographs. The initial phase segregation effaces over time while LSH platelets convert into the LDH phase. Fingerprinted by the change of in-plane cation accommodation, the associated topochemical reaction of the edge-sharing octahedral LSH platelets involves the transformation of metal lacunae, adjacently covered by one tetrahedral coordinated cation on each side to balance the negative surcharge, into fully occupied and monolayered platelets of edge-sharing octahedral LDH, the former voids being occupied by trivalent cations. This replenishing process of empty sites, coupled with the dissolution of tetrahedral sites is likely to be observed for the first time due to the presence of well separated, polymer intercalated platelets. TEM pictures vision crystal growth arising from the zone of the LSH edge-slab and by using solid state kinetics formalism the associated high activation energy of the first-order reaction agrees well with a plausible dissolution re-precipitation mechanism. The conversion of LSH into LDH platelets may be extended to others cations as Co(2+), Cu(2+), as well as the aluminum source (AlCl3) and the water-soluble polymer (NVP), thus indicating it is a new prevalent facet of LDH.

  17. Fenton-Like Catalysis and Oxidation/Adsorption Performances of Acetaminophen and Arsenic Pollutants in Water on a Multimetal Cu-Zn-Fe-LDH.

    Science.gov (United States)

    Lu, Hongtao; Zhu, Zhiliang; Zhang, Hua; Zhu, Jianyao; Qiu, Yanling; Zhu, Linyan; Küppers, Stephan

    2016-09-28

    Acetaminophen can increase the risk of arsenic-mediated hepatic oxidative damage; therefore, the decontamination of water polluted with coexisting acetaminophen and arsenic gives rise to new challenges for the purification of drinking water. In this work, a three-metal layered double hydroxide, namely, Cu-Zn-Fe-LDH, was synthesized and applied as a heterogeneous Fenton-like oxidation catalyst and adsorbent to simultaneously remove acetaminophen (Paracetamol, PR) and arsenic. The results showed that the degradation of acetaminophen was accelerated with decreasing pH or increasing H2O2 concentrations. Under the conditions of a catalyst dosage of 0.5 g·L(-1) and a H2O2 concentration of 30 mmol·L(-1), the acetaminophen in a water sample was completely degraded within 24 h by a Fenton-like reaction. The synthesized Cu-Zn-Fe-LDH also exhibited a high efficiency for arsenate removal from aqueous solutions, with a calculated maximum adsorption capacity of 126.13 mg·g(-1). In the presence of hydrogen peroxide, the more toxic arsenite can be gradually oxidized into arsenate and adsorbed at the same time by Cu-Zn-Fe-LDH. For simulated water samples with coexisting arsenic and acetaminophen pollutants, after treatment with Cu-Zn-Fe-LDH and H2O2, the residual arsenic concentration in water was less than 10 μg·L(-1), and acetaminophen was not detected in the solution. These results indicate that the obtained Cu-Zn-Fe-LDH is an efficient material for the decontamination of combined acetaminophen and arsenic pollution.

  18. Expression of Uptake Hydrogenase and Molybdenum Nitrogenase in Rhodobacter capsulatus Is Coregulated by the RegB-RegA Two-Component Regulatory System

    OpenAIRE

    2000-01-01

    Purple photosynthetic bacteria are capable of generating cellular energy from several sources, including photosynthesis, respiration, and H2 oxidation. Under nutrient-limiting conditions, cellular energy can be used to assimilate carbon and nitrogen. This study provides the first evidence of a molecular link for the coregulation of nitrogenase and hydrogenase biosynthesis in an anoxygenic photosynthetic bacterium. We demonstrated that molybdenum nitrogenase biosynthesis is under the control o...

  19. Synthesis, characterization and DFT studies of 1, 1′-Bis(diphenylphosphino)ferrocene substituted diiron complexes: Bioinspired [FeFe] hydrogenase model complexes

    Indian Academy of Sciences (India)

    Sandeep Kaur-Ghumaan; A Sreenithya; Raghavan B Sunoj

    2015-03-01

    The reaction of [Fe2(CO)6(-toluene-3, 4-benzenedithiolate)] 1 and bidentate diphosphine, 1, 1′-bis(diphenylphosphino)ferrocene (dppf) has been studied. New complexes obtained have been characterized by various spectroscopic techniques as bioinspired models of the iron hydrogenase active site. The crystal structure of [Fe2(CO)5(1-dppfO)(-toluene-3, 4-benzenedithiolate)] 4 is reported.

  20. Inactivation of uptake hydrogenase leads to enhanced and sustained hydrogen production with high nitrogenase activity under high light exposure in the cyanobacterium Anabaena siamensis TISTR 8012

    Directory of Open Access Journals (Sweden)

    Khetkorn Wanthanee

    2012-10-01

    Full Text Available Abstract Background Biohydrogen from cyanobacteria has attracted public interest due to its potential as a renewable energy carrier produced from solar energy and water. Anabaena siamensis TISTR 8012, a novel strain isolated from rice paddy field in Thailand, has been identified as a promising cyanobacterial strain for use as a high-yield hydrogen producer attributed to the activities of two enzymes, nitrogenase and bidirectional hydrogenase. One main obstacle for high hydrogen production by A. siamensis is a light-driven hydrogen consumption catalyzed by the uptake hydrogenase. To overcome this and in order to enhance the potential for nitrogenase based hydrogen production, we engineered a hydrogen uptake deficient strain by interrupting hupS encoding the small subunit of the uptake hydrogenase. Results An engineered strain lacking a functional uptake hydrogenase (∆hupS produced about 4-folds more hydrogen than the wild type strain. Moreover, the ∆hupS strain showed long term, sustained hydrogen production under light exposure with 2–3 folds higher nitrogenase activity compared to the wild type. In addition, HupS inactivation had no major effects on cell growth and heterocyst differentiation. Gene expression analysis using RT-PCR indicates that electrons and ATP molecules required for hydrogen production in the ∆hupS strain may be obtained from the electron transport chain associated with the photosynthetic oxidation of water in the vegetative cells. The ∆hupS strain was found to compete well with the wild type up to 50 h in a mixed culture, thereafter the wild type started to grow on the relative expense of the ∆hupS strain. Conclusions Inactivation of hupS is an effective strategy for improving biohydrogen production, in rates and specifically in total yield, in nitrogen-fixing cultures of the cyanobacterium Anabaena siamensis TISTR 8012.

  1. Expressão do Mg+2, CK, AST e LDH em equinos finalistas de provas de enduro

    Directory of Open Access Journals (Sweden)

    Juliana V.F. Sales

    2013-01-01

    Full Text Available Nos últimos anos, o equino atleta vem sendo cada vez mais requerido. Dessa forma, as exigências por alto desempenho têm fomentado o interesse pelo estudo das afecções relacionadas com a fisiopatologia de diversas enfermidades dos equinos. A relação entre o íon magnésio e o exercício físico tem recebido atenção significativa visto que este íon está intimamente relacionado ao tecido muscular estriado esquelético. Além disso, dentre as principais estratégias para a detecção e acompanhamento clínico de lesões musculares, destacam-se a avaliação das atividades das enzimas creatino quinase (CK, lactato desidrogenase (LDH e aspartato aminotransferase (AST. A busca pelo estabelecimento de parâmetros que se relacionam entre si é um fator determinante na compreensão de alterações fisiológicas encontradas diante do esforço em equinos atletas. Desta forma, o presente trabalho teve como objetivo determinar como as concentrações sanguíneas do íon magnésio e as atividades enzimáticas das enzimas CK, LDH e AST comportaram-se em equinos Puro Sangue Árabe finalistas de provas de enduro de 90km e relacionar as possíveis alterações com o tipo de esforço físico desempenhado pelos animais. Foram avaliadas a atividade enzimática das enzimas CK, LDH, AST e a concentração do íon magnésio no exercício em relação ao repouso de 14 equinos clinicamente hígidos da raça Puro Sangue Árabe, sendo 9 machos e 5 fêmeas, com idades variando entre 6 a 12 anos, submetidos a treinamento para enduro e participantes de provas de 90 km. Pode-se observar que as variáveis acima mencionadas sofreram aumento com diferença estatística em relação ao repouso. O exercício físico de enduro determinou a ocorrência de alterações nas atividades enzimáticas das enzimas CK (p≤0,001, LDH (p=0,0001, AST (p=0,0007 e na concentração do íon magnésio (p=0,0004, no exercício em relação ao repouso (p≤0,05. Fato que determinou altera

  2. Effects of Varying Particle Sizes and Different Types of LDH-Modified Anthracite in Simulated Test Columns for Phosphorous Removal.

    Science.gov (United States)

    Zhang, Xiangling; Chen, Qiaozhen; Guo, Lu; Huang, Hualing; Ruan, Chongying

    2015-06-16

    A comparative study was carried out for the removal of phosphorus in simulated unplanted vertical-flow constructed wetlands with different layered double hydroxide (LDHs) coated anthracite substrates. Three particle sizes of anthracites were selected and modified separately with nine kinds of LDH coating. The simulated substrates test columns loaded with the original and modified anthracites were constructed to treat the contaminated water. For the medium and large particle size modified anthracite substrates, the purification effects of total phosphorus, total dissolved phosphorus and phosphate were improved by various degrees, and the purification effect of the medium particle size anthracite is better than that of the large size one. The medium size anthracite modified by ZnCo-LDHs had optimal performance with average removal efficiencies of total phosphorus, total dissolved phosphorus and phosphate reaching 95%, 95% and 98%, respectively. The maximum adsorption capacity on ZnCo-LDHs and ZnAl-LDHs modified medium sizes anthracites were 65.79 (mg/kg) and 48.78 (mg/kg), respectively. In comparison, the small size anthracite is not suitable for LDHs modification.

  3. Effects of Varying Particle Sizes and Different Types of LDH-Modified Anthracite in Simulated Test Columns for Phosphorous Removal

    Directory of Open Access Journals (Sweden)

    Xiangling Zhang

    2015-06-01

    Full Text Available A comparative study was carried out for the removal of phosphorus in simulated unplanted vertical-flow constructed wetlands with different layered double hydroxide (LDHs coated anthracite substrates. Three particle sizes of anthracites were selected and modified separately with nine kinds of LDH coating. The simulated substrates test columns loaded with the original and modified anthracites were constructed to treat the contaminated water. For the medium and large particle size modified anthracite substrates, the purification effects of total phosphorus, total dissolved phosphorus and phosphate were improved by various degrees, and the purification effect of the medium particle size anthracite is better than that of the large size one. The medium size anthracite modified by ZnCo-LDHs had optimal performance with average removal efficiencies of total phosphorus, total dissolved phosphorus and phosphate reaching 95%, 95% and 98%, respectively. The maximum adsorption capacity on ZnCo-LDHs and ZnAl-LDHs modified medium sizes anthracites were 65.79 (mg/kg and 48.78 (mg/kg, respectively. In comparison, the small size anthracite is not suitable for LDHs modification.

  4. Cell-free H-cluster synthesis and [FeFe] hydrogenase activation: all five CO and CN⁻ ligands derive from tyrosine.

    Directory of Open Access Journals (Sweden)

    Jon M Kuchenreuther

    Full Text Available [FeFe] hydrogenases are promising catalysts for producing hydrogen as a sustainable fuel and chemical feedstock, and they also serve as paradigms for biomimetic hydrogen-evolving compounds. Hydrogen formation is catalyzed by the H-cluster, a unique iron-based cofactor requiring three carbon monoxide (CO and two cyanide (CN⁻ ligands as well as a dithiolate bridge. Three accessory proteins (HydE, HydF, and HydG are presumably responsible for assembling and installing the H-cluster, yet their precise roles and the biosynthetic pathway have yet to be fully defined. In this report, we describe effective cell-free methods for investigating H-cluster synthesis and [FeFe] hydrogenase activation. Combining isotopic labeling with FTIR spectroscopy, we conclusively show that each of the CO and CN⁻ ligands derive respectively from the carboxylate and amino substituents of tyrosine. Such in vitro systems with reconstituted pathways comprise a versatile approach for studying biosynthetic mechanisms, and this work marks a significant step towards an understanding of both the protein-protein interactions and complex reactions required for H-cluster assembly and hydrogenase maturation.

  5. Identification of an Isothiocyanate on the HypEF Complex Suggests a Route for Efficient Cyanyl-Group Channeling during [NiFe]-Hydrogenase Cofactor Generation.

    Directory of Open Access Journals (Sweden)

    Sven T Stripp

    Full Text Available [NiFe]-hydrogenases catalyze uptake and evolution of H2 in a wide range of microorganisms. The enzyme is characterized by an inorganic nickel/ iron cofactor, the latter of which carries carbon monoxide and cyanide ligands. In vivo generation of these ligands requires a number of auxiliary proteins, the so-called Hyp family. Initially, HypF binds and activates the precursor metabolite carbamoyl phosphate. HypF catalyzes removal of phosphate and transfers the carbamate group to HypE. In an ATP-dependent condensation reaction, the C-terminal cysteinyl residue of HypE is modified to what has been interpreted as thiocyanate. This group is the direct precursor of the cyanide ligands of the [NiFe]-hydrogenase active site cofactor. We present a FT-IR analysis of HypE and HypF as isolated from E. coli. We follow the HypF-catalyzed cyanation of HypE in vitro and screen for the influence of carbamoyl phosphate and ATP. To elucidate on the differences between HypE and the HypEF complex, spectro-electrochemistry was used to map the vibrational Stark effect of naturally cyanated HypE. The IR signature of HypE could ultimately be assigned to isothiocyanate (-N=C=S rather than thiocyanate (-S-C≡N. This has important implications for cyanyl-group channeling during [NiFe]-hydrogenase cofactor generation.

  6. Disclosure of key stereoelectronic factors for efficient H2 binding and cleavage in the active site of [NiFe]-hydrogenases.

    Science.gov (United States)

    Bruschi, Maurizio; Tiberti, Matteo; Guerra, Alessandro; De Gioia, Luca

    2014-02-05

    A comparative analysis of a series of DFT models of [NiFe]-hydrogenases, ranging from minimal NiFe clusters to very large systems including both the first and second coordination sphere of the bimetallic cofactor, was carried out with the aim of unraveling which stereoelectronic properties of the active site of [NiFe]-hydrogenases are crucial for efficient H2 binding and cleavage. H2 binding to the Ni-SIa redox state is energetically favored (by 4.0 kcal mol(-1)) only when H2 binds to Ni, the NiFe metal cluster is in a low spin state, and the Ni cysteine ligands have a peculiar seesaw coordination geometry, which in the enzyme is stabilized by the protein environment. The influence of the Ni coordination geometry on the H2 binding affinity was then quantitatively evaluated and rationalized analyzing frontier molecular orbitals and populations. Several plausible reaction pathways leading to H2 cleavage were also studied. It turned out that a two-step pathway, where H2 cleavage takes place on the Ni-SIa redox state of the enzyme, is characterized by very low reaction barriers and favorable reaction energies. More importantly, the seesaw coordination geometry of Ni was found to be a key feature for facile H2 cleavage. The discovery of the crucial influence of the Ni coordination geometry on H2 binding and activation in the active site of [NiFe]-hydrogenases could be exploited in the design of novel biomimetic synthetic catalysts.

  7. Effect of the culture media optimization, pH and temperature on the biohydrogen production and the hydrogenase activities by Klebsiella pneumoniae ECU-15.

    Science.gov (United States)

    Xiao, Yan; Zhang, Xu; Zhu, Minglong; Tan, Wensong

    2013-06-01

    The low yield of the biohydrogen production is the main constraint for its industrialization process. In order to improve its production, medium compositions of the hydrogen fermentation by Klebsiella pneumoniae ECU-15 were optimized through the response surface methodology (RSM). Experimental results showed that the optimum hydrogen production of 5363.8 ml/L was obtained when the concentration of glucose, the ammonium sulfate and the trace elements were 35.62 g/L, 2.78 g/L and 23.15 ml/L at temperature 37.0°C, pH 6.0. H2 evolving hydrogenase was greatly enhanced by the optimization of the medium compositions. The activity of H2 evolving hydrogenase increased with the temperature, and decreased with the pH, while the activity of the uptake hydrogenase increased with the temperature and the pH. So the biohydrogen production process of the K. pneumoniae ECU-15 was the comprehensive results of the evolution hydrogen process and the uptake hydrogen process.

  8. Anomalous self-reduction of layered double hydroxide (LDH): from α-Ni(OH)2 to hexagonal close packing (HCP) Ni/NiO by annealing without a reductant.

    Science.gov (United States)

    Ge, X; Gu, C D; Wang, X L; Tu, J P

    2015-01-21

    The traditional concept that nickel layered double hydroxide (Ni LDH, also known as α-Ni(OH)2) converts to NiO after annealing has been taken without doubt and utilized to fabricate NiO for years. This work reports that an anomalous self-reduction phenomenon can occur for Ni LDH synthesized from an ionic liquid system.

  9. Fractionation of Sulfur Isotopes by Desulfovibrio vulgaris Mutants Lacking Periplasmic Hydrogenases or the Type I Tetraheme Cytochrome c3

    Science.gov (United States)

    Sim, M.; Ono, S.; Bosak, T.

    2012-12-01

    A large fraction of anaerobic mineralization of organic compounds relies on microbial sulfate reduction. Sulfur isotope fractionation by these microbes has been widely used to trace the biogeochemical cycling of sulfur and carbon, but intracellular mechanisms behind the wide range of fractionations observed in nature and cultures are not fully understood. In this study, we investigated the influence of electron transport chain components on the fractionation of sulfur isotopes by culturing Desulfovibrio vulgaris Hildenborough mutants lacking hydrogenases or type I tetraheme cytochrome c3 (Tp1-c3). The mutants were grown both in batch and continuous cultures. All tested mutants grew on lactate or pyruvate as the sole carbon and energy sources, generating sulfide. Mutants lacking cytoplasmic and periplasmic hydrogenases exhibited similar growth physiologies and sulfur isotope fractionations to their parent strains. On the other hand, a mutant lacking Tp1-c3 (ΔcycA) fractionated the 34S/32S ratio more than the wild type, evolving H2 in the headspace and exhibiting a lower specific respiration rate. In the presence of high concentrations of pyruvate, the growth of ΔcycA relied largely on fermentation rather than sulfate reduction, even when sulfate was abundant, producing the largest sulfur isotope effect observed in this study. Differences between sulfur isotope fractionation by ΔcycA and the wild type highlight the effect of electron transfer chains on the magnitude of sulfur isotope fractionation. Because Tp1-c3 is known to exclusively shuttle electrons from periplasmic hydrogenases to transmembrane complexes, electron transfers in the absence of Tp1-c3 should bypass the periplasmic hydrogen cycling, and the loss of reducing equivalents in the form of H2 can impair the flow of electrons from organic acids to sulfur, increasing isotope fractionation. Larger fractionation by ΔcycA can inform interpretations of sulfur isotope data at an environmental scale as well

  10. High glutamate attenuates S100B and LDH outputs from rat cortical slices enhanced by either oxygen-glucose deprivation or menadione.

    Science.gov (United States)

    Demircan, Celaleddin; Gül, Zülfiye; Büyükuysal, R Levent

    2014-07-01

    One hour incubation of rat cortical slices in a medium without oxygen and glucose (oxygen-glucose deprivation, OGD) increased S100B release to 6.53 ± 0.3 ng/ml/mg protein from its control value of 3.61 ± 0.2 ng/ml/mg protein. When these slices were then transferred to a medium containing oxygen and glucose (reoxygenation, REO), S100B release rose to 344 % of its control value. REO also caused 192 % increase in lactate dehydrogenase (LDH) leakage. Glutamate added at millimolar concentration into the medium decreased OGD or REO-induced S100B release and REO-induced LDH leakage. Alpha-ketoglutarate, a metabolic product of glutamate, was found to be as effective as glutamate in decreasing the S100B and LDH outputs. Similarly lactate, 2-ketobutyrate and ethyl pyruvate, a lipophilic derivative of pyruvate, also exerted a glutamate-like effect on S100B and LDH outputs. Preincubation with menadione, which produces H2O2 intracellularly, significantly increased S100B and LDH levels in normoxic medium. All drugs tested in the present study, with the exception of pyruvate, showed a complete protection against menadione preincubation. Additionally, each OGD-REO, menadione or H2O2-induced mitochondrial energy impairments determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining and OGD-REO or menadione-induced increases in reactive oxygen substances (ROS) determined by 2,7-dichlorofluorescin diacetate (DCFH-DA) were also recovered by glutamate. Interestingly, H2O2-induced increase in fluorescence intensity derived from DCFH-DA in a slice-free physiological medium was attenuated significantly by glutamate and alpha-keto acids. All these drug actions support the conclusion that high glutamate, such as alpha-ketoglutarate and other keto acids, protects the slices against OGD- and REO-induced S100B and LDH outputs probably by scavenging ROS in addition to its energy substrate metabolite property.

  11. Fe(II) sorption on pyrophyllite: Effect of structural Fe(III) (impurity) in pyrophyllite on nature of layered double hydroxide (LDH) secondary mineral formation

    Energy Technology Data Exchange (ETDEWEB)

    Starcher, Autumn N.; Li, Wei; Kukkadapu, Ravi K.; Elzinga, Evert J.; Sparks, Donald L.

    2016-11-01

    Fe(II)-Al(III)-LDH (layered double hydroxide) phases have been shown to form from reactions of aqueous Fe(II) with Fe-free Al-bearing minerals (phyllosilicate/clays and Al-oxides). To our knowledge, the effect of small amounts of structural Fe(III) impurities in “neutral” clays on such reactions, however, were not studied. In this study to understand the role of structural Fe(III) impurity in clays, laboratory batch studies with pyrophyllite (10 g/L), an Al-bearing phyllosilicate, containing small amounts of structural Fe(III) impurities and 0.8 mM and 3 mM Fe(II) (both natural and enriched in 57Fe) were carried out at pH 7.5 under anaerobic conditions (4% H2 – 96% N2 atmosphere). Samples were taken up to 4 weeks for analysis by Fe-X-ray absorption spectroscopy and 57Fe Mössbauer spectroscopy. In addition to the precipitation of Fe(II)-Al(III)-LDH phases as observed in earlier studies with pure minerals (no Fe(III) impurities in the minerals), the analyses indicated formation of small amounts of Fe(III) containing solid(s), most probably hybrid a Fe(II)-Al(III)/Fe(III)-LDH phase. The mechanism of Fe(II) oxidation was not apparent but most likely was due to interfacial electron transfer from the sorbed Fe(II) to the structural Fe(III) and/or surface-sorption-induced electron-transfer from the sorbed Fe(II) to the clay lattice. Increase in the Fe(II)/Al ratio of the LDH with reaction time further indicated the complex nature of the samples. This research provides evidence for the formation of both Fe(II)-Al(III)-LDH and Fe(II)-Fe(III)/Al(III)-LDH-like phases during reactions of Fe(II) in systems that mimic the natural environments. Better understanding Fe phase formation in complex laboratory studies will improve models of natural redox systems.

  12. Models of the Ni-L and Ni-SIa States of the [NiFe]-Hydrogenase Active Site.

    Science.gov (United States)

    Chambers, Geoffrey M; Huynh, Mioy T; Li, Yulong; Hammes-Schiffer, Sharon; Rauchfuss, Thomas B; Reijerse, Edward; Lubitz, Wolfgang

    2016-01-19

    A new class of synthetic models for the active site of [NiFe]-hydrogenases are described. The Ni(I/II)(SCys)2 and Fe(II)(CN)2CO sites are represented with (RC5H4)Ni(I/II) and Fe(II)(diphos)(CO) modules, where diphos = 1,2-C2H4(PPh2)2(dppe) or cis-1,2-C2H2(PPh2)2(dppv). The two bridging thiolate ligands are represented by CH2(CH2S)2(2-) (pdt(2-)), Me2C(CH2S)2(2-) (Me2pdt(2-)), and (C6H5S)2(2-). The reaction of Fe(pdt)(CO)2(dppe) and [(C5H5)3Ni2]BF4 affords [(C5H5)Ni(pdt)Fe(dppe)(CO)]BF4 ([1a]BF4). Monocarbonyl [1a]BF4 features an S = 0 Ni(II)Fe(II) center with five-coordinated iron, as proposed for the Ni-SIa state of the enzyme. One-electron reduction of [1a](+) affords the S = 1/2 derivative [1a](0), which, according to density functional theory (DFT) calculations and electron paramagnetic resonance and Mössbauer spectroscopies, is best described as a Ni(I)Fe(II) compound. The Ni(I)Fe(II) assignment matches that for the Ni-L state in [NiFe]-hydrogenase, unlike recently reported Ni(II)Fe(I)-based models. Compound [1a](0) reacts with strong acids to liberate 0.5 equiv of H2 and regenerate [1a](+), indicating that H2 evolution is catalyzed by [1a](0). DFT calculations were used to investigate the pathway for H2 evolution and revealed that the mechanism can proceed through two isomers of [1a](0) that differ in the stereochemistry of the Fe(dppe)CO center. Calculations suggest that protonation of [1a](0) (both isomers) affords Ni(III)-H-Fe(II) intermediates, which represent mimics of the Ni-C state of the enzyme.

  13. Investigation of hydrogenase molecular marker to optimize hydrogen production from organic wastes and effluents of agro-food industries [abstract

    Directory of Open Access Journals (Sweden)

    Hamilton, C.

    2010-01-01

    Full Text Available In recent years policy makers have started looking for alternatives to fossil fuels, not only to counter the threat of global warming, but also to reduce the risk of overdependence on imported oil and gas supplies. By contrast with hydrocarbon fuels, hydrogen (H2, whether burned directly or used in fuel cells, is intrinsically a clean energy vector with near zero emission. However the main current method of producing hydrogen, steam reforming of methane, involves the release of large quantities of greenhouse gases. So although hydrogen already accounts for around 2% of world consumption of energy, its more widespread adoption is limited by several challenges. Therefore new processes are investigated, especially those using renewable raw material, e.g. woods and organic wastes, and/or involving microorganisms. Indeed, for some algae and bacteria, the generation of molecular hydrogen is an essential part of their energy metabolism. The approach with the greatest commercial potential is fermentative hydrogen generation (dark fermentation by bacteria from the Clostridium genus. This biological process, as a part of the methane-producing anaerobic digestion process, is very promising since it allows the production of hydrogen from a wide variety of renewable resources such as carbohydrate waste from the agricultural and agro-food industries or processed urban waste and sewage. To date most publications on hydrogen production by Clostridium strains have focused on the effects of operating parameters (such as temperature, pH, dilution rate, etc.. We now need to extend this knowledge by identifying and monitoring the various different metabolic agents involved in high H2 activity. Consequently the aim of this research at the CWBI in the University of Liege is to investigate the role of [Fe] hydrogenases, the key enzymes that remove excess electrons accumulating during fermentation. Clostridium butyricum CWBI1009, the strain used for these investigations

  14. EFFECT OF TRIGONELLA FOENUM GRAECUM ON LACTATE DEHYDROGENASE (LDH ACTIVITY OF BLOOD, LIVER AND PANCREAS IN NORMAL AND ALLOXAN- INDUCED DIABETIC MICE

    Directory of Open Access Journals (Sweden)

    Sekaran Sridhar et al.

    2012-02-01

    Full Text Available The effect of aqueous seeds extract of Trigonella foenum graecum Linn was studied on Lactate dehydrogenase (LDH activity of blood, liver and pancreas in normal and alloxan- induced diabetic mice. Our study showed that aqueous seeds extract, Oral administration of 50 mg/animal (0.5 ml of extract in alternative days up to 7 days (1st, 3rd, 5th & 7th day. In alloxan induced diabetic mice, there was a significant increase in LDH activity of all the three tissues. The enzyme Lactate dehydrogenase showed significant decrease in the diabetic group treated with aqueous extract of tested plant when compared with the diabetic group. It is clear from the current data in this study that ginseng aqueous extract was the most efficient of the tested plant.

  15. An alpha-proteobacterial type malate dehydrogenase may complement LDH function in Plasmodium falciparum. Cloning and biochemical characterization of the enzyme.

    Science.gov (United States)

    Tripathi, Abhai K; Desai, Prashant V; Pradhan, Anupam; Khan, Shabana I; Avery, Mitchell A; Walker, Larry A; Tekwani, Babu L

    2004-09-01

    Malate dehydrogenase (MDH) may be important in carbohydrate and energy metabolism in malarial parasites. The cDNA corresponding to the MDH gene, identified on chromosome 6 of the Plasmodium falciparum genome, was amplified by RT-PCR, cloned and overexpressed in Escherichia coli. The recombinant Pf MDH was purified to homogeneity and biochemically characterized as an NAD(+)(H)-specific MDH, which catalysed reversible interconversion of malate to oxaloacetate. Pf MDH could not use NADP/NADPH as a cofactor, but used acetylpyridine adenine dinucleoide, an analogue of NAD. The enzyme exhibited strict substrate and cofactor specificity. The highest levels of Pf MDH transcripts were detected in trophozoites while the Pf MDH protein level remained high in trophozoites as well as schizonts. A highly refined model of Pf MDH revealed distinct structural characteristics of substrate and cofactor binding sites and important amino acid residues lining these pockets. The active site amino acid residues involved in substrate binding were conserved in Pf MDH but the N-terminal glycine motif, which is involved in nucleotide binding, was similar to the GXGXXG signature sequence found in Pf LDH and also in alpha-proteobacterial MDHs. Oxamic acid did not inhibit Pf MDH, while gossypol, which interacts at the nucleotide binding site of oxidoreductases and shows antimalarial activity, inhibited Pf MDH also. Treatment of a synchronized culture of P. falciparum trophozoites with gossypol caused induction in expression of Pf MDH, while expression of Pf LDH was reduced and expression of malate:quinone oxidoreductase remained unchanged. Pf MDH may complement Pf LDH function of NAD/NADH coupling in malaria parasites. Thus, dual inhibitors of Pf MDH and Pf LDH may be required to target this pathway and to develop potential new antimalarial drugs.

  16. Aerobic Oxidation of Alcohols to Carbonyl Compounds Catalyzed by N-Hydroxyphthalimide (NHPI) Combined with CoTPP-Zn2sub>Al-LDH

    Indian Academy of Sciences (India)

    WEIYOU ZHOU; DONGWEI CHEN; AIJUN CUI; JUNFENG QIAN; MINGYANG HE; QUN CHEN

    2017-03-01

    A catalytic system for the aerobic oxidation of alcohols by N-hydroxyphthalimide (NHPI) combined with cobalt porphyrin intercalated heterogeneous hybrid catalyst (CoTPP-Zn2Al-LDH) has been developed. The results showed that this catalytic system can effectively catalyze the oxidation of alcohols to thecorresponding carbonyl compounds. And the hybrid catalyst can be reused for five times with no appreciable reduction of activity.

  17. Effect of HEMADO on Level of CK-MB and LDH Enzymes after Ischemia/Reperfusion Injury in Isolated Rat Heart

    Directory of Open Access Journals (Sweden)

    Mohammad Amani

    2013-01-01

    Full Text Available Introduction: Ischemia/Reperfusion (IR injury mainly causes the increase of enzymes involved in myocytes injury including CK-MB (creatine kinase-MB isoenzyme and LDH (lactate dehydrogenase. Leakage of CK-MB isoenzyme and LDH from myocardial tissues to blood is indicator of acute myocardial infarction. The aim of this study was to assess the effect of HEMADO on IR injury and its relationship with mitochondrial ATP-sensitive K+ channels (mitoKATP in rat heart. Methods: Twenty eight male Wistar rats (250-300g were divided into four groups (seven members in each group: control (without ischemia, I/R (with ischemia+without HEMADO, ischemia received HEMADO (HEMADO, ischemia received HEMADO and 5-HD (5-hydroxydecanoate, specific mitoKATP channel blocker (HEMADO+5-HD. The animals were anesthetized and the hearts were quickly removed and mounted on Langendorff apparatus and perfused by Krebs-Henseleit solution under constant pressure and temperature of 37ºC. After 20 minutes of stabilization, ischemic groups were exposed to 40 minutes of global ischemia and consecutive 90 minutes of reperfusion. Results: IR injury increased the level of LDH and CK-MB in the collected coronary flow during 5 minutes since start of reperfusion. HEMADO reduced the enzymes’ levels and using 5-HD abolished the effect of HEMADO. Conclusion: Our findings indicated that HEMADO could protect the heart against ischemia-reperfusion injury by decreasing the CK-MB and LDH levels. The cardioprotective effect of HEMADO may be mediated in part by mitoKATP.

  18. Comparative characterization of a temperature responsive gene (lactate dehydrogenase-B, ldh-b in two congeneric tropical fish, Lates calcarifer and Lates niloticus

    Directory of Open Access Journals (Sweden)

    Richard C. Edmunds, Lynne van Herwerden, Carolyn Smith-Keune, Dean R. Jerry

    2009-01-01

    Full Text Available The characterization of candidate loci is a critical step in obtaining insight into adaptation and acclimation of organisms. In this study of two non-model tropical (to sub-tropical congeneric perciformes (Lates calcarifer and Lates niloticus we characterized both coding and non-coding regions of lactate dehydrogenase-B (ldh-b, a locus which exhibits temperature-adaptive differences among temperate and sub-tropical populations of the North American killifish Fundulus heteroclitus. Ldh-b was 5,004 and 3,527 bp in length in L. calcarifer and L. niloticus, respectively, with coding regions comprising 1,005 bp in both species. A high level of sequence homology existed between species for both coding and non-coding regions of ldh-b (> 97% homology, corresponding to a 98.5% amino acid sequence homology. All six known functional sites within the encoded protein sequence (LDH-B were conserved between the two Lates species. Ten simple sequence repeat (SSR motifs (mono-, di-, tri- and tetranucleotide and thirty putative microRNA elements (miRNAs were identified within introns 1, 2, 5 and 6 of both Lates species. Five single nucleotide polymorphisms (SNPs were also identified within miRNA containing intron regions. Such SNPs are implicated in several complex human conditions and/or diseases (as demonstrated by extensive genome-wide association studies. This novel characterization serves as a platform to further examine how non-model species may respond to changes in their native temperatures, which are expected to increase by up to 6°C over the next century.

  19. From nicotinate-containing layered double hydroxides (LDHs) to NAD coenzyme-LDH nanocomposites - Syntheses and structural characterization by various spectroscopic methods

    Science.gov (United States)

    Muráth, Szabolcs; Dudás, Csilla; Kukovecz, Ákos; Kónya, Zoltán; Sipos, Pál; Pálinkó, István

    2017-07-01

    The syntheses of nicotinate anion- and NAD coenzyme-layered double hydroxide (LDH) composites were performed with the aim of having the organic component among the layers. In-house prepared CaAl-LDHs were the host materials. Intercalation was attempted by direct ion exchange or by the dehydration-rehydration method applying aqueous solvent mixtures (containing ethanol, propanol, acetone, N,N-dimethylformamide). For structural characterization, beside X-ray diffractometry, X-ray photoelectron and IR spectroscopies, transmission and scanning electron microscopies as well as energy-dispersive X-ray analysis were used. Molecular modelling served for the visualization of the arrangements of the intercalated ions among the layers of the LDH samples. Although not all the intercalation methods and solvent mixtures led to intercalated composite materials, successful ones could be identified. The combination of spectroscopic methods helped in proposing sensible spatial arrangements for the intercalated anions. The NAD-CaAl-LDH composite proved to be an active catalyst in the oxidation of hydroquinone to 1,4-bezoquinoe in the presence of H2O2.

  20. A simple protocol for using a LDH-based cytotoxicity assay to assess the effects of death and growth inhibition at the same time.

    Directory of Open Access Journals (Sweden)

    Shilo M Smith

    Full Text Available Analyzing the effects on cell growth inhibition and/or cell death has been an important component of biological research. The MTS assay and LDH-based cytotoxicity assays are two of the most commonly used methods for this purpose. However, data here showed that MTS cell proliferation assay could not distinguish the effects of cell death or cell growth inhibition. In addition, the original LDH-based cytotoxicity protocol grossly underestimated the proportion of dead cells in conditions with growth inhibition. To overcome the limitation, we present here a simple modified LDH-based cytotoxicity protocol by adding additional condition-specific controls. This modified protocol thus can provide more accurate measurement of killing effects in addition to the measurement of overall effects, especially in conditions with growth inhibition. In summary, we present here a simple, modified cytotoxicity assay, which can determine the overall effects, percentage of cell killing and growth inhibition in one 96-well based assay. This is a viable option for primary screening for many laboratories, and could be adapted for high throughput screening.

  1. Highly biocompatible behaviour and slow degradation of a LDH (layered double hydroxide)-coating on implants in the middle ear of rabbits.

    Science.gov (United States)

    Duda, Franziska; Kieke, Marc; Waltz, Florian; Schweinefuß, Maria E; Badar, Muhammad; Müller, Peter Paul; Esser, Karl-Heinz; Lenarz, Thomas; Behrens, Peter; Prenzler, Nils Kristian

    2015-01-01

    Chronic inflammation can irreversibly damage components of the ossicular chain which may lead to sound conduction deafness. The replacement of impaired ossicles with prostheses does not reduce the risk of bacterial infections which may lead to loss of function of the implant and consequently to additional damage of the connected structures such as inner ear, meninges and brain. Therefore, implants that could do both, reconstruct the sound conduction and in addition provide antibacterial protection are of high interest for ear surgery. Layered double hydroxides (LDHs) are promising novel biomaterials that have previously been used as an antibiotic-releasing implant coating to curb bacterial infections in the middle ear. However, animal studies of LDHs are scarce and there exist only few additional data on the biocompatibility and hardly any on the biodegradation of these compounds. In this study, middle ear prostheses were coated with an LDH compound, using suspensions of nanoparticles of an LDH containing Mg and Al as well as carbonate ions. These coatings were characterized and implanted into the middle ear of healthy rabbits for 10 days. Analysis of the explanted prostheses showed only little signs of degradation. A stable health constitution was observed throughout the whole experiment in every animal. The results show that LDH-based implant coatings are biocompatible and dissolve only slowly in the middle ear. They, therefore, appear as promising materials for the construction of controlled drug delivery vehicles.

  2. Elevation of serum GGT and LDH levels, together with higher BCLC staging are associated with poor overall survival from hepatocellular carcinoma: a retrospective analysis.

    Science.gov (United States)

    Yang, Zongguo; Ye, Peiyan; Xu, Qingnian; Lu, Yunfei; Tang, Bozong; Wang, Qiang; Chen, Shishi; Chen, Xiaorong

    2015-06-01

    Serum biomarkers predicting prognosis have not been adequately explored in HCC patients. The aim of this study was to investigate prognostic significance of parameters of liver function, tumor markers, and other clinicopathological features in HCC patients. Medical records of HCC patients were retrospectively extracted and overall survival was evaluated with the Kaplan-Meier method. Significant difference was estimated with the Log rank method. Univariate and multivariate analyses were used for the study of significance of prognostic factor. A total of 273 HCC patients were included in this analysis. According to the Cox regression analysis and Kaplan-Meier event analysis, GGT and LDH levels of liver function tests were significantly associated with HCC overall survival. Elevated serum CEA level was a risk factor related to poor HCC overall survival. And advanced BCLC staging contributed to a lower overall survival in HCC patients. HCC could benefit from surgical resection, TACE, and radiotherapy. ROC curves demonstrated that different from CEA, elevated GGT and LDH could accurately predict HCC overall survival. In conclusion, serum GGT and LDH together with higher BCLC staging should be potential predictive factors for HCC overall survival.

  3. Immobilization of hydrogenase on carbon nanotube polyelectrolytes as heterogeneous catalysts for electrocatalytic interconversion of protons and hydrogen

    Science.gov (United States)

    Liu, Jiang; Wu, Wen-Jie; Fang, Fang; Zorin, Nikolay A.; Chen, Meng; Qian, Dong-Jin

    2016-08-01

    Immobilization of active enzymes on the surfaces of electrodes and nanomaterials is important in the fields of bioscience, and biotechnology. In this study, we investigated electrocatalytic properties of the interconversion of protons and hydrogen by means of hydrogenase (H2ase)-functionalized carbon nanotube polyelectrolyte composites. Multiwalled carbon nanotube polyelectrolytes (MWNT-PEs) were synthesized through a diazonium and an addition reaction with poly(4-vinylpyridine) (P4VP), followed by another addition reaction with either methyl iodide (CH3I) or N-methyl- N'-benzyl bromide bipyridinium (VBenBr) to produce MWNT-P4VPMe or MWNT-P4VPBenV polyelectrolytes, respectively. The MWNT-PE@H2ase bio-nanocomposites were then prepared by means of MWNT-PEs as substrates to bind with H2ase. The redox current density of the MWNT-PE@H2ase-modified electrodes increased with a decrease in pH values of the Ar-saturated electrolyte solution owing to the catalytic reduction of protons (H2 production); further, it increased with the increasing pH values of the H2-saturated solution owing to the catalytic oxidation of hydrogen. The reversible color change between blue-colored and colorless viologen (catalyzed by the MWNT-PE@H2ase bio-nanocomposites) suggested that they may be developed as nano-biosensors for molecular H2. The as-synthesized bio-nanocomposites showed strong long-term stability and high bioactivity.

  4. Iron Hydride Detection and Intramolecular Hydride Transfer in a Synthetic Model of Mono-Iron Hydrogenase with a CNS Chelate.

    Science.gov (United States)

    Durgaprasad, Gummadi; Xie, Zhu-Lin; Rose, Michael J

    2016-01-19

    We report the identification and reactivity of an iron hydride species in a synthetic model complex of monoiron hydrogenase. The hydride complex is derived from a phosphine-free CNS chelate that includes a Fe-C(NH)(═O) bond (carbamoyl) as a mimic of the active site iron acyl. The reaction of [((O═)C(HN)N(py)S(Me))Fe(CO)2(Br)] (1) with NaHBEt3 generates the iron hydride intermediate [((O═)C(HN)N(py)S(Me))Fe(H)(CO)2] (2; δFe-H = -5.08 ppm). Above -40 °C, the hydride species extrudes CH3S(-) via intramolecular hydride transfer, which is stoichiometrically trapped in the structurally characterized dimer μ2-(CH3S)2-[((O═)C(HN)N(Ph))Fe(CO)2]2 (3). Alternately, when activated by base ((t)BuOK), 1 undergoes desulfurization to form a cyclometalated species, [((O═)C(NH)NC(Ph))Fe(CO)2] (5); derivatization of 5 with PPh3 affords the structurally characterized species [((O═)C(NH)NC)Fe(CO)(PPh3)2] (6), indicating complex 6 as the common intermediate along each pathway of desulfurization.

  5. Electrochemistry of metalloproteins: protein film electrochemistry for the study of E. coli [NiFe]-hydrogenase-1.

    Science.gov (United States)

    Evans, Rhiannon M; Armstrong, Fraser A

    2014-01-01

    Protein film electrochemistry is a technique which allows the direct control of redox-active enzymes, providing particularly detailed information on their catalytic properties. The enzyme is deposited onto a working electrode tip, and through control of the applied potential the enzyme activity is monitored as electrical current, allowing for direct study of inherent activity as electrons are transferred to and from the enzyme redox center(s). No mediators are used. Because the only enzyme present in the experiment is bound at the electrode surface, gaseous and liquid phase inhibitors can be introduced and removed whilst the enzyme remains in situ. Potential control means that kinetics and thermodynamics are explored simultaneously; the kinetics of a reaction can be studied as a function of potential. Steady-state catalytic rates are observed directly as current (for a given potential) and non-steady-state rates (such as interconversions between different forms of the enzyme) are observed from the change in current with time. The more active the enzyme, the higher the current and the better the signal-to-noise. In this chapter we outline the practical aspects of PFE for studying electroactive enzymes, using the Escherichia coli [NiFe]-hydrogenase 1 (Hyd-1) as an example.

  6. A threonine stabilizes the NiC and NiR catalytic intermediates of [NiFe]-hydrogenase.

    Science.gov (United States)

    Abou-Hamdan, Abbas; Ceccaldi, Pierre; Lebrette, Hugo; Gutiérrez-Sanz, Oscar; Richaud, Pierre; Cournac, Laurent; Guigliarelli, Bruno; De Lacey, Antonio L; Léger, Christophe; Volbeda, Anne; Burlat, Bénédicte; Dementin, Sébastien

    2015-03-27

    The heterodimeric [NiFe] hydrogenase from Desulfovibrio fructosovorans catalyzes the reversible oxidation of H2 into protons and electrons. The catalytic intermediates have been attributed to forms of the active site (NiSI, NiR, and NiC) detected using spectroscopic methods under potentiometric but non-catalytic conditions. Here, we produced variants by replacing the conserved Thr-18 residue in the small subunit with Ser, Val, Gln, Gly, or Asp, and we analyzed the effects of these mutations on the kinetic (H2 oxidation, H2 production, and H/D exchange), spectroscopic (IR, EPR), and structural properties of the enzyme. The mutations disrupt the H-bond network in the crystals and have a strong effect on H2 oxidation and H2 production turnover rates. However, the absence of correlation between activity and rate of H/D exchange in the series of variants suggests that the alcoholic group of Thr-18 is not necessarily a proton relay. Instead, the correlation between H2 oxidation and production activity and the detection of the NiC species in reduced samples confirms that NiC is a catalytic intermediate and suggests that Thr-18 is important to stabilize the local protein structure of the active site ensuring fast NiSI-NiC-NiR interconversions during H2 oxidation/production.

  7. 大豆和花生根瘤菌氢酶的研究%Studies on Hydrogenase from Rhizobium japonicum and Rhizobium arachis

    Institute of Scientific and Technical Information of China (English)

    许良树; 张凤章; 龙敏南; 曾定; 黄河清; 刘月英; 刘广发

    2001-01-01

    根瘤菌在共生固氮过程中因放H2所消耗的能量约占固氮总能量的40%~60%.吸氢酶则能回收和利用固氮过程所放的H2,减少能量损失,从而提高共生固氮效率.在厌氧条件下,加入防止酶蛋白聚合的试剂,利用DEAE-纤维素和SephacrylS-200柱层析,从自养性大豆根瘤菌和花生根瘤菌类菌体中分离并提纯膜结合态氢酶.纯化的两种氢酶表现相近的分子特征:均含有大(60 kD,65kD)、小(30 kD,35 kD)两个亚基;均为NiFe-氢酶,并具有较高的吸H2活性.大豆根瘤菌氢酶的纯酶组分不含Cyt b559.花生根瘤菌L8-3菌株能进行化能自养生长,诱导出高吸H2活性.根瘤菌的吸H2能明显提高固氮活性.从具有高吸H2活性的花生根瘤菌中分离并克隆吸氢基因,采用PCR和探针杂交技术,获得含有吸氢基因的质粒pZ-55.利用多种限制性内切酶构建了质粒pZ-55的物理图谱.通过三亲本杂交,将含吸氢基因的重组质粒转移到不吸H2的花生和毛豆根瘤菌中,所获得的结合株在自生和共生条件下均表达吸H2活性.以结合株接种大田花生,获得的共生根瘤的吸H2活性比接种受体株提高4倍,花生叶片和种子的含N量、产量分别提高1.7%、8.9%和9.6%.%Hydrogen produced by nitrogenase consumed 40~60%0 of energy of symbiotic nitro gen-fixation. Hydrogenase can uptake and reuse the H2 produced by nitrogenase, which results in decreasing the loss of energy and increasing the efficiency of nitrogen fixation. The membranebound hydrogenase from autotrophical cultured Rhizobium japanicum and from the bacteroids of peanut nodule have been purified and characterized. The hydrogenase from R. japonicum consists of two subunits (60 kD, 30 kD). The molecular weight of large and small subunits of hydrogenase from R. arachis is about 65 kD and 35 kD. Both hydrogenases are NiFe-hydrogenase. No cytochrome b(559) could

  8. 肺腺癌中LDH-V、AKT1及Glut1的表达及其与18氟-2-脱氧葡萄糖摄取的相关性研究%Expression of LDH-V, AKT1, Glut1 and Their Relationships with the Uptake of 18F-FDG in Lung Adenocarcinoma

    Institute of Scientific and Technical Information of China (English)

    李倩; 黄钢; 刘建军; 孙晓光

    2013-01-01

    目的:探讨肺腺癌中乳酸脱氢酶-V(lactate dehydrogenase V,LDH-V)、丝氨酸/苏氨酸蛋白激酶1(serine-threonine kinase 1,AKT1)及葡萄糖转运蛋白1(glucose transporter 1,Glut1)的表达及它们与18氟-2-脱氧葡萄糖(18F-FDG)摄取的关系.方法:54名肺腺癌患者,术前1周进行PET-CT显像.采用免疫组织化学SP法对LDH-V、AKT1、Glut1的表达进行半定量分析,并与术前PET-CT所得的18 F-FDG最大标准化摄取值(maximal standardized uptake value,SUVmax)进行相关性分析.结果:在肺腺癌中,LDH-V、AKT1及Glut1表达的阳性率为88.89%、88.89%、68.52%,LDH-V表达与AKT1及Glut1表达呈正相关(r分别为0.381和0.270,P<0.01和0.05);SUVmax与肿瘤最大直径(maximal diameter,Dmax)呈正相关(r=0.524,P<0.01),Dmax>2 cm患者(n=30)的SUVmax与Glut1表达呈正相关(r=0.407,P<0.05);SUVmax与病理分级、LDH-V及AKT1表达无关.结论:LDH-V、AKT1及Glut1在肺腺癌中广泛表达且LDH-V表达与AKT1及Glut1表达密切相关,Glut1在肺腺癌18F-FDG摄取中发挥重要作用.

  9. Determination of lactate dehydrogenase (LDH and Bcr-Abl transcript in the follow-up of patients with chronic myeloid leukemia = Determinação da lactate desidrogenase (LDH e do transcrito Bcr-Abl em pacientes com leucemia mielóide crônica

    Directory of Open Access Journals (Sweden)

    Roberto Iemitsu Tatakihara

    2010-07-01

    Full Text Available Chronic myeloid leukemia (CML is a malignant myeloproliferative disorder that originates from a pluripotent stem cell characterized by abnormal release of the expanded, malignant stem cell clone from the bone marrow into the bloodstream. The vast majority of patients with CML present Bcr-Abl transcripts. Lactate dehydrogenase (LDH is considered a biochemical marker common for tumor growth, anaerobic glycolysis and has been considered a poor prognostic factor for acute myeloid leukemia. Therefore, this study aimed to evaluate the concentration of LDH in plasma and the detection of the Bcr-Abl transcripts in patients with CML and healthy donors. We analyzed 22 patients demonstrably diagnosed with CML and 56 healthy donors. LDH concentration in plasma was higher in patients with CML. All patients with CML in this study were under treatment, but even so four patients had the Bcr-Abl (b3a2 transcript in peripheral blood. Two out of the four patients with b3a2 showed higher LDH (486 U L-1 and 589 U L-1. Thus, although the study was conducted with small numbers of samples, it is possible to suggest therapy alteration for two patients who presented transcript b3a2 in the peripheral blood samples and whose LDH concentration was high, in order to improve the disease. Leucemia mieloide crônica (LMC é uma desordem mieloproliferativa maligna que é originada de célula-tronco pluripotente caracterizada por expansão anormal, maligna de clones de células tronco da medula óssea na circulação. A grande maioria dos pacientes com LMC apresentam transcritos Bcr-Abl. Lactato desidrogenase (LDH,considerado um marcador bioquímico para crescimento tumoral, glicólise anaeróbica, e tem sido considerado um fator de pior prognóstico da LMC. Portanto, este estudo visa avaliar a concentraçãode LDH no plasma e a detecção do transcrito Bcr-Abl em 22 pacientes com LMC e 56 indivíduos saudáveis. Foram avaliados 22 pacientes com LMC e 56 doadores saudáveis. A

  10. [NiFe] hydrogenase structural and functional models: new bio-inspired catalysts for hydrogen evolution; Modeles structuraux et fonctionnels du site actif des hydrogenases [NiFe]: de nouveaux catalyseurs bio-inspires pour la production d'hydrogene

    Energy Technology Data Exchange (ETDEWEB)

    Oudart, Y

    2006-09-15

    Hydrogenase enzymes reversibly catalyze the oxidation and production of hydrogen in a range close to the thermodynamic potential. The [NiFe] hydrogenase active site contains an iron-cyano-carbonyl moiety linked to a nickel atom which is in an all sulphur environment. Both the active site originality and the potential development of an hydrogen economy make the synthesis of functional and structural models worthy. To take up this challenge, we have synthesised mononuclear ruthenium models and more importantly, nickel-ruthenium complexes, mimicking some structural features of the [NiFe] hydrogenase active site. Ruthenium is indeed isoelectronic to iron and some of its complexes are well-known to bear hydrides. The compounds described in this study have been well characterised and their activity in proton reduction has been successfully tested. Most of them are able to catalyze this reaction though their electrocatalytic potentials remain much more negative compared to which of platinum. The studied parameters point out the importance of the complexes electron richness, especially of the nickel environment. Furthermore, the proton reduction activity is stable for several hours at good rates. The ruthenium environment seems important for this stability. Altogether, these compounds represent the very first catalytically active [NiFe] hydrogenase models. Important additional results of this study are the synergetic behaviour of the two metals in protons reduction and the evidence of a protonation step as the limiting step of the catalytic cycle. We have also shown that a basic site close to ruthenium improves the electrocatalytic potential of the complexes. (author)

  11. Reduction of unusual iron-sulfur clusters in the H2-sensing regulatory Ni-Fe hydrogenase from Ralstonia eutropha H16.

    Science.gov (United States)

    Buhrke, Thorsten; Löscher, Simone; Lenz, Oliver; Schlodder, Eberhard; Zebger, Ingo; Andersen, Lars K; Hildebrandt, Peter; Meyer-Klaucke, Wolfram; Dau, Holger; Friedrich, Bärbel; Haumann, Michael

    2005-05-20

    The regulatory Ni-Fe hydrogenase (RH) from Ralstonia eutropha functions as a hydrogen sensor. The RH consists of the large subunit HoxC housing the Ni-Fe active site and the small subunit HoxB containing Fe-S clusters. The heterolytic cleavage of H(2) at the Ni-Fe active site leads to the EPR-detectable Ni-C state of the protein. For the first time, the simultaneous but EPR-invisible reduction of Fe-S clusters during Ni-C state formation was demonstrated by changes in the UV-visible absorption spectrum as well as by shifts of the iron K-edge from x-ray absorption spectroscopy in the wild-type double dimeric RH(WT) [HoxBC](2) and in a monodimeric derivative designated RH(stop) lacking the C-terminal 55 amino acids of HoxB. According to the analysis of iron EXAFS spectra, the Fe-S clusters of HoxB pronouncedly differ from the three Fe-S clusters in the small subunits of crystallized standard Ni-Fe hydrogenases. Each HoxBC unit of RH(WT) seems to harbor two [2Fe-2S] clusters in addition to a 4Fe species, which may be a [4Fe-3S-3O] cluster. The additional 4Fe-cluster was absent in RH(stop). Reduction of Fe-S clusters in the hydrogen sensor RH may be a first step in the signal transduction chain, which involves complex formation between [HoxBC](2) and tetrameric HoxJ protein, leading to the expression of the energy converting Ni-Fe hydrogenases in R. eutropha.

  12. A hybrid photocatalytic system comprising ZnS as light harvester and an [Fe(2)S(2)] hydrogenase mimic as hydrogen evolution catalyst.

    Science.gov (United States)

    Wen, Fuyu; Wang, Xiuli; Huang, Lei; Ma, Guijun; Yang, Jinhui; Li, Can

    2012-05-01

    Photo opportunity: A highly efficient and stable hybrid artificial photosynthetic H(2) evolution system is assembled by using a semiconductor (ZnS) as light-harvester and an [Fe(2)S(2)] hydrogenase mimic ([(μ-SPh-4-NH(2) )(2) Fe(2) (CO)(6)]) as catalyst for H(2) evolution. Photocatalytic H(2) production is achieved with more than 2607 turnovers (based on [Fe(2)S(2)]) and an initial turnover frequency of 100 h(-1) through the efficient transfer of photogenerated electrons from ZnS to the [Fe(2)S(2)] complex.

  13. Immobilization of FeFe hydrogenase mimics onto carbon and gold electrodes by controlled aryldiazonium salt reduction: an electrochemical, XPS and ATR-IR study

    Energy Technology Data Exchange (ETDEWEB)

    Le Goff, Alan; Metaye, Romain; Moggia, Fabrice; Jousselme, Bruno; Palacin, Serge [CEA, IRAMIS, SPCSI, Chemistry of Surfaces and Interfaces group, F-91191 Gif sur Yvette Cedex (France); Artero, Vincent; Razavet, Mathieu; Tran, Phong D.; Fontecave, Marc [Laboratoire de Chimie et Biologie des Metaux, Universite Joseph Fourier, CNRS UMR 5249, CEA DSV/iRTSV, 17 rue des Martyrs, F-38054 Grenoble cedex 9 (France)

    2010-10-15

    A dithiolate-bridged hexacarbonyldiiron complex was synthesized from the reaction of the N-hydroxysuccinimide (NHS) ester of lipoic acid with Fe{sub 3}(CO){sub 12} in toluene. This mimic of the active site of FeFe hydrogenases could be covalently attached, using an NHS ester route, to carbon or gold electrode first decorated with amino functions. Once grafted this complex catalyzes hydrogen electro-evolution under strongly acidic conditions but is rapidly inactivated. We could evidence that the activity loss was due to the elimination of the carbonyl ligands rather than a leaching of the catalyst as a consequence of hydrolysis of the amide linkages. (author)

  14. New redox states observed in [FeFe] hydrogenases reveal redox coupling within the H-cluster.

    Science.gov (United States)

    Adamska-Venkatesh, Agnieszka; Krawietz, Danuta; Siebel, Judith; Weber, Katharina; Happe, Thomas; Reijerse, Edward; Lubitz, Wolfgang

    2014-08-13

    Active [FeFe] hydrogenases can be obtained by expressing the unmaturated enzyme in Escherichia coli followed by incubation with a synthetic precursor of the binuclear [2Fe] subcluster, namely: [NEt4]2[Fe2(adt)(CO)4(CN)2] (adt = [S-CH2-NH-CH2-S](2-)). The binuclear subsite Fe2(adt)(CO)3(CN)2 is attached through a bridging cysteine side chain to a [4Fe-4S] subcluster already present in the unmaturated enzyme thus yielding the intact native "H-cluster". We present FTIR electrochemical studies of the [FeFe] hydrogenase from Chlamydomonas reinhardtii, CrHydA1, maturated with the precursor of the native cofactor [Fe2(adt)(CO)4(CN)2](2-) as well as a non-natural variant [Fe2(pdt)(CO)4(CN)2](2-) in which the bridging amine functionality is replaced by CH2. The obtained active enzyme CrHydA1(adt) shows the same redox states in the respective potential range as observed for the native system (E(ox/red) = -400 mV, E(red/sred) = -470 mV). For the Hox → Hred transition the reducing equivalent is stored on the binuclear part, ([4Fe-4S](2+)Fe(II)Fe(I) → [4Fe-4S](2+)Fe(I)Fe(I)), while the Hred → Hsred transition is characterized by a reduction of the [4Fe-4S] part of the H-cluster ([4Fe-4S](2+)Fe(I)Fe(I) → [4Fe-4S](+)Fe(I)Fe(I)). A similar transition is reported here for the CO inhibited state of the H-cluster: ([4Fe-4S](2+)Fe(I)Fe(II)CO → [4Fe-4S](+)Fe(I)Fe(II)CO). An FTIR electrochemical study of the inactive variant with the pdt ligand, CrHydA1(pdt), identified two redox states H(pdt)-ox and H(pdt)-"red". Both EPR and FTIR spectra of H(pdt)-ox are virtually identical to those of the H(adt)-ox and the native Hox state. The H(pdt)-"red" state is also characterized by a reduced [4Fe-4S] subcluster. In contrast to CrHydA1(adt), the H(pdt)-ox state of CrHydA1(pdt) is stable up to rather high potentials (+200 mV). This study demonstrates the distinct redox coupling between the two parts of the H-cluster and confirms that the [4Fe-4S]H subsite is also redox active and as

  15. Characterization of Photochemical Processes for H2 Production by CdS Nanorod-[FeFe] Hydrogenase Complexes

    Energy Technology Data Exchange (ETDEWEB)

    Brown, K. A.; Wilker, M. B.; Boehm, M.; Dukovic, G.; King, P. W.

    2012-03-28

    We have developed complexes of CdS nanorods capped with 3-mercaptopropionic acid (MPA) and Clostridium acetobutylicum [FeFe]-hydrogenase I (CaI) that photocatalyze reduction of H{sup +} to H{sub 2} at a CaI turnover frequency of 380-900 s{sup -1} and photon conversion efficiencies of up to 20% under illumination at 405 nm. In this paper, we focus on the compositional and mechanistic aspects of CdS:CaI complexes that control the photochemical conversion of solar energy into H{sub 2}. Self-assembly of CdS with CaI was driven by electrostatics, demonstrated as the inhibition of ferredoxin-mediated H{sub 2} evolution by CaI. Production of H{sub 2} by CdS:CaI was observed only under illumination and only in the presence of a sacrificial donor. We explored the effects of the CdS:CaI molar ratio, sacrificial donor concentration, and light intensity on photocatalytic H{sub 2} production, which were interpreted on the basis of contributions to electron transfer, hole transfer, or rate of photon absorption, respectively. Each parameter was found to have pronounced effects on the CdS:CaI photocatalytic activity. Specifically, we found that under 405 nm light at an intensity equivalent to total AM 1.5 solar flux, H{sub 2} production was limited by the rate of photon absorption ({approx}1 ms{sup -1}) and not by the turnover of CaI. Complexes were capable of H{sub 2} production for up to 4 h with a total turnover number of 106 before photocatalytic activity was lost. This loss correlated with inactivation of CaI, resulting from the photo-oxidation of the CdS capping ligand MPA.

  16. Model of the iron hydrogenase active site covalently linked to a ruthenium photosensitizer: synthesis and photophysical properties.

    Science.gov (United States)

    Ott, Sascha; Borgström, Magnus; Kritikos, Mikael; Lomoth, Reiner; Bergquist, Jonas; Akermark, Björn; Hammarström, Leif; Sun, Licheng

    2004-07-26

    A model of the iron hydrogenase active site with the structure [(mu-ADT)Fe2(CO)6] (ADT = azadithiolate (S-CH2-NR-CH2-S), (2: R = 4-bromophenyl, 3: R = 4-iodophenyl)) has been assembled and covalently linked to a [Ru(terpy)2]2+ photosensitizer. This trinuclear complex 1 represents one synthetic step toward the realization of our concept of light-driven proton reduction. A rigid phenylacetylene tether has been incorporated as the linking unit in 1 in order to prolong the lifetime of the otherwise short-lived [Ru(terpy)2]2+ excited state. The success of this strategy is demonstrated by comparison of the photophysical properties of 1 and of two related ruthenium complexes bearing acetylenic terpyridine ligands, with those of [Ru(terpy)2]2+. IR and electrochemical studies reveal that the nitrogen heteroatom of the ADT bridge has a marked influence on the electronic properties of the [Fe2(CO)6] core. Using the Rehm-Weller equation, the driving force for an electron transfer from the photoexcited *[Ru(terpy)2]2+ to the diiron site in 1 was calculated to be uphill by 0.59 eV. During the construction of the trinuclear complex 1, n-propylamine has been identified as a decarbonylation agent on the [(mu-ADT)Fe2(CO)6] portion of the supermolecule. Following this procedure, the first azadithiolate-bridged dinuclear iron complex coordinated by a phosphine ligand [(mu-ADT)Fe2(CO)5PPh3] (4, R = 4-bromophenyl) was synthesized. Copyright 2004 American Chemical Society

  17. Analysis of hydrogenase 1 levels reveals an intimate link between carbon and hydrogen metabolism in Escherichia coli K-12.

    Science.gov (United States)

    Pinske, Constanze; McDowall, Jennifer S; Sargent, Frank; Sawers, R Gary

    2012-03-01

    Two of the three [NiFe]-hydrogenases (Hyd) of Escherichia coli have a hydrogen-uptake function in anaerobic metabolism. While Hyd-2 is maximally synthesized when the bacterium grows by fumarate respiration, Hyd-1 synthesis shows a correlation with fermentation of sugar substrates. In an attempt to advance our knowledge on the physiological function of Hyd-1 during fermentative growth, we examined Hyd-1 activity and levels in various derivatives of E. coli K-12 MC4100 with specific defects in sugar utilization. MC4100 lacks a functional fructose phosphotransferase system (PTS) and therefore grows more slowly under anaerobic conditions in rich medium in the presence of d-fructose compared with d-glucose. Growth in the presence of fructose resulted in an approximately 10-fold increase in Hyd-1 levels in comparison with growth under the same conditions with glucose. This increase in the amount of Hyd-1 was not due to regulation at the transcriptional level. Reintroduction of a functional fruBKA-encoded fructose PTS into MC4100 restored growth on d-fructose and reduced Hyd-1 levels to those observed after growth on d-glucose. Reducing the rate of glucose uptake by introducing a mutation in the gene encoding the cAMP receptor protein, or consumption through glycolysis, by introducing a mutation in phosphoglucose isomerase, increased Hyd-1 levels during growth on glucose. These results suggest that the ability to oxidize hydrogen by Hyd-1 shows a strong correlation with the rate of carbon flow through glycolysis and provides a direct link between hydrogen, carbon and energy metabolism.

  18. Coordination and conformational isomers in mononuclear iron complexes with pertinence to the [FeFe] hydrogenase active site.

    Science.gov (United States)

    Orthaber, Andreas; Karnahl, Michael; Tschierlei, Stefanie; Streich, Daniel; Stein, Matthias; Ott, Sascha

    2014-03-21

    A series of six mononuclear iron complexes of the type [Fe(X-bdt)(P(R)2N(Ph)2)(CO)] (P(R)2N(Ph)2 = 1,5-diaza-3,7-diphosphaoctane, bdt = benzenedithiolate with X = H, Cl2 or Me and R = Ph, Bn, Cyc or tert-Bu) was prepared. This new class of penta-coordinate iron complexes contains a free coordination site and a pendant base as essential structural features of the [FeFe]-hydrogenase active site. The bidentate nature of the P(R)2N(Ph)2 ligands was found to be crucial for the preferential formation of coordinatively unsaturated penta-coordinate complexes, which is supported by first principle calculations. IR-spectroscopic data suggest the presence of coordination isomers around the metal center, as well as multiple possible conformers of the P(R)2N(Ph)2 ligand. This finding is further corroborated by X-ray crystallographic and computational studies. (31)P{(1)H}-NMR- and IR-spectroscopic as well as electrochemical measurements show that the electronic properties of the complexes are strongly, and independently, influenced by the P-substituents at the P(R)2N(Ph)2 ligand as well as by modifications of the bdt bridge. These results illustrate the advantages of this modular platform, which allows independent and selective tuning through site specific modifications. Potential catalytic intermediates, namely singly reduced and protonated complexes, have been further investigated by spectroscopic methods and exhibit remarkable stability. Finally, their general capacity for electro-catalytic reduction of protons to molecular hydrogen was verified.

  19. Catalytic properties of the isolated diaphorase fragment of the NAD-reducing [NiFe]-hydrogenase from Ralstonia eutropha.

    Directory of Open Access Journals (Sweden)

    Lars Lauterbach

    Full Text Available The NAD+-reducing soluble hydrogenase (SH from Ralstonia eutropha H16 catalyzes the H₂-driven reduction of NAD+, as well as reverse electron transfer from NADH to H+, in the presence of O₂. It comprises six subunits, HoxHYFUI₂, and incorporates a [NiFe] H+/H₂ cycling catalytic centre, two non-covalently bound flavin mononucleotide (FMN groups and an iron-sulfur cluster relay for electron transfer. This study provides the first characterization of the diaphorase sub-complex made up of HoxF and HoxU. Sequence comparisons with the closely related peripheral subunits of Complex I in combination with UV/Vis spectroscopy and the quantification of the metal and FMN content revealed that HoxFU accommodates a [2Fe2S] cluster, FMN and a series of [4Fe4S] clusters. Protein film electrochemistry (PFE experiments show clear electrocatalytic activity for both NAD+ reduction and NADH oxidation with minimal overpotential relative to the potential of the NAD+/NADH couple. Michaelis-Menten constants of 56 µM and 197 µM were determined for NADH and NAD+, respectively. Catalysis in both directions is product inhibited with K(I values of around 0.2 mM. In PFE experiments, the electrocatalytic current was unaffected by O₂, however in aerobic solution assays, a moderate superoxide production rate of 54 nmol per mg of protein was observed, meaning that the formation of reactive oxygen species (ROS observed for the native SH can be attributed mainly to HoxFU. The results are discussed in terms of their implications for aerobic functioning of the SH and possible control mechanism for the direction of catalysis.

  20. 白念珠菌与LDH抗体的交叉免疫研究%Cross-reactive immune response of Candida albicans with anti-LDH antibody

    Institute of Scientific and Technical Information of China (English)

    钟毅; 黄怀球; 赵静; 袁立燕; 张静; 张晓辉; 李美荣

    2012-01-01

    To identify the conserved epitopes of Candida albicans malate dehydrogenase (CaMDH), the structure, function and phylogenetic analysis of the CaMDH sequence were predicted by using tools of bioinformatics. The phylogenetic a-nalysis showed the amino acid sequence of Schistosoma japonic um lactic dehydrogenase (SjLDH) had lowest identity (18. 2%) with CaMDH than that of other species (43%). The structure prediction result showed that CaMDH contained LDH conserved domain and 7 main B cell epitopes, in which 3 epitopes displayed high levels of similarity with that of SjI.DH. The MDH active site located at the NAD (P) binding domain. Furthermore, the Sj'LDH recombinant protein immune serum which had lowest i-dentity with CaMDH in study was chosen to make cross-reactive immune response with Candida albicans protein. The positive result with the anti-LDH body identify that the interaction point was highly conserved epitopes of CaMDH homologous sequence and supply new methods for the vaccine, diagnosis and drug target study of the CaMDH.%目的 识别白念珠菌苹果酸脱氢酶(Candida albicans malate dehydrogenase,CaMDH)保守表位结构.方法 本研究利用生物信息学方法,预测CaMDH的结构、功能和进化关系,并选取同源序列中与CaMDH序列一致性较低的日本血吸虫乳酸脱氢酶(Schistosoma japonicum lactate dehydrogenase,SjLDH)免疫血清与白念珠菌总蛋白进行交叉免疫反应.结果 发现CaMDH与常见病原生物的同源序列一致性可达43%,其中与SjLDH一致性最低(18.2%).CaMDH具有乳酸脱氢酶保守区域,7个主要的B细胞表位,其中3处与SjLDH表位相似.苹果酸脱氢酶活化点包含于NAD(P)结合部分中.白念珠菌总蛋白与SjLDH免疫血清交叉免疫反应阳性.结论 交叉免疫反应阳性提示识别结合位点为LDH/MDH同源序列高度保守的表位结构,为进一步从高度保守的抗原决定簇中筛选真菌疫苗表位、诊断特异抗体、抗真菌药物治疗靶点提供了实验基础.

  1. Androgens enhance the glycolytic metabolism and lactate export in prostate cancer cells by modulating the expression of GLUT1, GLUT3, PFK, LDH and MCT4 genes.

    Science.gov (United States)

    Vaz, Cátia V; Marques, Ricardo; Alves, Marco G; Oliveira, Pedro F; Cavaco, José E; Maia, Cláudio J; Socorro, Sílvia

    2016-01-01

    The present study aims to investigate the role of androgens in controlling the glycolytic metabolism and lactate efflux in prostate cancer (PCa) cells. Androgen-responsive LNCaP cells were treated with 5α-dihydrotestosterone (DHT, 10 nM) for 12-48 h, and their glycolytic metabolism, lactate production and viability were analyzed. Intracellular and extracellular levels of glucose and lactate were determined spectrophotometrically, and the expression of glucose transporters (GLUT1/GLUT3), phosphofructokinase 1, lactate dehydrogenase (LDH) and monocarboxylate transporter (MCT4) was analyzed by real-time PCR and Western blot. The enzymatic activity of LDH was determined by means of a colorimetric assay. Experiments were reproduced in androgen-non-responsive DU145 and PC3 cells. Androgens stimulated glucose consumption in LNCaP cells by increasing the expression of GLUT3, GLUT1 and PFK, which was underpinned by increased cell viability. Accordingly, lactate production by LNCaP cells was enhanced upon DHT stimulation as evidenced by the increased levels of lactate found in cell culture medium. Although LDH enzymatic activity decreased in LNCaP cells treated with DHT, the expression of MCT4 was significantly increased with androgenic treatment, which sustains the increase on lactate export. Glucose consumption and the expression of GLUTs and PFK remained unchanged in DHT-treated DU145 and PC3 cells. The results obtained establish androgens as modulators of glycolytic metabolism in PCa cells by stimulating glucose consumption, as well as the production and export of lactate, which may represent a crucial issue-driven prostate tumor development. These findings also highlight the importance of PCa therapies targeting AR and metabolism-related proteins.

  2. Plasma IL-6/IL-10 Ratio and IL-8, LDH, and HBDH Level Predict the Severity and the Risk of Death in AIDS Patients with Pneumocystis Pneumonia.

    Science.gov (United States)

    Sun, Jia; Su, Junwei; Xie, Yirui; Yin, Michael T; Huang, Ying; Xu, Lijun; Zhou, Qihui; Zhu, Biao

    2016-01-01

    Objective. To identify blood biomarkers to predict severity and mortality in AIDS PCP patients. Methods. Biomarkers including clinical parameters and plasma inflammatory cytokines were assessed in 32 HIV-infected patients with Pneumocystis pneumonia (PCP) at time of admission. Predictive value of the biomarkers for clinical severity and in-hospital mortality was evaluated by corresponding ROC curve. Results. Levels of CRP, WBC, LDH, HBDH, and Ferritin were significantly higher in the severe and nonsurvivor AIDS PCP patients. These important biochemical indicators have inverse correlation with oxygenation index, especially levels of LDH (P = 0.008, R (2) = 0.258), HBDH (P = 0.001, R (2) = 0.335), and Ferritin (P = 0.005, R (2) = 0.237). Plasma IL-8 and IL-6 levels were significantly higher in patients with PaO2/FiO2 ≤ 200 mmHg and nonsurvivors than in those with PaO2/FiO2 > 200 mmHg and survivors. Severe and nonsurvival groups showed higher ratio of mean IL-6/IL-10 level (1.78 ± 1.56, P < 0.001; 1.11 ± 0.72, P = 0.043), larger AUC (95% CI 0.781-1.000, P < 0.001; 95% CI 0.592-0.917, P = 0.043), and more significantly inverse correlation with the oxygenation index. Conclusion. Plasma IL-8, LDH, and HBDH levels and IL-6/IL-10 ratio could be helpful for early evaluation of the severity and predicting fatal outcomes in AIDS PCP patients.

  3. Plasma IL-6/IL-10 Ratio and IL-8, LDH, and HBDH Level Predict the Severity and the Risk of Death in AIDS Patients with Pneumocystis Pneumonia

    Directory of Open Access Journals (Sweden)

    Jia Sun

    2016-01-01

    Full Text Available Objective. To identify blood biomarkers to predict severity and mortality in AIDS PCP patients. Methods. Biomarkers including clinical parameters and plasma inflammatory cytokines were assessed in 32 HIV-infected patients with Pneumocystis pneumonia (PCP at time of admission. Predictive value of the biomarkers for clinical severity and in-hospital mortality was evaluated by corresponding ROC curve. Results. Levels of CRP, WBC, LDH, HBDH, and Ferritin were significantly higher in the severe and nonsurvivor AIDS PCP patients. These important biochemical indicators have inverse correlation with oxygenation index, especially levels of LDH (P=0.008, R2=0.258, HBDH (P=0.001, R2=0.335, and Ferritin (P=0.005, R2=0.237. Plasma IL-8 and IL-6 levels were significantly higher in patients with PaO2/FiO2 ≤ 200 mmHg and nonsurvivors than in those with PaO2/FiO2 > 200 mmHg and survivors. Severe and nonsurvival groups showed higher ratio of mean IL-6/IL-10 level (1.78 ± 1.56, P<0.001; 1.11 ± 0.72, P=0.043, larger AUC (95% CI 0.781–1.000, P<0.001; 95% CI 0.592–0.917, P=0.043, and more significantly inverse correlation with the oxygenation index. Conclusion. Plasma IL-8, LDH, and HBDH levels and IL-6/IL-10 ratio could be helpful for early evaluation of the severity and predicting fatal outcomes in AIDS PCP patients.

  4. Breathing air to save energy--new insights into the ecophysiological role of high-affinity [NiFe]-hydrogenase in Streptomyces avermitilis.

    Science.gov (United States)

    Liot, Quentin; Constant, Philippe

    2016-02-01

    The Streptomyces avermitilis genome encodes a putative high-affinity [NiFe]-hydrogenase conferring the ability to oxidize tropospheric H2 in mature spores. Here, we used a combination of transcriptomic and mutagenesis approaches to shed light on the potential ecophysiological role of the enzyme. First, S. avermitilis was either exposed to low or hydrogenase-saturating levels of H2 to investigate the impact of H2 on spore transcriptome. In total, 1293 genes were differentially expressed, with 1127 and 166 showing lower and higher expression under elevated H2 concentration, respectively. High H2 exposure lowered the expression of the Sec protein secretion pathway and ATP-binding cassette-transporters, with increased expression of genes encoding proteins directing carbon metabolism toward sugar anabolism and lower expression of NADH dehydrogenase in the respiratory chain. Overall, the expression of relA responsible for the synthesis of the pleiotropic alarmone ppGpp decreased upon elevated H2 exposure, which likely explained the reduced expression of antibiotic synthesis and stress response genes. Finally, deletion of hhySL genes resulted in a loss of H2 uptake activity and a dramatic loss of viability in spores. We propose that H2 is restricted to support the seed bank of Streptomyces under a unique survival-mixotrophic energy mode and discuss important ecological implications of this finding.

  5. Direct Comparison of the Performance of a Bio-inspired Synthetic Nickel Catalyst and a [NiFe]-Hydrogenase, Both Covalently Attached to Electrodes

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez-Macia, Patricia; Dutta, Arnab; Lubitz, Wolfgang; Shaw, Wendy J.; Rudiger, Olaf

    2015-10-12

    The active site of hydrogenases has been a source of inspiration for the development of molecular catalysts. However, direct comparisons between molecular catalysts and enzymes have not been possible because different techniques are used to evaluate both types of catalysts, minimizing our ability to determine how far we’ve come in mimicking the impressive enzymatic performance. Here we directly compare the catalytic properties of the [Ni(PCy2NGly2)2]2+ complex with the [NiFe]-hydrogenase from Desulfobivrio vulgaris Miyazaki F (DvMF) immobilized to a functionalized electrode under identical conditions. At pH=7, the enzyme has higher performance in both activity and overpotential, and is more stable, while at low pH, the molecular catalyst outperforms the enzyme in all respects. The Ni complex also has increased tolerance to CO. This is the first direct comparison of enzymes and molecular complexes, enabling a unique understanding of the benefits and detriments of both systems, and advancing our understanding of the utilization of these bioinspired complexes in fuel cells. AD and WJS acknowledge the Office of Science Early Career Research Program through the US Department of Energy (US DOE), Office of Science, Office of Basic Energy Sciences (BES), and Pacific Northwest National Laboratory (PNNL). PNNL is operated by Battelle for the US DOE.

  6. Hydrogen bioelectrooxidation on gold nanoparticle-based electrodes modified by Aquifex aeolicus hydrogenase: Application to hydrogen/oxygen enzymatic biofuel cells.

    Science.gov (United States)

    Monsalve, Karen; Roger, Magali; Gutierrez-Sanchez, Cristina; Ilbert, Marianne; Nitsche, Serge; Byrne-Kodjabachian, Deborah; Marchi, Valérie; Lojou, Elisabeth

    2015-12-01

    For the first time, gold nanoparticle-based electrodes have been used as platforms for efficient immobilization of the [NiFe] hydrogenase from the hyperthermophilic bacterium Aquifex aeolicus. AuNPs were characterized by electronic microscopy, dynamic light scattering and UV-Vis spectroscopy. Two sizes around 20.0±5.3 nm and 37.2±4.3 nm nm were synthesized. After thiol-based functionalization, the AuNPs were proved to allow direct H2 oxidation over a large range of temperatures. A high current density up to 1.85±0.15 mA·cm(-2) was reached at the smallest AuNPs, which is 170 times higher than the one recorded at the bare gold electrode. The catalytic current was especially studied as a function of the AuNP size and amount, and procedure for deposition. A synergetic effect between the AuNP porous deposit and the increase surface area was shown. Compared to previously used nanomaterials such as carbon nanofibers, the covalent grafting of the enzyme on the thiol-modified gold nanoparticles was shown to enhance the stability of the hydrogenase. This bioanode was finally coupled to a biocathode where BOD from Myrothecium verrucaria was immobilized on AuNP-based film. The performance of the so-mounted H2/O2 biofuel cell was evaluated, and a power density of 0.25 mW·cm(-2) was recorded.

  7. Effects of Iron on Hydrogen-producing Capacity,Hydrogenase and NADH-fd Reductase Activities of a Fermentative Hydrogen-producing Bacterial Strain B49

    Institute of Scientific and Technical Information of China (English)

    Wang Xiangjing(王相晶); Ren Nanqi; Xiang Wensheng

    2004-01-01

    Iron plays an important role in hydrogen production, cell growth, hydrogenase and NADH-fd reductase activities of hydrogen-producing bacterial strain B49 (AF481148 in EMBL). At the end of fermentation from 10 g/L glucose, for the culture containing 10 mg/L FeSO4*7H2O the cell growth in terms of optical density (OD) at 600nm was 1.13, the ratio of ethanol amount (mg/L) to acetate amount (mg/L) was 1.55, and the accumulated hydrogen volume was 1816.3 ml H2/L culture; whereas for the culture of 80 mg/L FeSO4*7H2O OD600nm was increased to 1.34, the accumulated hydrogen volume was increased to 2360.5 ml H2/L culture, and the ratio of ethanol amount (mg/L) to acetate amount (mg/L) decreased to 1.31. Moreover, the iron addition to the medium at different fermentation time could affect hydrogen-producing ability. However, the later the addition time of FeSO4*7H2O was postponed, the less the effect on hydrogen evolution was. In the course of fermentation, the specific activities of hydrogenase and NADH-fd reductase of hydrogen-producing bacterial strain B49 decreased with the consumption of iron.

  8. Biochemical Characterization of HydF, a Scaffolding Enzyme, in the Synthesis of the Hydrogenase Active Site Metal Center: Implications Towards the Evolution of Biocatalysts from Mineral-based Components on Early Earth

    Science.gov (United States)

    Duffus, B. R.; Shepard, E. M.; McGlynn, S. E.; Bueling, A. L.; Winslow, M. A.; Peters, J. W.; Broderick, J. B.

    2010-04-01

    [FeFe]-hydrogenase active site biosynthesis utilizes radical chemistry on a scaffold protein whose ancestor may have been one of the earliest examples of a protein that couples the chemistry of an Fe-S peptide nest with a nucleotide binding nest.

  9. Contributions of the [NiFe]- and [FeFe]-hydrogenase to H2 production in Shewanella oneidensis MR-1 as revealed by isotope ratio analysis of evolved H2

    Energy Technology Data Exchange (ETDEWEB)

    Kreuzer, Helen W.; Hill, Eric A.; Moran, James J.; Bartholomew, Rachel A.; Hui, Yang; Hegg, Eric L.

    2014-03-01

    Shewanella oneidensis MR-1 encodes both a [NiFe]- and an [FeFe]-hydrogenase. While the output of these proteins has been characterized in mutant strains expressing only one of the enzymes, the contribution of each to H2 synthesis in the wild-type organism is not clear. Here we use stable isotope analysis of H2 in the culture headspace, along with transcription data and measurements of the concentrations of gases in the headspace, to characterize H2 production in the wild-type strain. After most of the O2 in the headspace had been consumed, H2 was produced and then consumed by the bidirectional [NiFe]-hydrogenase. Once the cultures were completely anaerobic, a new burst of H2 synthesis catalyzed by both enzymes took place. Our data is consistent with the hypothesis that at this point in the culture cycle, a pool of electrons is shunted toward both hydrogenases in the wild-type organism, but that in the absence of one of the hydrogenases, the flux is redirected to the available enzyme. To our knowledge, this is the first use of stable isotope analysis of a metabolic product to elucidate substrate flux through two alternative enzymes in the same cellular system.

  10. [Nitrogenase, hydrogenase and nitrate reductase activities, oxygen consumption, and ATP content in nodules formed by strains of Rhizobium leguminosarum 128C53 and 300 in symbiosis with pea plants].

    Science.gov (United States)

    Bedmar, E J; Olivares, J

    1986-10-01

    The nitrogenase activity, nitrate reductase activity and oxygen uptake as well as the hydrogen incorporation and ATP content were examined in the root nodules and bacteroids, respectively, formed by Rhizobium leguminosarum strains 128C53 (hydrogenase positive) and 300 (hydrogenase negative) in symbiosis with Pisum sativum plants grown in the presence of 2 mM KNO3. The strain 128C53 showed the greatest values for all parameters analyzed, except for the nitrate reductase activity, which was higher for the strain 300. Similarly, nodule nitrate reductase activity in strain 300 was greater than that in strain 128C53 when plants grew in the absence of combined nitrogen. In general, the highest values were obtained when determinations were made after 7 hours of plant illumination. However, the hydrogenase activity of strain 128C53 and the nitrate reductase activities of both strains increased with the light period, reaching a maximum after 14 hours of illumination. These results suggest that the benefits derived from the superior symbiotic properties and from the presence of hydrogenase activity in strain 128C53 could be counteracted by the higher rates of the nodule nitrate reductase activity in strain 300.

  11. Photoactivation of the Ni-SIr state to the Ni-SIa state in [NiFe] hydrogenase: FT-IR study on the light reactivity of the ready Ni-SIr state and as-isolated enzyme revisited.

    Science.gov (United States)

    Tai, Hulin; Xu, Liyang; Inoue, Seiya; Nishikawa, Koji; Higuchi, Yoshiki; Hirota, Shun

    2016-08-10

    The Ni-SIr state of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F was photoactivated to its Ni-SIa state by Ar(+) laser irradiation at 514.5 nm, whereas the Ni-SL state was light induced from a newly identified state, which was less active than any other identified state and existed in the "as-isolated" enzyme.

  12. Controlled leaching with prolonged activity for Co-LDH supported catalyst during treatment of organic dyes using bicarbonate activation of hydrogen peroxide.

    Science.gov (United States)

    Jawad, Ali; Li, Yibing; Lu, Xiaoyan; Chen, Zhuqi; Liu, Weidong; Yin, Guochuan

    2015-05-30

    The effluents from industries are commonly non-biodegradable and produce various hazardous intermediate products by chemical reactions that have direct impact on environment. In the present investigation, a series of Co-Mg/AL ternary LDH catalysts with fixed Mg/Al ratio were prepared by co-precipitation method. The effect of Co on the activity of the catalyst was monitored on the degradation of methylene blue (MB) as model compound at batch level using bicarbonate activation of H2O2 (BAP) system. On bench level, the best CoMgAl-4 catalyst can completely decolorize both methylene blue (MB) and methylene orange (MO) in short time, while in fixed bed, the catalyst was found stable for over 300 h with nearly 100% decolorization and excellent chemical oxygen demand (COD) removal. No leaching of Co was detected for the entire fixed experiment which may be accounted for long life stability and good activity of the catalyst. The ternary LDH catalysts were characterized by AES, XRD, FTIR, BET, and SEM for its compositional, phase structure, optical properties, textural, and surface morphology respectively. The XRD analysis confirmed characteristic pattern of hydrotalcite like structures without impurity phases. The formation of superoxide and hydroxyl radical as ROS was proposed with CoMgAl-4 by radical's scavengers.

  13. 110 Cases of LDH Treated with Round Sharp Needling%员利针治疗腰椎间盘突出症110例

    Institute of Scientific and Technical Information of China (English)

    孙海花; 何良志; 董延芬; 庄平; 于娟

    2012-01-01

    目的:观察员利针疗法治疗腰椎间盘突出症的疗效.方法:治疗组110例腰椎间盘突出症患者采用员利针疗法治疗,常规针灸组(以夹脊穴为主)作为对照组.结果:以员利针为主的治疗组即时疗效及远期综合疗效优于常规针刺组.结论:员利针疗法治疗腰椎间盘突症出具有见效快、疗程短、无创伤、痛苦小等优点,适宜在临床推广应用.%Objective;To observe the treatment efficacy with the round sharp needling on lumbar disc hernia-tion(LDH). Methods; 110 LDH patients were treated with the round sharp needling as the treatment group. And others were treated with the traditional acupuncture as the control group. Results;The treatment group was better than the control group in the immediate effect and the comprehensive long - term efficacy. Conclusion: The round sharp needling in the treatment of lumbar disc herniation has many benefits such as rapid effects, short courses, non - invasion, little pain, etc. It is suitable for clinical application.

  14. Reactive oxygen species (ROS) production triggered by prostaglandin D2 (PGD2) regulates lactate dehydrogenase (LDH) expression/activity in TM4 Sertoli cells.

    Science.gov (United States)

    Rossi, Soledad P; Windschüttl, Stefanie; Matzkin, María E; Rey-Ares, Verónica; Terradas, Claudio; Ponzio, Roberto; Puigdomenech, Elisa; Levalle, Oscar; Calandra, Ricardo S; Mayerhofer, Artur; Frungieri, Mónica B

    2016-10-15

    Reactive oxygen species (ROS) regulate testicular function in health and disease. We previously described a prostaglandin D2 (PGD2) system in Sertoli cells. Now, we found that PGD2 increases ROS and hydrogen peroxide (H2O2) generation in murine TM4 Sertoli cells, and also induces antioxidant enzymes expression suggesting that defense systems are triggered as an adaptive stress mechanism that guarantees cell survival. ROS and specially H2O2 may act as second messengers regulating signal transduction pathways and gene expression. We describe a stimulatory effect of PGD2 on lactate dehydrogenase (LDH) expression via DP1/DP2 receptors, which is prevented by the antioxidant N-acetyl-L-cysteine and the PI3K/Akt pathway inhibitor LY 294002. PGD2 also enhances Akt and CREB/ATF-1 phosphorylation. Our results provide evidence for a role of PGD2 in the regulation of the oxidant/antioxidant status in Sertoli cells and, more importantly, in the modulation of LDH expression which takes place through ROS generation and the Akt-CREB/ATF-1 pathway.

  15. Determination of lactate dehydrogenase (LDH and Bcr-Abl transcript in the follow-up of patients with chronic myeloid leukemia - doi: 10.4025/actascihealthsci.v32i2.6408 Determination of lactate dehydrogenase (LDH and Bcr-Abl transcript in the follow-up of patients with chronic myeloid leukemia - doi: 10.4025/actascihealthsci.v32i2.6408

    Directory of Open Access Journals (Sweden)

    Thiago Cezar Fujita

    2010-09-01

    Full Text Available Chronic myeloid leukemia (CML is a malignant myeloproliferative disorder that originates from a pluripotent stem cell characterized by abnormal release of the expanded, malignant stem cell clone from the bone marrow into the bloodstream. The vast majority of patients with CML present Bcr-Abl transcripts. Lactate dehydrogenase (LDH is considered a biochemical marker common for tumor growth, anaerobic glycolysis and has been considered a poor prognostic factor for acute myeloid leukemia. Therefore, this study aimed to evaluate the concentration of LDH in plasma and the detection of the Bcr-Abl transcripts in patients with CML and healthy donors. We analyzed 22 patients demonstrably diagnosed with CML and 56 healthy donors. LDH concentration in plasma was higher in patients with CML. All patients with CML in this study were under treatment, but even so four patients had the Bcr-Abl (b3a2 transcript in peripheral blood. Two out of the four patients with b3a2 showed higher LDH (486 U L-1 and 589 U L-1. Thus, although the study was conducted with small numbers of samples, it is possible to suggest therapy alteration for two patients who presented transcript b3a2 in the peripheral blood samples and whose LDH concentration was high, in order to improve the disease.Chronic myeloid leukemia (CML is a malignant myeloproliferative disorder that originates from a pluripotent stem cell characterized by abnormal release of the expanded, malignant stem cell clone from the bone marrow into the bloodstream. The vast majority of patients with CML present Bcr-Abl transcripts. Lactate dehydrogenase (LDH is considered a biochemical marker common for tumor growth, anaerobic glycolysis and has been considered a poor prognostic factor for acute myeloid leukemia. Therefore, this study aimed to evaluate the concentration of LDH in plasma and the detection of the Bcr-Abl transcripts in patients with CML and healthy donors. We analyzed 22 patients demonstrably diagnosed

  16. 1,2,3,4,6-Penta-O-galloylglucose within Galla Chinensis Inhibits Human LDH-A and Attenuates Cell Proliferation in MDA-MB-231 Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Shihab Deiab

    2015-01-01

    Full Text Available A characteristic feature of aggressive malignancy is the overexpression of lactic acid dehydrogenase- (LDH- A, concomitant to pericellular accumulation of lactate. In a recent high-throughput screening, we identified Rhus chinensis (Mill. gallnut (RCG (also known as Galla Chinensis extract as a potent (IC50 < 1 µg/mL inhibitor of human LDH-A (hLDH-A. In this study, through bioactivity guided fractionation of the crude extract, the data demonstrate that penta-1,2,3,4,6-O-galloyl-β-D-glucose (PGG was a primary constituent responsible for hLDH-A inhibition, present at ~9.95 ± 0.34% dry weight. Theoretical molecular docking studies of hLDH-A indicate that PGG acts through competitive binding at the NADH cofactor site, effects confirmed by functional enzyme studies where the IC50 = 27.32 nM was reversed with increasing concentration of NADH. Moreover, we confirm protein expression of hLDH-A in MDA-231 human breast carcinoma cells and show that PGG was toxic (LC50 = 94.18 µM, parallel to attenuated lactic acid production (IC50 = 97.81 µM. In a 72-hour cell proliferation assay, PGG was found to be a potent cytostatic agent with ability to halt cell division (IC50 = 1.2 µM relative to paclitaxel (IC50 < 100 nM. In summary, these findings demonstrate that PGG is a potent hLDH-A inhibitor with significant capacity to halt proliferation of human breast cancer cells.

  17. 1,2,3,4,6-Penta-O-galloylglucose within Galla Chinensis Inhibits Human LDH-A and Attenuates Cell Proliferation in MDA-MB-231 Breast Cancer Cells.

    Science.gov (United States)

    Deiab, Shihab; Mazzio, Elizabeth; Eyunni, Suresh; McTier, Oshlii; Mateeva, Nelly; Elshami, Faisel; Soliman, Karam F A

    2015-01-01

    A characteristic feature of aggressive malignancy is the overexpression of lactic acid dehydrogenase- (LDH-) A, concomitant to pericellular accumulation of lactate. In a recent high-throughput screening, we identified Rhus chinensis (Mill.) gallnut (RCG) (also known as Galla Chinensis) extract as a potent (IC50 < 1 µg/mL) inhibitor of human LDH-A (hLDH-A). In this study, through bioactivity guided fractionation of the crude extract, the data demonstrate that penta-1,2,3,4,6-O-galloyl-β-D-glucose (PGG) was a primary constituent responsible for hLDH-A inhibition, present at ~9.95 ± 0.34% dry weight. Theoretical molecular docking studies of hLDH-A indicate that PGG acts through competitive binding at the NADH cofactor site, effects confirmed by functional enzyme studies where the IC50 = 27.32 nM was reversed with increasing concentration of NADH. Moreover, we confirm protein expression of hLDH-A in MDA-231 human breast carcinoma cells and show that PGG was toxic (LC50 = 94.18 µM), parallel to attenuated lactic acid production (IC50 = 97.81 µM). In a 72-hour cell proliferation assay, PGG was found to be a potent cytostatic agent with ability to halt cell division (IC50 = 1.2 µM) relative to paclitaxel (IC50 < 100 nM). In summary, these findings demonstrate that PGG is a potent hLDH-A inhibitor with significant capacity to halt proliferation of human breast cancer cells.

  18. Single-Amino Acid Modifications Reveal Additional Controls on the Proton Pathway of [FeFe]-Hydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Cornish, Adam J.; Ginovska, Bojana; Thelen, Adam; da Silva, Julio C. S.; Soares, Thereza A.; Raugei, Simone; Dupuis, Michel; Shaw, Wendy J.; Hegg, Eric L.

    2016-06-07

    The proton pathway of [FeFe]-hydrogenase is essential for enzymatic H2 production and oxidation and is composed of four residues and a modeled water molecule. Recently, a computational analysis of this pathway revealed that the solvent-exposed residue of the pathway (Glu282) could form hydrogen bonds to two residues outside of the pathway (Arg286 and Ser320), implicating that these residues could function in regulating proton transfer. Substituting Arg286 with leucine eliminates hydrogen bonding with Glu282 and results in a 2.5-fold enhancement in H2 production activity, suggesting that Arg286 serves an important role in controlling the rate of proton delivery. In contrast, substitution of Ser320 with alanine reduces the rate approximately 5-fold, implying that it either acts as a member of the pathway or influences Glu282 to enable proton transfer. Interestingly, QM/MM and molecular dynamics calculations indicate that Ser320 does not play an electronic or structural role. QM calculations also estimate that including Ser320 in the pathway does not significantly change the barrier to proton movement, providing further support for its role as a member of the proton pathway. While further studies are needed to quantify the role of Ser320, collectively, these data provide evidence that the enzyme scaffold plays a significant role in modulating the activity of the enzyme, demonstrating that the rate of intraprotein proton transfer can be accelerated, particularly in a non-biological context. This work was supported by the DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science, DE-FC02-07ER64494). In addition, support from the DOE Office of Science Early Career Research Program through the Office of Basic Energy Sciences (WJS, BGP, SR) is gratefully acknowledged. Computational resources were provided at W. R. Wiley Environmental Molecular Science Laboratory (EMSL), a national scientific user facility sponsored by the Department of Energy’s Office of

  19. Is a Plasmodium lactate dehydrogenase (pLDH) enzyme-linked immunosorbent (ELISA)-based assay a valid tool for detecting risky malaria blood donations in Africa?

    Science.gov (United States)

    Atchade, Pascal S; Doderer-Lang, Cécile; Chabi, Nicodème; Perrotey, Sylvie; Abdelrahman, Tamer; Akpovi, Casimir D; Anani, Ludovic; Bigot, André; Sanni, Ambaliou; Candolfi, Ermanno

    2013-08-08

    Malaria is a leading cause of mortality in southern Benin. The main causative agent, Plasmodium falciparum, poses a threat on critical transfusions in pregnant women and children. This study's objective was to compare the performance of different malaria screening methods in blood donors in southern Benin, a malaria-endemic country. Blood from 2,515 voluntary blood donors in Benin was collected over a period of 10 months in ethylenediaminetetraacetic acid (EDTA) tubes, which were then classified according to extraction time: long rainy season, short dry season, short rainy season, and long dry season. Microscopic examination was used to count parasites. Parasite density (PD) was expressed as the number of parasites per μL of blood. Pan Plasmodium pLDH detection was assessed by an ELISA-malaria antigen test. Using crude soluble P. falciparum antigens, an ELISA-malaria antibody test detected anti-Plasmodium antibodies. Among the 2,515 blood donors (2,025 males and 488 females) screened, the rate of asymptomatic Plasmodium carriage was 295/2,515 (11.72%, 95% CI: 10.5-13.1%). Males had a higher infection rate (12.4%) than did females (8.8%). Parasite density was very low: between seven and100 parasites per μL of blood was reported in 80% of donors with parasitaemia. Three Plasmodium species were diagnosed: P. falciparum in 280/295 patients (95.0%), Plasmodium malariae in 14/295 (5.0%), and Plasmodium ovale in 1/295 (0.34%). Malaria prevalence in donors was higher during the rainy seasons (13.7%) compared with the dry seasons (9.9%). The use of a highly sensitive assay enabled pan Plasmodium pLDH detection in 966/2,515 (38.4%, 95% CI: 36.5%-40.3%). Malaria antibody prevalence was 1,859/2,515 (73.9%, 95% CI: 72.16-75.6%). Donors' antigenaemia and antibody levels varied significantly (P Plasmodium can transmit malaria to donation recipients. Malaria diagnostic methods are currently available, but the feasibility criteria for mass screening in endemic areas become

  20. The application of layered double hydroxide clay (LDH)-poly(lactide-co-glycolic acid) (PLGA) film composites for the controlled release of antibiotics

    DEFF Research Database (Denmark)

    Chakraborti, Michelle; Jackson, John K.; Plackett, David

    2012-01-01

    and quantitation of the unbound fraction by UV/Vis absorbance or HPLC analysis. Drug release from layered double hydroxide clay/drug complexes dispersed in polymeric films was measured by incubation in phosphate-buffered saline (pH 7.4) at 37 °C using absorbance or HPLC analysis. Antimicrobial activity of drug......Many sites of bacterial infection such as in-dwelling catheters and orthopedic surgical sites require local rather than systemic antibiotic administration. However, currently used controlled release vehicles, such as polymeric films, release water-soluble antibiotics too quickly, whereas nonporous...... at sites requiring long-term antibiotic exposure as they maintain the drug in a non-degraded state and release effective amounts of drug over long time periods. LDH clay/drug complexes are amenable to homogenous dispersion in polymeric films where implant coating may be optimal or required....

  1. Hydrogen production at high Faradaic efficiency by a bio-electrode based on TiO2 adsorption of a new [FeFe]-hydrogenase from Clostridium perfringens.

    Science.gov (United States)

    Morra, Simone; Valetti, Francesca; Sarasso, Veronica; Castrignanò, Silvia; Sadeghi, Sheila J; Gilardi, Gianfranco

    2015-12-01

    The [FeFe]-hydrogenase CpHydA from Clostridium perfringens was immobilized by adsorption on anatase TiO2 electrodes for clean hydrogen production. The immobilized enzyme proved to perform direct electron transfer to and from the electrode surface and catalyses both H2 oxidation (H2 uptake) and H2 production (H2 evolution) with a current density for H2 evolution of about 2 mA cm(-1). The TiO2/CpHydA bioelectrode remained active for several days upon storage and when a reducing potential was set, H2 evolution occurred with a mean Faradaic efficiency of 98%. The high turnover frequency of H2 production and the tight coupling of electron transfer, resulting in a Faradaic efficiency close to 100%, support the exploitation of the novel TiO2/CpHydA stationary electrode as a powerful device for H2 production.

  2. Correlation of LDH with mosquitoes deltamethrin-resistance%乳酸脱氢酶与蚊虫对溴氰菊酯抗性相关性的研究

    Institute of Scientific and Technical Information of China (English)

    张利; 叶钰亭; 孙林春; 洪善超; 沈波; 朱昌亮

    2012-01-01

    目的 研究乳酸脱氢酶(LDH)与蚊溴氰菊酯抗性之间的关系. 方法 应用RT- PCR及cDNA末端快速扩增(rapid amplification of cDNA end,RACE)技术扩增白纹伊蚊LDH全长基因;用实时荧光定量PCR检测敏感和抗性蚊C6/36细胞中LDH基因的表达水平;用乳酸脱氢酶活性检测试剂盒检测敏感和抗性蚊细胞中LDH酶活性.结果 LDH基因全长共1831 bp,其中ORF为996 bp,共编码331个氨基酸.该基因与埃及伊蚊、黑腹果蝇、小家鼠和人的乳酸脱氢酶的同源性分别为89%、72%、66%和65%.实时荧光定量PCR结果表明,LDH基因在抗性蚊细胞中高表达,为敏感蚊细胞的1.87倍(P<0.05).LDH酶活性检测结果显示,LDH在敏感蚊细胞中的酶活性范围为1~ 95 U/gprot,均值为40 U/gprot;在抗性蚊细胞中的酶活性范围为20 ~ 150 U/gprot,均值为75 U/gprot(n=15,P<0.01).结论 抗性蚊细胞中的LDH表达量和酶活性均高于敏感蚊细胞,提示LDH可能与蚊溴氰菊酯抗性相关.%Objective To investigate the correlation between LDH and deltamethrin( DM) -resistance in mosquitoes. Methods Cloning the full-length of LDH from mosquito C6/36 cell line and cheeking the expression level of LDH between DM-susceptible and resistant mosquito cells using quantitative PCR. Then, assaying the enzyme activities of LDH between DM-susceptible and resistant mosquito cells using lactate dehydrogenase assay kit. Results The full-length of LDH( 1 831 bp) was cloned from Aedes albopictus using RT-PCR and RACE-PCR. An open reading frame ( ORF) of 996 bp was found to encode 331 amino acids. The sequence shared 89% , 72% , 66% and 65% ho-mology with LDH of Aedes aegypti,Drosophila melanogaster,Mus musculus and Homo sapiens. Quantitative PCR demonstrated that the expression level of LDH was 1.87 folds (P<0. 05) higher in DM-resistant cells than that in-susceptible cells. The enzyme activities of LDH in DM-susceptible cells was 1-95 U/gprot,the average level was 40 U

  3. The AbrB2 autorepressor, expressed from an atypical promoter, represses the hydrogenase operon to regulate hydrogen production in Synechocystis strain PCC6803.

    Science.gov (United States)

    Dutheil, Jérémy; Saenkham, Panatda; Sakr, Samer; Leplat, Christophe; Ortega-Ramos, Marcia; Bottin, Hervé; Cournac, Laurent; Cassier-Chauvat, Corinne; Chauvat, Franck

    2012-10-01

    We have thoroughly investigated the abrB2 gene (sll0822) encoding an AbrB-like regulator in the wild-type strain of the model cyanobacterium Synechocystis strain PCC6803. We report that abrB2 is expressed from an active but atypical promoter that possesses an extended -10 element (TGTAATAT) that compensates for the absence of a -35 box. Strengthening the biological significance of these data, we found that the occurrence of an extended -10 promoter box and the absence of a -35 element are two well-conserved features in abrB2 genes from other cyanobacteria. We also show that AbrB2 is an autorepressor that is dispensable to cell growth under standard laboratory conditions. Furthermore, we demonstrate that AbrB2 also represses the hox operon, which encodes the Ni-Fe hydrogenase of biotechnological interest, and that the hox operon is weakly expressed even though it possesses the two sequences resembling canonical -10 and -35 promoter boxes. In both the AbrB2-repressed promoters of the abrB2 gene and the hox operon, we found a repeated DNA motif [TT-(N(5))-AAC], which could be involved in AbrB2 repression. Supporting this hypothesis, we found that a TT-to-GG mutation of one of these elements increased the activity of the abrB2 promoter. We think that our abrB2-deleted mutant with increased expression of the hox operon and hydrogenase activity, together with the reporter plasmids we constructed to analyze the abrB2 gene and the hox operon, will serve as useful tools to decipher the function and the regulation of hydrogen production in Synechocystis.

  4. X-ray crystallographic and EPR spectroscopic analysis of HydG, a maturase in [FeFe]-hydrogenase H-cluster assembly.

    Science.gov (United States)

    Dinis, Pedro; Suess, Daniel L M; Fox, Stephen J; Harmer, Jenny E; Driesener, Rebecca C; De La Paz, Liliana; Swartz, James R; Essex, Jonathan W; Britt, R David; Roach, Peter L

    2015-02-03

    Hydrogenases use complex metal cofactors to catalyze the reversible formation of hydrogen. In [FeFe]-hydrogenases, the H-cluster cofactor includes a diiron subcluster containing azadithiolate, three CO, and two CN(-) ligands. During the assembly of the H cluster, the radical S-adenosyl methionine (SAM) enzyme HydG lyses the substrate tyrosine to yield the diatomic ligands. These diatomic products form an enzyme-bound Fe(CO)x(CN)y synthon that serves as a precursor for eventual H-cluster assembly. To further elucidate the mechanism of this complex reaction, we report the crystal structure and EPR analysis of HydG. At one end of the HydG (βα)8 triosephosphate isomerase (TIM) barrel, a canonical [4Fe-4S] cluster binds SAM in close proximity to the proposed tyrosine binding site. At the opposite end of the active-site cavity, the structure reveals the auxiliary Fe-S cluster in two states: one monomer contains a [4Fe-5S] cluster, and the other monomer contains a [5Fe-5S] cluster consisting of a [4Fe-4S] cubane bridged by a μ2-sulfide ion to a mononuclear Fe(2+) center. This fifth iron is held in place by a single highly conserved protein-derived ligand: histidine 265. EPR analysis confirms the presence of the [5Fe-5S] cluster, which on incubation with cyanide, undergoes loss of the labile iron to yield a [4Fe-4S] cluster. We hypothesize that the labile iron of the [5Fe-5S] cluster is the site of Fe(CO)x(CN)y synthon formation and that the limited bonding between this iron and HydG may facilitate transfer of the intact synthon to its cognate acceptor for subsequent H-cluster assembly.

  5. Effects of budC gene knockout and ldhA overexpression on D-lactic acid production by Klebsiella pneumoniae%budC敲除及ldhA过表达对Klebsiella pneumoniae合成D-乳酸的影响

    Institute of Scientific and Technical Information of China (English)

    王凤寰; 孟青青

    2012-01-01

    为了提高K.pneumoniae中D-乳酸的合成效率,本文以BUD和LDH为改造目标,扩增丁二醇脱氢酶基因budC,并在其中插入四环素抗性基因tet,构建了基因敲除载体pTBT,转化K.pneumoniae,利用同源重组技术,敲除K.pneumoniae染色体上的budC基因,得到重组菌K.pneumoniae B-;在此基础上,构建了表达载体pKP-ldhA,转化K.pneumoniae B-,过量表达乳酸脱氢酶基因ldhA,得到重组菌Kpneumoniae B-L+.摇瓶发酵结果显示,重组菌K.pneumoniae B-L+的丁二醇合成浓度比原始菌降低了90%以上,D-乳酸合成浓度比K.pneumoniae B-和原始菌分别提高了77.1%和41.4%,发酵罐实验D-乳酸产量68.4 g/L,转化率0.78,生产强度1.22 g/(L·h).结果表明,敲除budC及过表达ldhA有利于改善克雷伯肺炎杆菌中D-乳酸的合成.%In the metabolic pathway of Klebsiella pneumoniae, 2,3-butanediol is a byproduct and its accumulation decreases the yield of main product, D-lactic acid. Butanediol dehydrogenase (BUD) is one of the key enzymes for butanediol biosynthesis, which competes with lactate dehydrogenase (LDH) for reducing equivalents of nicotinam-ide adenine dinueleotide (NADH). In order to reduce the butanediol accumulation and improve the D-lactic acid production, in this study, a homologous recombination vector pTBT was constructed and transformed into K. pneumoniae, resulting in K. pneumoniae B-, in which the budC gene encoding butanediol dehydrogenase was disrupted by inserting a tetracycline resistant gene (tet). Simultaneously, the expression vector pKP-ldhA harboring the ldhA gene was constructed and transformed into K. pneumoniae B- to overexpress lactate dehydrogenase. The resulting recombinant strain K. pneumoniae B-L + exhibited a nearly abolished butanediol formation ( decreased by 90% ) but a significantly improved NADH availability and D-lactic acid production. In flask culture, the D-lactic acid concentrations were 77. 1% and 41.4% , respectively, higher than those of

  6. Ni l-edge soft x-ray spectroscopy of ni-fe hydrogenases and modelcompounds--evidence for high-spin ni(ii) in the active enzyme

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Hongxin; Ralston, C.Y.; Patil, D.S.; Jones, R.M.; Gu, M.; Verhagen, M.; Adams, M.; Ge, P.; Riordan, C.; Marganian, C.A.; Mascharak,P.; Kovacs, J.; Miller, C.G.; Collins, T.J.; Brooker, S.; Croucher, P.D.; Wang, Kun; Stiefel, E.I.; Cramer, S.P.

    2000-03-15

    L-edge X-ray absorption spectroscopy has been used to study, under a variety of conditions, the electronic structure of Ni in the Ni-Fe hydrogenases from Desulfovibrio gigas, Desulfovibrio baculatus, and Pyrococcus furiosus. The status of the enzyme films used for these measurements was monitored by FT-IR spectroscopy. The L-edge spectra were interpreted by ligand field multiplet simulations and by comparison with data for Ni model complexes. The spectrum for Ni in D. gigas enzyme ''form A'' is consistent with a covalent Ni(III) species. In contrast, all of the reduced enzyme samples exhibited high spin Ni(II) spectra. The significance of the Ni(II) spin state for the structure of the hydrogenase active site is discussed.

  7. Synthesis of MgAl-LDH/CoFe2O4 and MgAl-CLDH/CoFe2O4 nanofibres for the removal of Congo Red from aqueous solution

    Indian Academy of Sciences (India)

    Zhigang Jia; Shengbiao Li; Jianhong Liu; Qi Qin; Rongsun Zhu

    2015-12-01

    MgAl-LDH/CoFe2O4 and MgAl-CLDH/CoFe2O4 nanofibres were prepared by urea-hydrolysed hydrothermal reaction and the subsequent calcinations. The morphology and structure of the products were characterized by X-ray diffraction, scanning electron microscopy and transmission electron microscope and Fourier transformed infrared. The adsorption performance of MgAl-LDH/CoFe2O4 and MgAl-CLDH/CoFe2O4 nanofibres for the removal of an anionic dye (Congo Red, CR) from aqueous solution was investigated. The results showed that MgAl-LDH/CoFe2O4 and MgAl-CLDH/CoFe2O4 nanofibres are particularly efficient in removing CR. The adsorption follows a pseudo-second-order kinetic model and best fits the Langmuir isotherm model. The maximum adsorption capacities of MgAl-LDH/CoFe2O4 and MgAl-CLDH/CoFe2O4 nanofibres for CR were found to be 213.2 and 49.8 mg g−1, respectively. The both adsorption processes were found to be spontaneous and exothermic in nature.

  8. Concentração sérica das enzimas creatinoquinase, aspartato aminotransferase e dehidrogenase lática em equinos da raça crioula CK, ASTand LDH seric concentration in crioulo breed horses

    Directory of Open Access Journals (Sweden)

    Elisiane Lourdes Da Cás

    2000-08-01

    Full Text Available A concentração de creatinoquinase (CK, aspartato aminotransferase (AST e dehidrogenase lática (LDH foi determinada em amostras de soro obtidas de 60 equinos da raça Crioula: 20 éguas mantidas no pasto (Grupo A, 20 equinos em treinamento (Grupo B e 20 participantes da competição do "Freio de Ouro de 1997" em Esteio - RS (Grupo C, dos quais foram colhidas amostras 24 a 48 horas antes do início da competição e 24 e 48 horas após a mesma. Não houve variação significativa na LDH. O grupo B apresentou concentrações de CK e AST mais elevadas (pCreatine Kinase (CK, Aspartate Aminotransferase (AST and Lactic Dehidrogenase (LDH concentration was determined in serum samples obtained from 60 horses of the Criollo breed: 20 mares managed on pasture (Group A, 20 horses in training (Group B and 20 horses participating of the Freio de Ouro 1997 competition (Group C, where samples were collected 24-48 hours before competition and 24 and 48 hours there after. There was no difference in LDH values between groups. Group B horses had higher (p<0.05 CK and AST serum concentrations than horses in groups A and C, indicating an adaptation to exercise. Forty eight hours after competition, CK values were lowest and AST highest. CK and AST were more informative than LDH in evaluating muscular function. Females had higher CK activity (p<0.05 than males. There were significam diferences related to final outcome of competition with a trena of lower values in the first places, indicating the athletic condition of the best horses.

  9. Sudden onset of facial edema and serum LDH elevation after radiation therapy for malignant lymphoma of the left parotid gland. A case report

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Gen; Ogo, Etuyo; Toda, Yukihiro; Suefuji, Hiroaki; Hayabuchi, Naofumi [Kurume Univ., Fukuoka (Japan). School of Medicine

    2001-12-01

    A report of a 48 year-old male with non-Hodgkin's lymphoma of the left parotid gland (clinical stage I EA, follicular medium-sized B cell type) is presented. He was solely treated with 30 Gy of radiation to the whole neck region, bilateral paraclavicular region and the left axilla, and 10.6 Gy boost was given to the primary lesion. Five months later, facial edema and serum LDH elevation developed suddenly. Relapse of the malignant lymphoma was suspected, but a whole body CT scan failed to show this. On the contrary, the CT scan showed a diffuse hypoattenuated area of the thyroid gland. In addition to positive antibodies, i.e, antithyroglobulin and antimicrosomal antibodies, total cholesterol and other serum markers also suggested hypofunction of the thyroid due to acute exacerbation of chronic thyroiditis. Immediately after hormone-replacement therapy, his symptoms disappeared and the abnormal serum data improved. Although the relationship between chronic thyroiditis and radiation injury has not been clearly demonstrated, it seems necessary to evaluate thyroid function before radiotherapy for head and neck tumors. Patients with chronic thyroiditis should be followed carefully after radiotherapy. (author)

  10. Metabolic markers in relation to hypoxia; staining patterns and colocalization of pimonidazole, HIF-1α, CAIX, LDH-5, GLUT-1, MCT1 and MCT4

    Directory of Open Access Journals (Sweden)

    Bussink Johan

    2011-05-01

    Full Text Available Abstract Background The cellular response of malignant tumors to hypoxia is diverse. Several important endogenous metabolic markers are upregulated under hypoxic conditions. We examined the staining patterns and co-expression of HIF-1α, CAIX, LDH-5, GLUT-1, MCT1 and MCT4 with the exogenous hypoxic cell marker pimonidazole and the association of marker expression with clinicopathological characteristics. Methods 20 biopsies of advanced head and neck carcinomas were immunohistochemically stained and analyzed. All patients were given the hypoxia marker pimonidazole intravenously 2 h prior to biopsy taking. The tumor area positive for each marker, the colocalization of the different markers and the distribution of the markers in relation to the blood vessels were assessed by semiautomatic quantitative analysis. Results MCT1 staining was present in hypoxic (pimonidazole stained as well as non-hypoxic areas in almost equal amounts. MCT1 expression showed a significant overall correlation (r = 0.75, p Conclusions Colocalization and staining patterns of metabolic and hypoxia-related proteins provides valuable additional information over single protein analyses and can improve the understanding of their functions and environmental influences.

  11. An innovative cloning platform enables large-scale production and maturation of an oxygen-tolerant [NiFe]-hydrogenase from Cupriavidus necator in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Johannes Schiffels

    Full Text Available Expression of multiple heterologous genes in a dedicated host is a prerequisite for approaches in synthetic biology, spanning from the production of recombinant multiprotein complexes to the transfer of tailor-made metabolic pathways. Such attempts are often exacerbated, due in most cases to a lack of proper directional, robust and readily accessible genetic tools. Here, we introduce an innovative system for cloning and expression of multiple genes in Escherichia coli BL21 (DE3. Using the novel methodology, genes are equipped with individual promoters and terminators and subsequently assembled. The resulting multiple gene cassettes may either be placed in one vector or alternatively distributed among a set of compatible plasmids. We demonstrate the effectiveness of the developed tool by production and maturation of the NAD(+reducing soluble [NiFe]-hydrogenase (SH from Cupriavidus necator H16 (formerly Ralstonia eutropha H16 in E. coli BL21Star™ (DE3. The SH (encoded in hoxFUYHI was successfully matured by co-expression of a dedicated set of auxiliary genes, comprising seven hyp genes (hypC1D1E1A2B2F2X along with hoxW, which encodes a specific endopeptidase. Deletion of genes involved in SH maturation reduced maturation efficiency substantially. Further addition of hoxN1, encoding a high-affinity nickel permease from C. necator, considerably increased maturation efficiency in E. coli. Carefully balanced growth conditions enabled hydrogenase production at high cell-densities, scoring mg·(Liter culture(-1 yields of purified functional SH. Specific activities of up to 7.2±1.15 U·mg(-1 were obtained in cell-free extracts, which is in the range of the highest activities ever determined in C. necator extracts. The recombinant enzyme was isolated in equal purity and stability as previously achieved with the native form, yielding ultrapure preparations with anaerobic specific activities of up to 230 U·mg(-1. Owing to the combinatorial power

  12. An innovative cloning platform enables large-scale production and maturation of an oxygen-tolerant [NiFe]-hydrogenase from Cupriavidus necator in Escherichia coli.

    Science.gov (United States)

    Schiffels, Johannes; Pinkenburg, Olaf; Schelden, Maximilian; Aboulnaga, El-Hussiny A A; Baumann, Marcus E M; Selmer, Thorsten

    2013-01-01

    Expression of multiple heterologous genes in a dedicated host is a prerequisite for approaches in synthetic biology, spanning from the production of recombinant multiprotein complexes to the transfer of tailor-made metabolic pathways. Such attempts are often exacerbated, due in most cases to a lack of proper directional, robust and readily accessible genetic tools. Here, we introduce an innovative system for cloning and expression of multiple genes in Escherichia coli BL21 (DE3). Using the novel methodology, genes are equipped with individual promoters and terminators and subsequently assembled. The resulting multiple gene cassettes may either be placed in one vector or alternatively distributed among a set of compatible plasmids. We demonstrate the effectiveness of the developed tool by production and maturation of the NAD(+)reducing soluble [NiFe]-hydrogenase (SH) from Cupriavidus necator H16 (formerly Ralstonia eutropha H16) in E. coli BL21Star™ (DE3). The SH (encoded in hoxFUYHI) was successfully matured by co-expression of a dedicated set of auxiliary genes, comprising seven hyp genes (hypC1D1E1A2B2F2X) along with hoxW, which encodes a specific endopeptidase. Deletion of genes involved in SH maturation reduced maturation efficiency substantially. Further addition of hoxN1, encoding a high-affinity nickel permease from C. necator, considerably increased maturation efficiency in E. coli. Carefully balanced growth conditions enabled hydrogenase production at high cell-densities, scoring mg·(Liter culture)(-1) yields of purified functional SH. Specific activities of up to 7.2±1.15 U·mg(-1) were obtained in cell-free extracts, which is in the range of the highest activities ever determined in C. necator extracts. The recombinant enzyme was isolated in equal purity and stability as previously achieved with the native form, yielding ultrapure preparations with anaerobic specific activities of up to 230 U·mg(-1). Owing to the combinatorial power exhibited by the

  13. Preparation of nano composite latex of poly(butyl acrylate-co-methyl methacrylate) P (BA-co-MMA) and layered double hydroxide (LDH) by mini emulsion polymerization; Preparacao de latex nanocomposito de poli(acrilato de butila-co-metacrilato de metila) P (BA-co-MMA) e hidroxido duplo lamelar (HDL) por meio da tecnica de polimerizacao em miniemulsao

    Energy Technology Data Exchange (ETDEWEB)

    Silva, Rodrigo D.; Lona, Liliane M.F., E-mail: liliane@feq.unicamp.br [Universidade Estadual de Campinas - Unicamp, Faculdade de Engenharia Quimica, SP (Brazil); Dube, Marc A. [Universidade de Ottawa. Departamento de Engenharia Quimica e Biologica, Ottawa, ON (Canada)

    2011-07-01

    In the present work, the synthesis of polymeric nonconsumption (PNCs) of P(BA-coMMA) and layered LDH through mini emulsion polymerization (MEP) was studied. The commercial organically modified LDH Perkalite F100S was used as filler and octadecyl acrylate (ODA) as costabilizer of the mini emulsions. Two types of surfactant, a cationic and nonionic one, were investigated and the cationic one could not stabilize the system when the LDH was present. The polymerization kinetics was not significantly affected by the presence of LDH which kept the pH of the system constant during the reaction. The dispersion of the inorganic material in the polymeric matrix was evaluated by X-ray diffraction which suggested exfoliation of the LDH. (author)

  14. MA和AIHA患者LDH和RET%检测的临床意义%Detection and clinical significance of serum LDH level and reticulocyte percentage in the megaloblastic anemia and autoimmune hemolytic anemia patients

    Institute of Scientific and Technical Information of China (English)

    马丽娜; 李兴; 杨晓阳; 崔晓红; 吴和弟

    2012-01-01

    目的 通过检测巨幼细胞贫血(MA)和自身免疫性溶血性贫血(AIHA)患者血清乳酸脱氢酶(LDH)和网织红细胞百分比(RET%),探讨LDH和RET%在MA和AIHA诊断中的作用.方法 对40例MA组、30例AIHI组以及40例正常对照组LDH、RET%的水平变化作回顾性分析比较.结果 MA组、AIHI组血清LDH水平明显高于正常组,差异有统计学意义(均P<0.01);MA组LDH明显高于AIHI组,差异有统计学意义(P<0.01); AIHI组RET%明显高于MA组和正常组,差异有统计学意义(P<0.01);MA组RET%与正常组对比无差异(P>0.05).结论 血清LDH和RET%水平对临床鉴别MA和AIHA具有临床指导意义.%Objective In older to identify the clinical diagnosis significance of serum lactate dehydrogenase (LDH) and reticulocyte percentage (RET%)in patients of megaloblastic anemia (MA)and autoimmune hemolytic anemia (AIHA). Methods The serum lactate dehydrogenase(LDH)and reticulocyte percentage(RET%)levels of the 40 MA group,30 AIHI group and 40 normal group,were retrospectively compared and analyzed. Results LDH levels were significantly higher in the MA and AIHI group than the normal group's (P<0.0l). Meanwhile,the levels of LDH were also significantly different between the MA group and the AIHA group (P<0.0l). RET% levels were significantly higher in the AIHA group than the levels of MA and normal group (P<0.01) but RET% levels were not different between the MA group and the normal group (P>0.05). Conclusion The levels of Serum LDH and RET% are important for the clinical diagnosis of MA and AIHA patients.

  15. Effects of CXCR4 siRNA/dextran-spermine nanoparticles on CXCR4 expression and serum LDH levels in a mouse model of colorectal cancer metastasis to the liver

    Directory of Open Access Journals (Sweden)

    Abedini F

    2011-09-01

    Full Text Available Fatemeh Abedini1, Maznah Ismail1,4, Hossein Hosseinkhani2, Tengku Azmi Tengku Ibrahim1,3, Abdul Rahman Omar1,3, Pei Pei Chong4, Mohd Hair Bejo3, Abraham J Domb51Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, Selangor Darul Ehsan, Malaysia; 2Graduate Institute of Biomedical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan; 3Faculty of Veterinary Medicine, 4Faculty of Medicine and Health Science, Universiti Putra Malaysia, Selangor Darul Ehsan, Malaysia; 5Department of Medicinal Chemistry and Natural Products, School of Pharmacy, Hebrew University-Hadassah Medical School, Jerusalem, IsraelAbstract: Liver metastasis is the main cause of mortality related to colorectal cancer. CXCR4 is necessary for the outgrowth of colon cancer micrometastases. In oncology, it has been demonstrated that several human tumors release lactate dehydrogenase (LDH into the circulation. CXCR4 gene expression and serum LDH levels are often increased in patients with colorectal cancer. Despite technological advances in cancer therapy, five-year overall survival is still around 50%. Therefore, better treatment needs to be developed. RNA interference (RNAi is a modern and powerful tool for inhibition of gene expression. However, the rate-limiting step in this technology is effective delivery of RNAi agents. We have investigated a novel strategy of CXCR4 siRNA therapy and its effect on serum LDH levels in a BALB/C mouse model of colorectal cancer metastasis to the liver. Hepatic metastasis was established by injecting a CT26.WT mouse colon carcinoma cell line via the tail vein. Our results demonstrated that CXCR4 siRNA/dextran-spermine nanoparticles achieved high silencing efficiency with low toxicity. Favorable localization of the nanoparticles was confirmed with CXCR4 gene expression in the liver, that was correlated with serum LDH levels. More research will be needed to determine the effect of CXCR4

  16. Computational Design of Iron Diphosphine Complexes with Pendant Amines for Hydrogenation of CO2 to Methanol: A Mimic of [NiFe] Hydrogenase.

    Science.gov (United States)

    Chen, Xiangyang; Jing, Yuanyuan; Yang, Xinzheng

    2016-06-20

    Inspired by the active-site structure of the [NiFe] hydrogenase, we have computationally designed the iron complex [P(tBu) 2 N(tBu) 2 )Fe(CN)2 CO] by using an experimentally ready-made diphosphine ligand with pendant amines for the hydrogenation of CO2 to methanol. Density functional theory calculations indicate that the rate-determining step in the whole catalytic reaction is the direct hydride transfer from the Fe center to the carbon atom in the formic acid with a total free energy barrier of 28.4 kcal mol(-1) in aqueous solution. Such a barrier indicates that the designed iron complex is a promising low-cost catalyst for the formation of methanol from CO2 and H2 under mild conditions. The key role of the diphosphine ligand with pendent amine groups in the reaction is the assistance of the cleavage of H2 by forming a Fe-H(δ-) ⋅⋅⋅H(δ+) -N dihydrogen bond in a fashion of frustrated Lewis pairs.

  17. Molecular recognition and self-assembly special feature: Self-assembled biomimetic [2Fe2S]-hydrogenase-based photocatalyst for molecular hydrogen evolution.

    Science.gov (United States)

    Kluwer, A M; Kapre, R; Hartl, F; Lutz, M; Spek, A L; Brouwer, A M; van Leeuwen, P W N M; Reek, J N H

    2009-06-30

    The large-scale production of clean energy is one of the major challenges society is currently facing. Molecular hydrogen is envisaged as a key green fuel for the future, but it becomes a sustainable alternative for classical fuels only if it is also produced in a clean fashion. Here, we report a supramolecular biomimetic approach to form a catalyst that produces molecular hydrogen using light as the energy source. It is composed of an assembly of chromophores to a bis(thiolate)-bridged diiron ([2Fe2S]) based hydrogenase catalyst. The supramolecular building block approach introduced in this article enabled the easy formation of a series of complexes, which are all thoroughly characterized, revealing that the photoactivity of the catalyst assembly strongly depends on its nature. The active species, formed from different complexes, appears to be the [Fe(2)(micro-pdt)(CO)(4){PPh(2)(4-py)}(2)] (3) with 2 different types of porphyrins (5a and 5b) coordinated to it. The modular supramolecular approach was important in this study as with a limited number of building blocks several different complexes were generated.

  18. X-ray crystal structure of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum to 1.8 angstrom resolution.

    Science.gov (United States)

    Peters, J W; Lanzilotta, W N; Lemon, B J; Seefeldt, L C

    1998-12-01

    A three-dimensional structure for the monomeric iron-containing hydrogenase (CpI) from Clostridium pasteurianum was determined to 1.8 angstrom resolution by x-ray crystallography using multiwavelength anomalous dispersion (MAD) phasing. CpI, an enzyme that catalyzes the two-electron reduction of two protons to yield dihydrogen, was found to contain 20 gram atoms of iron per mole of protein, arranged into five distinct [Fe-S] clusters. The probable active-site cluster, previously termed the H-cluster, was found to be an unexpected arrangement of six iron atoms existing as a [4Fe-4S] cubane subcluster covalently bridged by a cysteinate thiol to a [2Fe] subcluster. The iron atoms of the [2Fe] subcluster both exist with an octahedral coordination geometry and are bridged to each other by three non-protein atoms, assigned as two sulfide atoms and one carbonyl or cyanide molecule. This structure provides insights into the mechanism of biological hydrogen activation and has broader implications for [Fe-S] cluster structure and function in biological systems.

  19. Comparison of H{sub 2} accumulation by Rhodobacter sphaeroides KD131 and its uptake hydrogenase and PHB synthase deficient mutant

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Mi-Sun; Baek, Jin-Sook [Biomass Research Center, Korea Institute of Energy Research, 71-2 Jang-dong, Yuseong-gu, Daejeon 305-343 (Korea, Republic of); Lee, Jeong K. [Department of Life Science, Sogang University, Shinsu-dong, Mapo-gu, Seoul 121-742 (Korea, Republic of)

    2006-01-15

    Rhodobacter sphaeroides KD131 and its mutant strain lacking uptake hydrogenase (Hup{sup -}) and PHB synthase (Phb{sup -}) have been studied on H{sub 2} production and cell growth under different culture conditions. Both strains started producing H{sub 2} from the middle of the logarithmic growth phase and continued until the cell concentration leveled out. The rates of H{sub 2} production were 1.32 and 3.34ml H{sub 2}/mg-dcw for the wild-type and Hup{sup -}/Phb{sup -} mutant strain, respectively, at the optimum conditions. Malate and lactate were better carbon sources than starch, sucrose or glycerol. Approximately 60% of acetic acid was degraded in 48h by the wild-type strain and pH increased to 9.4. However, the Hup{sup -}/Phb{sup -} mutant strain did not grow well and degraded only 19% of acetic acid. The pH ranges of 7.0 were the optimum for the cell growth and pH 7.5 for the H{sub 2} production. Both strains grew and produced hydrogen under the irradiance of 12-120W/m{sup 2}, but cell growth was inhibited over 400W/m{sup 2}. (author)

  20. Secondary coordination sphere accelerates hole transfer for enhanced hydrogen photogeneration from [FeFe]-hydrogenase mimic and CdSe QDs in water

    Science.gov (United States)

    Wen, Min; Li, Xu-Bing; Jian, Jing-Xin; Wang, Xu-Zhe; Wu, Hao-Lin; Chen, Bin; Tung, Chen-Ho; Wu, Li-Zhu

    2016-07-01

    Achieving highly efficient hydrogen (H2) evolution via artificial photosynthesis is a great ambition pursued by scientists in recent decades because H2 has high specific enthalpy of combustion and benign combustion product. [FeFe]-Hydrogenase ([FeFe]-H2ase) mimics have been demonstrated to be promising catalysts for H2 photoproduction. However, the efficient photocatalytic H2 generation system, consisting of PAA-g-Fe2S2, CdSe QDs and H2A, suffered from low stability, probably due to the hole accumulation induced photooxidation of CdSe QDs and the subsequent crash of [FeFe]-H2ase mimics. In this work, we take advantage of supramolecular interaction for the first time to construct the secondary coordination sphere of electron donors (HA‑) to CdSe QDs. The generated secondary coordination sphere helps realize much faster hole removal with a ~30-fold increase, thus leading to higher stability and activity for H2 evolution. The unique photocatalytic H2 evolution system features a great increase of turnover number to 83600, which is the highest one obtained so far for photocatalytic H2 production by using [FeFe]-H2ase mimics as catalysts.

  1. Activation Thermodynamics and H/D Kinetic Isotope Effect of the Hox to HredH+ Transition in [FeFe] Hydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    King, Paul W [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Ratzloff, Michael W [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Mulder, David W [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Lubner, Carolyn E [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Brown, Katherine A [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Wilker, Molly B. [University of Colorado; Hamby, Hayden [University of Colorado; Dukovic, Gordana [University of Colorado

    2017-08-29

    Molecular complexes between CdSe nanocrystals and Clostridium acetobutylicum [FeFe] hydrogenase I (CaI) enabled light-driven control of electron transfer for spectroscopic detection of redox intermediates during catalytic proton reduction. Here we address the route of electron transfer from CdSe->CaI and activation thermodynamics of the initial step of proton reduction in CaI. The electron paramagnetic spectroscopy of illuminated CdSe:CaI showed how the CaI accessory FeS cluster chain (F-clusters) functions in electron transfer with CdSe. The Hox->HredH+ reduction step measured by Fourier-transform infrared spectroscopy showed an enthalpy of activation of 19 kJ mol-1 and a ~2.5-fold kinetic isotope effect. Overall these results support electron injection from CdSe into CaI involving F-clusters, and that the Hox->HredH+ step of catalytic proton reduction in CaI proceeds by a proton-dependent process.

  2. Atomic model of the F420-reducing [NiFe] hydrogenase by electron cryo-microscopy using a direct electron detector.

    Science.gov (United States)

    Allegretti, Matteo; Mills, Deryck J; McMullan, Greg; Kühlbrandt, Werner; Vonck, Janet

    2014-02-25

    The introduction of direct electron detectors with higher detective quantum efficiency and fast read-out marks the beginning of a new era in electron cryo-microscopy. Using the FEI Falcon II direct electron detector in video mode, we have reconstructed a map at 3.36 Å resolution of the 1.2 MDa F420-reducing hydrogenase (Frh) from methanogenic archaea from only 320,000 asymmetric units. Videos frames were aligned by a combination of image and particle alignment procedures to overcome the effects of beam-induced motion. The reconstructed density map shows all secondary structure as well as clear side chain densities for most residues. The full coordination of all cofactors in the electron transfer chain (a [NiFe] center, four [4Fe4S] clusters and an FAD) is clearly visible along with a well-defined substrate access channel. From the rigidity of the complex we conclude that catalysis is diffusion-limited and does not depend on protein flexibility or conformational changes. DOI: http://dx.doi.org/10.7554/eLife.01963.001.

  3. Investigating the Role of the Outer-Coordination Sphere in [Ni(PPh2NPh-R2)2]2+ Hydrogenase Mimics

    Energy Technology Data Exchange (ETDEWEB)

    Jain, Avijita; Reback, Matthew L.; Lindstrom, Mary L.; Thogerson, Colleen E.; Helm, Monte L.; Appel, Aaron M.; Shaw, Wendy J.

    2012-06-18

    A series of dipeptide nickel complexes with the general formula, [Ni(PPh2NNNA-amino acid/ester2)2](BF4)2, have been synthesized and characterized (P2N2= 1,5-diaza-3,7-diphosphacyclooctane, amino acid/esters = glutamic acid, alanine, lysine, and aspartic acid). Each of these complexes is an efficient electrocatalyst for H2 production. The contribution of the outer-coordination sphere, specifically the impact of sterics, the ability to protonate and the pKa of amino acid side chain on the hydrogen production activity of these complexes, was investigated. The rates of all of the catalysts ranged over an order of magnitude. The amino acid containing complexes display 2-3 times higher rates of hydrogen production than the corresponding ester complexes, suggesting the significance of protonated species (side chains/backbone of amino acids) in the outer-coordination sphere. The largest had the fastest rates suggesting that catalytic activity is not hindered by sterics. However, the shapes of catalytic waves are indicative of hindered electron transfer and may suggest a competing mechanism for catalysis than that observed for the unsubstituted parent complex. These studies demonstrate the significant contribution that the outer-coordination sphere can have in tuning the catalytic activity of small molecule hydrogenase mimics.

  4. A new cumulene diiron complex related to the active site of Fe-only hydrogenases and its phosphine substituted derivatives: synthesis, electrochemistry and structural characterization.

    Science.gov (United States)

    Wen, Na; Xu, Fenfen; Feng, Yanan; Du, Shaowu

    2011-09-01

    A new cumulene diiron complex related to the Fe-only hydrogenase active site [(μ-SCH(2)C(S)CCH(2))Fe(2)(CO)(6)] (1) was obtained by treatment of (μ-LiS)(2)Fe(2)(CO)(6) with excess 1,4-dichloro-2-butyne. By controllable CO displacement of 1 with PPh(3) and bis(diphenylphosphino)methane (dppm), mono- and di-substituted complexes, namely [(μ-SCH(2)C(S)CCH(2))Fe(2)(CO)(5)L] (2: L=PPh(3); 3: L=dppm) and [(μ-SCH(2)C(S)CCH(2))Fe(2)(CO)(4)L(2)] (4: L=PPh(3); 5: L=dppm) could be prepared in moderate yields. Treatment of 1 with bis(diphenylphosphino)ethane (dppe) afforded a double butterfly complex [(μ-SCH(2)C(S)CCH(2))Fe(2)(CO)(5)](2)(μ-dppe) (7). With dppm in refluxing toluene, a dppm-bridged complex [(μ-SCH(2)C(S)CCH(2))Fe(2)(CO)(4)(μ-dppm)] (6) was obtained. These model complexes were characterized by IR, (1)H, (31)P NMR spectra and the molecular structures of 1, 2 and 5-7 were determined by single crystal X-ray analyses. The electrochemistry of 1-3 was studied and the electrocatalytic property of 1 was investigated for proton reduction in the presence of HOAc. Copyright © 2011 Elsevier Inc. All rights reserved.

  5. Reduction Potentials of [FeFe]-Hydrogenase Accessory Iron–Sulfur Clusters Provide Insights into the Energetics of Proton Reduction Catalysis

    Energy Technology Data Exchange (ETDEWEB)

    Artz, Jacob H. [Institute; Mulder, David W. [Biosciences; Ratzloff, Michael W. [Biosciences; Lubner, Carolyn E. [Biosciences; Zadvornyy, Oleg A. [Institute; LeVan, Axl X. [Department; Williams, S. Garrett [School; Adams, Michael W. W. [B216B; Jones, Anne K. [School; King, Paul W. [Biosciences; Peters, John W. [Institute

    2017-07-06

    An [FeFe]-hydrogenase from Clostridium pasteurianum, CpI, is a model system for biological H2 activation. In addition to the catalytic H-cluster, CpI contains four accessory iron-sulfur [FeS] clusters in a branched series that transfer electrons to and from the active site. In this work, potentiometric titrations have been employed in combination with electron paramagnetic resonance (EPR) spectroscopy at defined electrochemical potentials to gain insights into the role of the accessory clusters in catalysis. EPR spectra collected over a range of potentials were deconvoluted into individual components attributable to the accessory [FeS] clusters and the active site H-cluster, and reduction potentials for each cluster were determined. The data suggest a large degree of magnetic coupling between the clusters. The distal [4Fe-4S] cluster is shown to have a lower reduction potential (~<-450 mV) than the other clusters, and molecular docking experiments indicate that the physiological electron donor, ferredoxin (Fd), most favorably interacts with this cluster. The low reduction potential of the distal [4Fe-4S] cluster thermodynamically restricts the Fdox/Fdred ratio at which CpI can operate, consistent with the role of CpI in recycling Fdred that accumulates during fermentation. Subsequent electron transfer through the additional accessory [FeS] clusters to the H-cluster is thermodynamically favorable.

  6. Polarized potential and electrode materials implication on electro-fermentative di-hydrogen production: Microbial assemblages and hydrogenase gene copy variation.

    Science.gov (United States)

    Arunasri, Kotakonda; Annie Modestra, J; Yeruva, Dileep Kumar; Vamshi Krishna, K; Venkata Mohan, S

    2016-01-01

    This study examined the changes in microbial diversity in response to different electrode materials viz., stainless steel mesh (SS) and graphite plate as anodes in two microbial electrolysis cell (MEC) each poised at 0.2V, 0.4V, 0.6V and 0.8V. Changes in microbiota prior to and after pretreatment along with microbiota enriched in response to various poised potentials with SS and graphite are monitored by 16S rRNA gene based DGGE profiling. Significant shifts in microbial community were noticed at all these experimental conditions. Correspondingly, the level of hydrogenase belonging to genera Bacillus, Pseudomonas, Rhodopseudomonas and Clostridium was studied by quantitative real time PCR (RT-PCR) at various applied potentials. DGGE based 16S rRNA gene profiling revealed enriched members belonging to phylum Firmicutes predominantly present at 0.8V in both MECs contributing to high hydrogen production. This study first time explored the growth behavior of mixed consortia in response to poised potentials and electrode materials.

  7. Synthesis, structures and electrochemical properties of nitro- and amino-functionalized diiron azadithiolates as active site models of Fe-only hydrogenases.

    Science.gov (United States)

    Liu, Tianbiao; Wang, Mei; Shi, Zhan; Cui, Hongguang; Dong, Weibing; Chen, Jiesheng; Akermark, Björn; Sun, Licheng

    2004-09-20

    Complex [[(mu-SCH2)2N(4-NO2C6H4)]Fe2(CO)6] (4) was prepared by the reaction of the dianionic intermediate [(mu-S)2Fe2(CO)6](2-) and N,N-bis(chloromethyl)-4-nitroaniline as a biomimetic model of the active site of Fe-only hydrogenase. The reduction of 4 by Pd-C/H2 under a neutral condition afforded complex [[(mu-SCH2)2N(4-NH2C6H4)]Fe2(CO)6] (5) in 67 % yield. Both complexes were characterized by IR, 1H and 13C NMR spectroscopy and MS spectrometry. The molecular structure of 4, as determined by X-ray analysis, has a butterfly 2Fe2S core and the aryl group on the bridged-N atom slants to the Fe(2) site. Cyclic voltammograms of 4 and 5 were studied to evaluate their redox properties. It was found that complex 4 catalyzed electrochemical proton reduction in the presence of acetic acid. A plausible mechanism of the electrocatalytic proton reduction is discussed.

  8. Spherical LDH-Ag°-montmorillonite heterocoagulated system with a pH-dependent sol-gel structure for controlled accessibility of AgNPs immobilized on the clay lamellae.

    Science.gov (United States)

    Deák, Ágota; Janovák, László; Tallósy, Szabolcs Péter; Bitó, Tamás; Sebők, Dániel; Buzás, Norbert; Pálinkó, István; Dékány, Imre

    2015-02-17

    Aqueous suspensions of spherical ZnMgAl-layered double hydroxides [LDH(sph)] and antibacterial silver nanoparticles (AgNPs) deposited on the lamellae of montmorillonite were used for the synthesis of composites, which behave like coherent gels at low pH (≲4.5) and incoherent sols at higher pH (≳4.5). The composition of the composite was chosen as LDH(sph)/Ag°-montm. = 25:75 wt % in order to ensure a sol-gel transition that can also be characterized by viscometry. This pH-sensitive heterocoagulated system consisting of oppositely charged colloid particles was suitable for the release of antimicrobial AgNPs immobilized on the clay lamellae via a pH-controlled gel-sol transition. The heterocoagulation process was also characterized by surface charge titration measurements. Spherical LDH/Ag°-montmorillonite composite samples were identified by X-ray diffraction (XRD) measurements. The morphological properties of the composites were studied, and the presence of the heterocoagulated structure was confirmed by scanning electron microscopy (SEM). The nanoscale structure of the LDH(sph)-Ag°-montmorillonite composite obtained was also verified by small-angle X-ray scattering (SAXS), and the rheological characteristics were studied at various pH values. The viscosity and yield value of the composite decreased by an order of magnitude upon increasing the pH from 3.0 to 5.5. The sol-gel transition of the composite suspension was reversible in the previously mentioned pH range.

  9. ATIVIDADE DA LACTATO DESIDROGENASE (LDH SÉRICA EM CÃES SUBMETIDOS À OXIGENAÇÃO EXTRACORPÓREA POR MEMBRANA (ECMO POR UM PERÍODO DE TRÊS HORAS

    Directory of Open Access Journals (Sweden)

    FELIPP SILVEIRA FERREIRA

    2011-09-01

    Full Text Available The extracorporeal membrane oxygenation (ECMO is a prolonged cardiopulmonary support technique, which aims to help the lungs and the heart when they do not respond to conventional non-invasive treatments. This research was carried out to determine the behavior oflactate dehydrogenase (LDH of five mongrel dogs undergoing ECMO for three hours. Under controlled ventilation, positive end-expiratory pressure (PEEP at 10mmHg and FiO2 at 21%, the animals were submitted to femoral cannulation for ECMO (artery and vein, by the arterial-venous (AV deviation. The LDH was measured and evaluated every thirty minutes for an uninterrupted period of three hours. The results were tabulated and statistically analyzed with ANOVA and Tukey tests, with p<0.05. The results showed an increase of serum LDH,featuring a muscle injury during the procedure due to a physiological response, similar to that caused by a hypovolemic shock. We concluded that ECMO is a viable technique for prolonged ventilatory support, but it needs some adjustments for clinical use in dogs.

  10. PARÁMETROS BIOQUÍMICOS ENZIMÁTICOS (ALT, AST, ALP, Γ-GT, LDH EN NIÑOS CON LEUCEMIA LINFOBLÁSTICA AGUDA ANTES DEL TRATAMIENTO ANTINEOPLÁSICO

    Directory of Open Access Journals (Sweden)

    Jeél Moya S

    2015-12-01

    Full Text Available Objective: To determine the enzymatic biochemical parameters (glutamic pyruvic transaminase (ALT, glutamic oxaloacetic transaminase (AST, alkaline phosphatase (ALP, gamma glutamyltransferase (γ-GT, and lactate dehydrogenase (LDH in children with acute lymphoblastic leukemia (ALL before cancer treatment. Material and Methods: A prospective experimental, observational, cross-sectional study was conducted in 30 children between 2 and 15 years old, from several Neoplastic Centers in Lima. Blood collection was performed in BD red cap Vacutainer tubes, processed in the semi-automated analyzer BIOTEC® EMP-168, with Wiener Lab Group enzyme reagents under the modified method Szaaz and UV-Optimized by IFCC, SSCC and SFBC. Finally, coding and tabulation was performed. Results: 60% were boys and 46.7% are between the ages of 2-6 years. Serum levels of AST were increased by 33.3% in boys and 50% in girls. Serum ALT values were increased in 33.3% of boys and 41.7% of girls; only 25% of girls showed increased levels of γ-GT values; ALP was increased in 44.4% of boys and 66.7% of girls. Moreover LDH levels were increased in 55.6% of boys and 41.7% of girls. Conclusions: The enzymatic tests LDH, AST, ALT and ALP are increased in children with ALL compared to normal values due to tumor lysis syndrome characterized by electrolyte abnormalities, and as a result of the massive destruction of tumor cells and rapid release of large amounts of intracellular elements.

  11. 变形链球菌荧光报告株的构建和鉴定%The construction and identification of the ldh-luc reporter strain of Streptococcus mutans and the reporter strain

    Institute of Scientific and Technical Information of China (English)

    霍丽琚; 刘红艳; 凌均檠; 黄湘雅

    2012-01-01

    目的 探讨变形链球菌(S.mutans)荧光素酶(luc)基因报告株构建方法,拟为探讨抗菌剂对多菌种生物膜中S.mutans的作用提供研究基础.方法 将S.mutans UA159乳酸脱氢酶(ldh)基因及其上游部分约1 100 000片段克隆至自杀质粒pFW5-luc的多克隆位点,构建重组质粒,并经酶切和测序证实.采用自然转化的方法,实现重组自杀质粒和S.mutans UA159同源序列的单次交换.结果 经过聚合酶链反应(PCR)和测序分析,筛选出具有报告活性的S.mutans ldh-luc 基因荧光报告株.结论 成功构建了S.mutans ldh-luc基因荧光报告株.

  12. Multivariate optimization of process parameters in the synthesis of calcined Ca‒Al (NO3) LDH for defluoridation using 3(3) factorial, central composite and Box-Behnken design.

    Science.gov (United States)

    Ghosal, Partha S; Gupta, Ashok K; Sulaiman, Ayoob

    2016-01-01

    Response surface methodology was applied for the first time in the optimization of the preparation of layered double hydroxide (LDH) for defluoridation. The influence of three vital process parameters (viz. pH, molar ratio and calcination temperature) in the synthesis of the adsorbent 'Calcined Ca‒Al (NO3) LDH' was thoroughly examined to maximize its fluoride scavenging potential. The process parameters were optimized using the 3(3) factorial, face centered central composite and Box-Behnken designs and a comparative assessment of the methods was conducted. The maximum fluoride removal efficiency was achieved at a calcination temperature of approximately 500ºC; however, the efficiency decreased with increasing pH and molar ratio. The outcome of the comparative assessment clearly delineates the case specific nature of the models. A better predictability over the entire experimental domain was obtained with the 3(3) factorial method, whereas the Box-Behnken design was found to be the most efficient model with lesser number of experimental runs. The desirability function technique was performed for optimizing the response, wherein face centered central composite design exhibited a maximum desirability. The calcined Ca‒Al (NO3) LDH, synthesized under the optimum conditions, demonstrated the removal efficiencies of 95% and 99% for the doses of 3 g L(-1) and 5 g L(-1), respectively.

  13. Homologous Cloning of Clostridium Hydrogenase Gene and Construction of Gene Knockout Vector%梭菌氢酶基因部分片段的同源克隆及敲除载体的构建

    Institute of Scientific and Technical Information of China (English)

    闫倩; 闫苗章; 王保莉; 曲东

    2012-01-01

    Dissimilatory iron-reducing bacteria grown in soil play an important role in bioremediation of organics and heavy metal pollution. The aim of this study is to explore the internal relationship between the iron-reducing ability and hydrogen-evolution of Clostridium, which is a typical iron-reducing bacteria. In this paper, a Clostridium strain isolated from paddy soil as the research object. Using homologous clone, a fragment (761 bp) of hydrogenase gene was obtained. Bioinformatics analysis showed that this gene fragment contained the active centers of hydrogenase, and was its major functional domain. Moreover, knockout vector (pMD-19-HTH) included tetracycline resistance gene was constructed by overlap PCR technique with the purpose of knockout hydrogenase function and lay the foundation for uncovering relationship between the iron-reducing ability and hydrogen-evolution.%为从分子水平探索典型铁还原菌一梭菌的铁还原能力与其氢酶产氢之间的关系,以从水稻土中分离得到一株具有高铁还原能力和高产氢能力的梭菌为材料,通过同源克隆获得长度为761 bp氢酶基因的部分序列.生物信息分析发现,该基因片段覆盖氢酶的活性中心,是氢酶的主要功能结构域.采用Overlap PCR的方法构建含有四环素抗性基因的氢酶基因敲除载体(pMD-19-HTH),以期进一步构建氢酶基因缺失的梭菌突变体,为分析氢酶产氢与铁还原的关系奠定基础.

  14. Adaptação enzimática da LDH em ratos submetidos a treinamento aeróbio em esteira e laser de baixa intensidade Adaptation of LDH enzyme in rats undergoing aerobic treadmill training and low intensity laser therapy

    Directory of Open Access Journals (Sweden)

    WHB Vieira

    2006-01-01

    Full Text Available OBJETIVO: Verificar a atividade e padrão eletroforético da LDH em resposta ao exercício em esteira em ratos submetidos à fotoestimulação por laser de baixa intensidade (780 etam. MÉTODO: Foram utilizados nesse estudo 54 ratos machos jovens (30dias, Winstar, com peso inicial médio de 112 ± 4,7g, os quais foram divididos em quatro grupos: dois permaneceram em repouso, GRC (grupo repouso controle, n= 9 e GRL (grupo repouso laser, n= 10, sendo o segundo irradiado por laser, e outros dois foram submetidos ao treinamento aeróbio e testes de esforço crescente em esteira (em degraus descontínuos visando à determinação do limiar de anaerobiose (LA durante 5 semanas, GEC (grupo exercício controle, n= 16 e GEL (grupo exercício laser, n= 19, sendo o último também irradiado por laser. O laser foi aplicado no quadríceps, glúteo máximo, sóleo e tibial anterior (TA, bilateralmente, imediatamente após cada sessão de treinamento, usando: 3,8J/cm², 15mW, 10s, modo contínuo, durante 5 semanas. Amostras de sóleo, TA e coração foram removidas 48 horas após a última sessão de exercício para análise eletroforética e espectrofotométrica. A estatística foi realizada através do teste ANOVA e post-hoc de TUKEY. O nível de significância foi considerado (pOBJECTIVE: To analyze the activity and electrophoretic pattern of the enzyme LDH in response to treadmill training, in rats undergoing photostimulation using low-level laser therapy (LLLT (780 etam. METHOD: Fifty-four 30-day-old male Winstar rats initially weighing 112 ± 4.7g were divided into four groups. Two groups remained at rest: resting control group (RCG, n= 9 and resting LLLT group (RLG, n= 10. The other two groups underwent a five-week treadmill program of aerobic training and non-continuous incremental effort tests, with the aim of determining the anaerobic limit. One of the latter was an exercise control group (ECG, n= 16 and the other was an exercise LLLT group (ELG, n

  15. Effective inhibition of colon cancer cell growth with MgAl-layered double hydroxide (LDH loaded 5-FU and PI3K/mTOR dual inhibitor BEZ-235 through apoptotic pathways

    Directory of Open Access Journals (Sweden)

    Chen J

    2014-07-01

    Full Text Available Jiezhong Chen,1,2 Renfu Shao,3 Li Li,4 Zhi Ping Xu,4 Wenyi Gu4 1School of Biomedical Sciences, University of Queensland, St Lucia, Queensland, 2Faculty of Science, Medicine and Health, University of Wollongong, Wollongong, New South Wales, 3GeneCology Research Centre, Faculty of Science, Health, Education and Engineering, University of the Sunshine Coast, Maroochydore, Queensland, 4Australian Institute of Bioengineering and Nanotechnology, University of Queensland, St Lucia, Queensland, Australia Abstract: Colon cancer is the third most common cancer and the third largest cause of cancer-related death. Fluorouracil (5-FU is the front-line chemotherapeutic agent for colon cancer. However, its response rate is less than 60%, even in combination with other chemotherapeutic agents. The side effects of 5-FU also limit its application. Nanoparticles have been used to deliver 5-FU, to increase its effectiveness and reduce side effects. Another common approach for colon cancer treatment is targeted therapy against the phosphoinositide 3-kinase (PI3K/protein kinase B (Akt pathway. A recently-invented inhibitor of this pathway, BEZ-235, has been tested in several clinical trials and has shown effectiveness and low side effects. Thus, it is a very promising drug for colon cancer treatment. The combination of these two drugs, especially nanoparticle-packed 5-FU and BEZ-235, has not been studied. In the present study, we demonstrated that nanoparticles of layered double hydroxide (LDH loaded with 5-FU were more effective than a free drug at inhibiting colon cancer cell growth, and that a combination treatment with BEZ-235 further increased the sensitivity of colon cancer cells to the treatment of LDH-packed 5-FU (LDH-5-FU. BEZ-235 alone can decrease colon cancer HCT-116 cell viability to 46% of the control, and the addition of LDH-5-FU produced a greater effect, reducing cell survival to 8% of the control. Our data indicate that the combination therapy of

  16. Hydrogen-producing microflora and Fe-Fe hydrogenase diversities in seaweed bed associated with marine hot springs of Kalianda, Indonesia.

    Science.gov (United States)

    Xu, Shou-Ying; He, Pei-Qing; Dewi, Seswita-Zilda; Zhang, Xue-Lei; Ekowati, Chasanah; Liu, Tong-Jun; Huang, Xiao-Hang

    2013-05-01

    Microbial fermentation is a promising technology for hydrogen (H(2)) production. H(2) producers in marine geothermal environments are thermophilic and halotolerant. However, no one has surveyed an environment specifically for thermophilic bacteria that produce H(2) through Fe-Fe hydrogenases (H(2)ase). Using heterotrophic medium, several microflora from a seaweed bed associated with marine hot springs were enriched and analyzed for H(2) production. A H(2)-producing microflora was obtained from Sargassum sp., 16S rRNA genes and Fe-Fe H(2)ase diversities of this enrichment were also analyzed. Based on 16S rRNA genes analysis, 10 phylotypes were found in the H(2)-producing microflora showing 90.0-99.5 % identities to known species, and belonged to Clostridia, Gammaproteobacteria, and Bacillales. Clostridia were the most abundant group, and three Clostridia phylotypes were most related to known H(2) producers such as Anaerovorax odorimutans (94.0 % identity), Clostridium papyrosolvens (98.4 % identity), and Clostridium tepidiprofundi (93.1 % identity). For Fe-Fe H(2)ases, seven phylotypes were obtained, showing 63-97 % identities to known Fe-Fe H(2)ases, and fell into four distinct clusters. Phylotypes HW55-3 and HM55-1 belonged to thermophilic and salt-tolerant H(2)-producing Clostridia, Halothermothrix orenii-like Fe-Fe H(2)ases (80 % identity), and cellulolytic H(2)-producing Clostridia, C. papyrosolvens-like Fe-Fe H(2)ases (97 % identity), respectively. The results of both 16S rRNA genes and Fe-Fe H(2)ases surveys suggested that the thermophilic and halotolerant H(2)-producing microflora in seaweed bed of hot spring area represented previously unknown H(2) producers, and have potential application for H(2) production.

  17. Characterization of the Fe site in iron-sulfur cluster-free hydrogenase (Hmd) and of a model compound via nuclear resonance vibrational spectroscopy (NRVS).

    Science.gov (United States)

    Guo, Yisong; Wang, Hongxin; Xiao, Yuming; Vogt, Sonja; Thauer, Rudolf K; Shima, Seigo; Volkers, Phillip I; Rauchfuss, Thomas B; Pelmenschikov, Vladimir; Case, David A; Alp, Ercan E; Sturhahn, Wolfgang; Yoda, Yoshitaka; Cramer, Stephen P

    2008-05-19

    We have used (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to study the iron site in the iron-sulfur cluster-free hydrogenase Hmd from the methanogenic archaeon Methanothermobacter marburgensis. The spectra have been interpreted by comparison with a cis-(CO)2-ligated Fe model compound, Fe(S2C2H4)(CO)2(PMe3)2, as well as by normal mode simulations of plausible active site structures. For this model complex, normal mode analyses both from an optimized Urey-Bradley force field and from complementary density functional theory (DFT) calculations produced consistent results. For Hmd, previous IR spectroscopic studies found strong CO stretching modes at 1944 and 2011 cm(-1), interpreted as evidence for cis-Fe(CO)2 ligation. The NRVS data provide further insight into the dynamics of the Fe site, revealing Fe-CO stretch and Fe-CO bend modes at 494, 562, 590, and 648 cm(-1), consistent with the proposed cis-Fe(CO)2 ligation. The NRVS also reveals a band assigned to Fe-S stretching motion at approximately 311 cm(-1) and another reproducible feature at approximately 380 cm(-1). The (57)Fe partial vibrational densities of states (PVDOS) for Hmd can be reasonably well simulated by a normal mode analysis based on a Urey-Bradley force field for a five-coordinate cis-(CO)2-ligated Fe site with additional cysteine, water, and pyridone cofactor ligands. A "truncated" model without a water ligand can also be used to match the NRVS data. A final interpretation of the Hmd NRVS data, including DFT analysis, awaits a three-dimensional structure for the active site.

  18. Pd(II)-Directed Encapsulation of Hydrogenase within the Layer-by-Layer Multilayers of Carbon Nanotube Polyelectrolyte Used as a Heterogeneous Catalyst for Oxidation of Hydrogen.

    Science.gov (United States)

    Liu, Jiang; Zorin, Nikolay A; Chen, Meng; Qian, Dong-Jin

    2015-06-16

    A metal-directed assembling approach has been developed to encapsulate hydrogenase (H2ase) within a layer-by-layer (LBL) multilayer of carbon nanotube polyelectrolyte (MWNT-PVPMe), which showed efficient biocatalytic oxidation of H2 gas. The MWNT-PVPMe was prepared via a diazonium process and addition reactions with poly(4-vinylpyridine) (PVP) and methyl iodide (MeI). The covalently attached polymers and organic substituents in the polyelectrolyte comprised 60-70% of the total weight. The polyelectrolyte was then used as a substrate for H2ase binding to produce MWNT-PVPMe@H2ase bionanocomposites. X-ray photoelectron spectra revealed that the bionanocomposites included the elements of Br, S, C, N, O, I, Fe, and Ni, which confirmed that they were composed of MWNT-PVPMe and H2ase. Field emission transmission electron microscope images revealed that the H2ase was adsorbed on the surface of MWNT-PVPMe with the domains ranging from 20 to 40 nm. Further, with the use of the bionanocomposites as nanolinkers and Na2PdCl4 as connectors, the (Pd/MWNT-PVPMe@H2ase)n multilayers were constructed on the quartz and gold substrate surfaces by the Pd(II)-directed LBL assembling technique. Finally, the as-prepared LBL multilayers were used as heterogeneous catalysts for hydrogen oxidation with methyl viologen (MV(2+)) as an electron carrier. The dynamic processes for the reversible color change between blue-colored MV(+) and colorless MV(2+) (catalyzed by the LBL multilayers) were video recorded, which confirmed that the H2ase encapsulated within the present LBL multilayers was of much stronger stability and higher biocatalytic activity of H2 oxidation resulting in potential applications for the development of H2 biosensors and fuel cells.

  19. Escherichia coli K-12 survives anaerobic exposure at pH 2 without RpoS, Gad, or hydrogenases, but shows sensitivity to autoclaved broth products.

    Directory of Open Access Journals (Sweden)

    Daniel P Riggins

    Full Text Available Escherichia coli and other enteric bacteria survive exposure to extreme acid (pH 2 or lower in gastric fluid. Aerated cultures survive via regulons expressing glutamate decarboxylase (Gad, activated by RpoS, cyclopropane fatty acid synthase (Cfa and others. But extreme-acid survival is rarely tested under low oxygen, a condition found in the stomach and the intestinal tract. We observed survival of E. coli K-12 W3110 at pH 1.2-pH 2.0, conducting all manipulations (overnight culture at pH 5.5, extreme-acid exposure, dilution and plating in a glove box excluding oxygen (10% H2, 5% CO2, balance N2. With dissolved O2 concentrations maintained below 6 µM, survival at pH 2 required Cfa but did not require GadC, RpoS, or hydrogenases. Extreme-acid survival in broth (containing tryptone and yeast extract was diminished in media that had been autoclaved compared to media that had been filtered. The effect of autoclaved media on extreme-acid survival was most pronounced when oxygen was excluded. Exposure to H2O2 during extreme-acid treatment increased the death rate slightly for W3110 and to a greater extent for the rpoS deletion strain. Survival at pH 2 was increased in strains lacking the anaerobic regulator fnr. During anaerobic growth at pH 5.5, strains deleted for fnr showed enhanced transcription of acid-survival genes gadB, cfa, and hdeA, as well as catalase (katE. We show that E. coli cultured under oxygen exclusion (<6 µM O2 requires mechanisms different from those of aerated cultures. Extreme acid survival is more sensitive to autoclave products under oxygen exclusion.

  20. Synthesis, structural characterization, and some properties of 2-acylmethyl-6-ester group-difunctionalized pyridine-containing iron complexes related to the active site of [Fe]-hydrogenase.

    Science.gov (United States)

    Song, Li-Cheng; Hu, Fu-Qiang; Wang, Miao-Miao; Xie, Zhao-Jun; Xu, Kai-Kai; Song, Hai-Bin

    2014-06-07

    As biomimetic models for [Fe]-hydrogenase, the 2-acylmethyl-6-ester group-difunctionalized pyridine-containing iron(II) complexes 1-4 have been successfully prepared via the following three separate steps. In the first step, the acylation or esterification of difunctionalized pyridine 2-(p-MeC6H4SO3CH2)-6-HOCH2C5H3N with acetyl chloride or benzoic acid gives the corresponding pyridine derivatives 2-(p-MeC6H4SO3CH2)-6-RCO2CH2C5H3N (A, R = Me; B, R = Ph). The second step involves reaction of A or B with Na2Fe(CO)4 followed by treatment of the intermediate Fe(0) complexes [Na(2-CH2-6-RCO2CH2C5H3N)Fe(CO)4] (M1, R = Me; M2, R = Ph) with iodine to afford 2-acylmethyl-6-acetoxymethyl or 6-benzoyloxymethyl-difunctionalized pyridine-containing Fe(II) iodide complexes [2-C(O)CH2-6-RCO2CH2C5H3N]Fe(CO)2I (1, R = Me; 3, R = Ph). Finally, when 1 or 3 is treated with sodium 2-mercaptopyridinate, the corresponding difunctionalized pyridine-containing Fe(ii) mercaptopyridinate complexes [2-C(O)CH2-6-RCO2C5H3N]Fe(CO)2(2-SC5H4N) (2, R = Me; 4, R = Ph) are produced. While the structures of model complexes 1-4 are confirmed by X-ray crystallography, the electrochemical properties of 2 and 4 are compared with those of the two previously reported models. In addition, complexes 2 and 4 have been found to be catalysts for H2 production in the presence of TFA under CV conditions.

  1. The Effect of Eccentric exercise to LDH and SDH on Skeletal Muscle of Developing Rats%不同持续时间离心运动对大鼠骨骼肌LDH和SDH的影响

    Institute of Scientific and Technical Information of China (English)

    陶霞; 张宇

    2013-01-01

    Objective: To explore the developmental period of eccentric exercise on skeletal muscle aerobic metabolism and glycolytic capacity. Methods: The developmental stages and adult rats were randomly divided into control group (NC group), 3W training group (T3 group), 6W training group (T6 group), 9W training group (T9 group), reference to the exercise load Bedford program. UV spectrophotometric determination of SDH activity, using semi-automatic biochemical analyzer determination of LDH activity. The results showed that eccentric exercise can cause developmental stages of rat SDH first and then decrease, LDH increased after the first reduction. Eccentric exercise on the developmental stages of rat SDH, LDH is greater than the impact of adult rats, and when the greatest difference in sports 6W.%目的:探讨离心运动对发育期大鼠骨骼肌有氧代谢和糖酵解能力的影响。方法:将发育期与成年大鼠随机分为对照组( NC组)、3W训练组(T3组)、6W训练组(T6组)、9W训练组(T9组),参照Bedford的运动负荷方案。采用紫外分光光度法测定 SDH活性,采用半自动生化分析仪测定中LDH活性。结果发现离心运动可引起发育期大鼠SDH先升高后降低,LDH先降低后升高。离心运动对发育期大鼠 SDH、LDH的影响大于成年大鼠,且在运动6W时差异最大。

  2. Self-assembled superamolecular switch based on liposome regulates LDH activities%脂质体超分子开关的自组装及其对酶活性的调控

    Institute of Scientific and Technical Information of China (English)

    刘宝全; 王剑锋; 李春斌; 范圣第

    2011-01-01

    以十六烷基胺与十六烷基溴为原料,通过取代反应与缩合反应等5步反应分别合成人工脂质(肽脂质)与人工受体.利用自组装技术在脂质体上构建了超分子开关体系,其中磷酸吡哆醛(PLP)为信号分子,铜离子做为中间信使.在没有调控信号时,铜离子与乳酸脱氢酶结合,抑制酶的活性;当加入信号分子PLP时,信号分子与人工受体作用形成席夫碱,席夫碱与乳酸脱氢酶竞争铜离子,解除铜离子对乳酸脱氢酶活性的抑制,恢复乳酸脱氢酶的催化活性.%A cationic peptide lipid named N, N-dihexadecyl-Na-[6-( trimethylammonio ) hexanoyl]-alaninamide bromide ( N+ C5Ala2C16 ) and a artificial receptor were synthesized with 1-hexadecylamine and 1 -bromohexadecane through five steps, including substitution and condensation reactions. A self assembled superamolecular switch system was obtained based on liposome. In this system, pyridoxal 5'-phosphate acts as artificial signal,Cu2+ acts as a mediator. Without signal molecules, Cu2+ bound LDH and inhibited LDH activities. When signal molecule--PLP was added to this system, PLP attached to liposome surface,and reacted with the amino group of the artificial receptor, and formed Schiff base, and captured copper cation, and led to the recovery of LDH activities which has been inhibited by copper cation.

  3. Cloning and knockout of formate hydrogen lyase and H{sub 2}-uptake hydrogenase genes in Enterobacter aerogenes for enhanced hydrogen production

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Hongxin; Ma, Kun; Lu, Yuan; Zhang, Chong; Wang, Liyan; Xing, Xin-Hui [Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Tsinghua Yuan, Beijing 100084 (China)

    2009-01-15

    A 5431-bp DNA fragment partially encoding the formate hydrogen lyase (FHL) gene cluster hycABCDE was isolated and identified from Enterobacter aerogenes IAM1183 chromosomal DNA. All the five putative gene products showed a high degree of homology to the reported bacterial FHL proteins. The gene hycA, encoding the FHL repressor protein, and hybO, encoding the small subunit of the uptake hydrogenase, were targeted for genetic knockout for improving the hydrogen production. The pYM-Red recombination system was adopted to form insertional mutations in the E. aerogenes genome, thereby creating mutant strains of IAM1183-A ({delta} hycA), IAM1183-O ({delta} hybO), and IAM1183-AO ({delta} hycA/ {delta} hybO double knockout). The hydrogen production experiments with these mutants showed that the maximum specific hydrogen productivities of IAM1183-A, IAM1183-O, and IAM1183-AO were 2879.466 {+-} 38.59, 2747.203 {+-} 13.25 and 3372.019 {+-} 4.39 (ml h{sup -1} g{sup -1}dry cell weight), respectively, higher than that of the wild strain (2321.861 {+-} 15.34 ml h{sup -1} g{sup -1}dry cell weight). The total H{sub 2} yields by the three mutants IAM1183-A, IAM1183-O and IAM1183-AO were 0.73, 0.78, and 0.83 mol-H{sub 2}/mol glucose, respectively, while the wild-type IAM1183 was only 0.65 mol-H{sub 2}/mol glucose. The metabolites of the mutants including acetate, ethanol, 2,3-butanediol and succinate were all increased compared with that of the wild type, implying the changed metabolic flux by the mutation. In the fermentor cultivation with IAM1183 {delta} hycA/ {delta} hybO, the total hydrogen volume after 16 h cultivation reached 4.4 L, while that for the wild type was only 2.9 L. (author)

  4. Formation and yield of multi-walled carbon nanotubes synthesized via chemical vapour deposition routes using different metal-based catalysts of FeCoNiAl, CoNiAl and FeNiAl-LDH.

    Science.gov (United States)

    Hussein, Mohd Zobir; Jaafar, Adila Mohamad; Yahaya, Asmah Hj; Masarudin, Mas Jaffri; Zainal, Zulkarnain

    2014-11-05

    Multi-walled carbon nanotubes (MWCNTs) were prepared via chemical vapor deposition (CVD) using a series of different catalysts, derived from FeCoNiAl, CoNiAl and FeNiAl layered double hydroxides (LDHs). Catalyst-active particles were obtained by calcination of LDHs at 800 °C for 5 h. Nitrogen and hexane were used as the carrier gas and carbon source respectively, for preparation of MWCNTs using CVD methods at 800 °C. MWCNTs were allowed to grow for 30 min on the catalyst spread on an alumina boat in a quartz tube. The materials were subsequently characterized through X-ray diffraction, Fourier transform infrared spectroscopy, surface area analysis, field emission scanning electron microscopy and transmission electron microscopy. It was determined that size and yield of MWCNTs varied depending on the type of LDH catalyst precursor that is used during synthesis. MWCNTs obtained using CoNiAl-LDH as the catalyst precursor showed smaller diameter and higher yield compared to FeCoNiAl and FeNiAl LDHs.

  5. Formation and Yield of Multi-Walled Carbon Nanotubes Synthesized via Chemical Vapour Deposition Routes Using Different Metal-Based Catalysts of FeCoNiAl, CoNiAl and FeNiAl-LDH

    Directory of Open Access Journals (Sweden)

    Mohd Zobir Hussein

    2014-11-01

    Full Text Available Multi-walled carbon nanotubes (MWCNTs were prepared via chemical vapor deposition (CVD using a series of different catalysts, derived from FeCoNiAl, CoNiAl and FeNiAl layered double hydroxides (LDHs. Catalyst-active particles were obtained by calcination of LDHs at 800 °C for 5 h. Nitrogen and hexane were used as the carrier gas and carbon source respectively, for preparation of MWCNTs using CVD methods at 800 °C. MWCNTs were allowed to grow for 30 min on the catalyst spread on an alumina boat in a quartz tube. The materials were subsequently characterized through X-ray diffraction, Fourier transform infrared spectroscopy, surface area analysis, field emission scanning electron microscopy and transmission electron microscopy. It was determined that size and yield of MWCNTs varied depending on the type of LDH catalyst precursor that is used during synthesis. MWCNTs obtained using CoNiAl-LDH as the catalyst precursor showed smaller diameter and higher yield compared to FeCoNiAl and FeNiAl LDHs.

  6. 恩再适注射液肌注治疗LDH术后残余神经痛的临床疗效观察%Observation on Clinical Curative Effect of Analgecine Muscle Injection Treating Postoperative Residual Neuralgia of LDH

    Institute of Scientific and Technical Information of China (English)

    徐昆; 郝佳颖; 肖天洁; 杜元良; 孙贺; 张义龙; 赵艳军; 张峥

    2016-01-01

    Objective:To observe the clinical curative effect of analgecine muscle injection treating postoper-ative residual neuralgia of LDH ( protrusion of lumbar intervertebral disc ) . Methods: 90 LDH patients be-tween 40 and 59 years old who were insensitive to regular conservative treatment ( four weeks) , with VAS 5 points,and still sufferedfrom lower limb pain or ( and) numb at the 2nd week after waist 4-5 or waist 5-sa-crum 1 nucleus pulposus removal and intervertebral fusion. They were randomized into three groups, with 30 cases in each group:group A ( VitaminB1+B12Injectiongroup) , group B ( Cobamamide injection group) , and group C ( analgecine injection group) . After being treated for 3-6 weeks by the listed three treatment meth-ods, the clinical curative effect of analgecine injection treating postoperative residual neuralgia of LDH were observed. Result:the clinical curative effect of group C ( analgecine injection group) was significantly better than that of group A and group B. Conclusions:analgecine injection can significantly improve the postoperative residualneuralgia of LDH and it has a significant clinical curative effect.%目的::观察恩再适注射液肌注治疗LDH(腰椎间盘突出症)术后残余神经痛的临床疗效。方法:选择年龄于40~59岁之间的LDH患者90例(男女各45例)经正规保守治疗4周无效且VAS≥5分患者,行腰4-5或腰5-骶1髓核摘除、椎间融合术后第2周仍存在下肢疼痛或(和)麻木者,按照完全随机等分法分三组、每组30例:A组(维生素B1+B12注射液组),B组(注射用腺苷钴胺组),C组(恩再适注射液组),通过上述三组治疗3~6周后,观察恩再适注射液组治疗LDH术后残余神经痛的临床疗效。结果:C组(恩再适注射液组)临床疗效显著优于A、B两组。结论:恩再适注射液可显著改善LDH术后残余神经痛症状,具有显著临床疗效。

  7. Relationship of proton motive force and the F(0)F (1)-ATPase with bio-hydrogen production activity of Rhodobacter sphaeroides: effects of diphenylene iodonium, hydrogenase inhibitor, and its solvent dimethylsulphoxide.

    Science.gov (United States)

    Hakobyan, Lilit; Gabrielyan, Lilit; Trchounian, Armen

    2012-08-01

    Rhodobacter sphaeroides MDC 6521 was able to produce bio-hydrogen (H(2)) in anaerobic conditions under illumination. In this study the effects of the hydrogenase inhibitor-diphenylene iodonium (Ph(2)I) and its solvent dimethylsulphoxide (DMSO) on growth characteristics and H(2) production by R. sphaeroides were investigated. The results point out the concentration dependent DMSO effect: in the presence of 10 mM DMSO H(2) yield was ~6 fold lower than that of the control. The bacterium was unable to produce H(2) in the presence of Ph(2)I. In order to examine the mediatory role of proton motive force (∆p) or the F(0)F(1)-ATPase in H(2) production by R. sphaeroides, the effects of Ph(2)I and DMSO on ∆p and its components (membrane potential (∆ψ) and transmembrane pH gradient), and ATPase activity were determined. In these conditions ∆ψ was of -98 mV and the reversed ∆pH was +30 mV, resulting in ∆p of -68 mV. Ph(2)I decreased ∆ψ in concentrations of 20 μM and higher; lower concentrations of Ph(2)I as DMSO had no valuable effect on ∆ψ. The R. sphaeroides membrane vesicles demonstrated significant ATPase activity sensitive to N,N'-dicyclohexylcarbodiimide. The 10-20 μM Ph(2)I did not affect the ATPase activity, whereas 40 μM Ph(2)I caused a marked inhibition (~2 fold) in ATPase activity. The obtained results provide novel evidence on the involvement of hydrogenase and the F(0)F(1)-ATPase in H(2) production by R. sphaeroides. Moreover, these data indicate the role of hydrogenase and the F(0)F(1)-ATPase in ∆p generation. In addition, DMSO might increase an interaction of nitrogenase with CO(2), decreasing nitrogenase activity and affecting H(2) production.

  8. 微反应器法制备ZnAl-LDH纳米片及LDH基疏水膜的组装%Preparation of Zn-Al layered double hydroxide nanosheets by microchannel reactor and fabrication of LDH-based hydrophobic films

    Institute of Scientific and Technical Information of China (English)

    庞秀江; 代娇娇; 刘源; 陈利; 迟铭君; 王延涛; 李再峰

    2016-01-01

    Layered double hydroxide( LDH) nanosheets can be used as basic building block or be modified with organic for the syn-thesis of LDH-based functional materials. But the current methods to prepare ultrathin LDH nanosheets have problems such as the utilization of toxic solvents,the adsorption of organic substances on the surface of LDH,or the difficulty in realizing macroscopic preparation,which all limited the practical application of ultrathin LDH. In this paper,ZnAl-LDH nanosheets were prepared using a microchannel reactor by controlling pH value during the co-precipitation process. The so-obtained nanosheets were naked( without organic absorbed) because water was the only solution during the preparation,and nanosheets can be prepared in great amount. The ZnAl-LDH nanosheets were characterized by X-ray diffraction,transmission electron microscopy and atomic force microscopy. The results showed that products were not chemically pure but contained zinc oxide and the morphology was irregular in shape when pH value was high. When pH value was reduced to 7. 9,LDH nanosheets with the thickness of 1. 0-1. 5nm was obtained and the distri-bution of particle was quite narrow. The surface of ZnAl-LDH nanosheets was easily modified with sodium laurate through the simple blend. The laurate-ZnAl-LDH film was composed of platelets lying perpendicular to the surface of the glass substrate and the water contact angle was 149°,which provided a simple method to fabricate LDH-based functional materials.%层状双金属氢氧化物( LDH)纳米片可作为基本的结构单元或利用有机物修饰其表面使其功能化来构筑LDH基功能材料,但现有的制备方法存在使用有机溶剂、所得LDH纳米片表面吸附有机物、难以宏量制备等问题,限制了其实际应用。本文利用微反应器通过控制共沉淀过程中的pH值制备了ZnAl-LDH ([ Zn2+0.67 Al3+0.33( OH)2] NO3·yH2 O)纳米片,由于利用微反应器法制备LDH纳米片的过

  9. Requirements for functional models of the iron hydrogenase active site: D2/H2O exchange activity in ((mu-SMe)(mu-pdt)[Fe(CO)2(PMe3)]2+)[BF4-].

    Science.gov (United States)

    Georgakaki, Irene P; Miller, Matthew L; Darensbourg, Marcetta Y

    2003-04-21

    Hydrogen uptake in hydrogenase enzymes can be assayed by H/D exchange reactivity in H(2)/D(2)O or H(2)/D(2)/H(2)O mixtures. Diiron(I) complexes that serve as structural models for the active site of iron hydrogenase are not active in such isotope scrambling but serve as precursors to Fe(II)Fe(II) complexes that are functional models of [Fe]H(2)ase. Using the same experimental protocol as used previously for ((mu-H)(mu-pdt)[Fe(CO)(2)(PMe(3))](2)(+)), 1-H(+) (Zhao et al. J. Am. Chem. Soc. 2001, 123, 9710), we now report the results of studies of ((mu-SMe)(mu-pdt)[Fe(CO)(2)(PMe(3))](2)(+)), 1-SMe(+), toward H/D exchange. The 1-SMe(+) complex can take up H(2) and catalyze the H/D exchange reaction in D(2)/H(2)O mixtures under photolytic, CO-loss conditions. Unlike 1-H(+), it does not catalyze H(2)/D(2) scrambling under anhydrous conditions. The molecular structure of 1-SMe(+) involves an elongated Fe.Fe separation, 3.11 A, relative to 2.58 A in 1-H(+). It is proposed that the strong SMe(-) bridging ligand results in catalytic activity localized on a single Fe(II) center, a scenario that is also a prominent possibility for the enzyme active site. The single requirement is an open site on Fe(II) available for binding of D(2) (or H(2)), followed by deprotonation by the external base H(2)O (or D(2)O).

  10. Synthesis of Molybdate Inhibitor Based on Intercalated Layered Double Hydroxide(LDH) and Its Anticorrosive Properties%LDH型MoO42-缓蚀剂的合成及防腐性能

    Institute of Scientific and Technical Information of China (English)

    于湘; 俞志东; 程丽华; 潘奇

    2013-01-01

    采用共沉淀法制备了镁铝氢氧化物为层板、MoO42-柱撑的LDH型MoO42-缓蚀剂(记为MoO42-LDH),利用XRD和Raman光谱对样品进行表征.通过缓释实验,讨论了LDH型缓蚀剂的释放能力以及缓蚀剂的缓蚀机理.SEM-EDS、ICP、N2吸附脱附和极化曲线测试结果表明,合成的LDH型MoO42-缓蚀剂具有很好的离子交换和吸附Cl-的性能,释放出MoO42-缓蚀剂进入电解液,24 h内对镁合金的腐蚀电流保持在9.129 μA/cm2,减缓了镁合金腐蚀.添加质量分数20% MoO42-LDH颜料的环氧涂层在质量分数3.5% NaCl溶液中的EIS测试体现出较好的耐蚀作用,耐盐雾腐蚀187 h以上.%Mg-Al layered double hydroxide ( LDH) loaded with molybdate anions were synthesized via coprecipitation method. The resulting compounds were characterized respectively by means of X-ray diffraction (XRD) and Raman spectra. The properties of the release of inhibitor and the inhibition mechanism were observed and analyzed by slow-release experiment. The SEM - EDS, ICP and N2 adsorption-desorption measurements demonstrated the excellent anion-exchange and adsorption capabilities for chloride ion. Polarization curves measurements showed that the filtrate as electrolyte exhibited a lower corrosion current density value due to the presence of MoO2-4 . The results indicate that the released inhibitor can provide adequate corrosion protection, maintaining a corrosion current density at approximately 9. 129 μA/cm2 for magnesium alloy within 24 h. The coatings containing 20% MoO2-4-LDH pigment keep excellent corrosion resistance by electrochemical impedance spectroscopy (EIS) during immersion in 3.5% NaCl solution with more than 187 hours of exposure to salt spray test.

  11. 中药外用药治疗腰椎间盘突出症的频次分析%Study on the properties of Chinese herb with external formulae in treatment of LDH

    Institute of Scientific and Technical Information of China (English)

    杨佳裕; 刘光明; 陈建华; 孙波

    2013-01-01

    Objective To summarize and analyze the properties of Chinese herb with External formulae in the treatment of LDH. Methods We retrieved medical periodicals from 1998 to 2008 and collected all the Chinese herb with External formulae in treating LDH. According to different efficacies,we calculated the frequencies of different kinds of herbs. Results total of 79 formulae were covered in this study, there were 27 herbs of Medicine For Rheumatism which were used 338 times,25 herbs of blood-activating and stasis-eliminating compound used 327 times,7 herbs of diaphoretic recipes to Divergent chill used 124 times,8 herbs of warm the body used 59 times, 11 herbs of inducing diuresis and reducing edema used 29 times,7 herbs of qi-regulating used 17 times,6 herbs of tonic used 45 times and 2 herbs of relieve dizziness used 2 times. Conclusion The External formulae for LDH mainly comprise Medicine For Rheumatism and blood-activating and stasis-eliminating compound.%目的 归纳分析中药外治治疗腰椎间盘突出症处方的用药特点.方法 检索1998-01-2008-12各类医学期刊,获取外治治疗腰椎间盘突出症的方剂;统计方剂中不同功效类别药物的使用频次.结果 共有79个方剂纳入研究.其中79个方剂中,祛风湿通络药27味338次,活血化瘀药25味327次,解表发散风寒药7味124次,温里药8味59次,利湿化痰药11味29次,理气药7味17次,补益药6味45次,息风止痉药3味4次.结论 祛风湿通络,活血化瘀是外治治疗腰椎间盘突出症的总则.

  12. Study on clinical significance of SF,LDH and gamma GT testing in diagnosis of breast cancer%SF、LDH和γ-GT检测在乳腺癌诊断治疗中的临床应用价值

    Institute of Scientific and Technical Information of China (English)

    张勇; 刘爱胜; 文艳

    2014-01-01

    目的:探讨血清铁蛋白( SF)、乳酸脱氢酶( LDH)和γ-谷氨酰转肽酶(γ-GT)检测在临床乳腺癌诊断及治疗疗效判断中的应用价值。方法收集2010年2月至2013年8月乳腺癌患者42例,乳腺良性肿瘤患者57例和正常健康女性85例,分别用化学发光免疫法测定SF,用生化速率法测定LDH和γ-GT的含量,并对所测数据进行分组比较分析。结果乳腺癌血清中SF、LDH和γ-GT含量显著高于乳腺良性肿瘤患者和正常健康女性,两组差异有显著性( P <0.01);SF、LDH和γ-GT含量随乳腺癌临床分期的增加而显著增加,随手术或治疗效果好转而迅速下降,复发时再次增高;SF、LDH和γ-GT对乳腺癌诊断的敏感度分别为64.9%、81.2%和87.5%。结论 SF、LDH和γ-GT含量检测对乳腺癌临床诊断有一定的参考价值,特别对乳腺癌治疗效果的判断有极为重要的临床应用价值。%Objective Toexplorethediagnosticsignificanceofserumferritin(SF),lactatedehydrogenase(LDH)andγ-glutamyl-transpeptidase(γ-GT)indiagnosisandjudgementofefficacyintreatmentofbreastcancer.Methods Theclinicaldataof42patientswith breast cancer,57 patients with breast benign tumour and 85 normal healthy women during February 2010 to August 2013 were collected for this study. Serum ferritin( SF)was measured by chemiluminescence immunoassay,the levels of LDH and γ-GT were determined by biochemical method,andtheirresultsweregroupedandcomparativelyanalyzed.Results TheserumlevelsofSF,LDHandγ-GTinpatientswithbreast cancer were significantly higher than those of patients with breast benign tumour and normal healthy women,and the difference in results between them was statistically significant( P <0. 01). The levels of SF,LDH andγ-GT were significantly increased followed with the increase of clinical staging of breast cancer,and they were rapidly dropped after surgery or improved after treatment,and they were increased again with the

  13. 意蜂工蜂、雄蜂和蜂王的LDH和EST同工酶比较%Study on LDH and EST isoenzymes of queen bee, spado and drone of Italian honeybee

    Institute of Scientific and Technical Information of China (English)

    邵邻相; 杜震宇

    2003-01-01

    用聚丙烯酰胺凝胶电泳的方法,对意蜂工蜂、雄蜂和蜂王以及两种不同大小幼虫之间的乳酸脱氢酶(LDH)和酯酶(EST)同工酶分别作了比较.结果表明,工蜂、雄蜂和蜂王之间LDH同工酶数目无差异,但活性上存在差异;而酯酶同工酶在幼虫阶段与成虫阶段及工蜂、雄蜂和蜂王之间则有着不同的同工酶谱型.说明意蜂同工酶在种内的表达受到发育程度、进食质量、分化方向等因素的影响.

  14. 常规方法测定LDH测量不确定度的评定%Evaluation of Uncertainty in Measurement of Detecting of LDH in Serum by Routine Method

    Institute of Scientific and Technical Information of China (English)

    郭玉红

    2014-01-01

    目的探讨测量不确定度评定在临床检验常规测量中的应用。方法以常规方法测定乳酸脱氢酶(LDH-L)为列,按测量不确定度评定过程,分析不确定度的来源并分别按A类和B类进行评定,合成标准不确定度,计算拓展不确定度。结果当LDH浓度为水平1时,A类不确定度UA=1.09,B类不确定度UB=3.32,合成标准不确定度UC=3.49,拓展不确定度U=6.84。当LDH浓度为水平2时,A类不确定度UA=0.58,B类不确定度UB=3.32,合成标准不确定度UC=3.37,拓展不确定度U=6.61。结论该研究只评估测量过程的不确定度,各实验室应参照叶测量不确定度表示指南》建立本实验室各项检测项目不确定度评定程序并进行评定,以确保实验结果的可靠性。%Objective To explore the application of evaluation for measurement uncertainty in routine clinical laboratory. Methods The routine test of lactate dehydrogenase (LDH) in serum was chosen as the example to evaluate the process of measurement uncertainty. The sources of uncertainty were identified, and the components of uncertainty were evaluated as type A and B respectively, combined standard uncertainty and calculating the expanded uncertainty. Results When the concentration of LDH is level one, the uncertainty of type A is 1.09, the uncertainty of type B is 3.32, the standard uncertainty is 3.49,the expanded uncertainty is 6.84. When the concentration of LDH is level two, the uncertainty of type A is 0.58, the uncertainty of type B is 3.32, the standard uncertainty is 3.37,the expanded uncertainty is 6.61. Conclusion This study only evaluates the measurement uncertainty during detection process, each laboratory should refer to the "Guide to the expression of uncertainty in measurement" established in their own test content's assessment procedures of measurement uncertainty and assessment it to ensure the reliability of the experimental results.

  15. Repercussões da L-alanil-glutamina sobre as concentrações de lactato e lactato desidrogenase (LDH em pacientes com isquemia crítica dos membros inferiores submetidos a revascularização distal Repercussions of l-alanyl-glutamine upon the concentrations of lactate and lactate dehydrogenase (LDH in patients with critical ischemia of lower limbs subjected to distal revascularization

    Directory of Open Access Journals (Sweden)

    Wellington Forte Alves

    2003-06-01

    Full Text Available OBJETIVO: Investigar efeitos da L-alanil-glutamina nas concentrações musculares de lactato, e nas concentrações sanguíneas de LDH, em pacientes com isquemia crítica dos membros inferiores submetidos à revascularização distal. MÉTODOS: Dezesseis adultos (12-homens/4-mulheres foram distribuídos em 2 grupos (1-controle/2-estudo. Três horas após injeção endovenosa de 250 ml de L-alanil-glutamina a 20% adicionados a 750 ml de soro fisiológico (Grupo 2, ou 1000 ml de solução salina (Grupo 1, iniciava-se a revascularização, sob raquianestesia. Amostras musculares e de sangue (arterial/venoso foram coletadas no início do procedimento (TI, no final (TF, e 10 e 20 minutos após isquemia (T1/T2. RESULTADOS: Observou-se redução significante (pPURPOSE: Investigate the repercussions of L-alanyl-glutamine in muscular tissue concentrations of lactate, and venous and arterial blood concentrations of LDH, in patients with critical ischemia of the lower limbs submitted to distal revascularization. METHODS: Sixteen adults (12 male/4 female were distributed in 2 groups (1-Control/2-Experiment. Three hours after the intravenous injection of 250 ml of a 20% solution of L-alanyl-glutamine added to 750 ml of saline solution (Group 2; or 1000 ml of saline solution (Group 1, distal bypass was carried out under spinal anesthesia. Muscle and blood samples (arterial/venous were collected at the beginning of the surgical procedure (TI, at the end (TF, and 10 and 20 minutes after re-establishment of blood flow. RESULTS: Significant reduction (p<0,05 of lactate concentration was observed in healthy muscle tissue in L-alanyl-glutamine treated patients in comparison to control group, at all times studied. There was a significant reduction (p <0,05 in venous concentrations of LDH in treated patients at all times studied (TI/TFV/T1V/T2V; and in arterial blood during reperfusion (T1A/T2A. CONCLUSIONS: 1. Decreased lactate concentrations in healthy skeletal

  16. Utility of NSE, ProGRP and LDH in Diagnosis and Treatment in Patients with Small Cell Lung Cancer%血清NSE、ProGRP和LDH在小细胞肺癌诊断治疗中的作用

    Institute of Scientific and Technical Information of China (English)

    彭彦; 王燕; 李峻岭; 郝学志; 胡兴胜

    2016-01-01

    Background and objective Small cell lung cancer (SCLC) is a rapidly growing tumor with character-istic of neuroendocrine cellular function. Neuron speciifc enolase (NSE), pro-gastrin-releasing peptide (ProGRP) and lactic dehydrogenase (LDH) are valuable in diagnosis and treatment of SCLC. By analyzing the variation of NSE, ProGRP and LDH before and atfer treatment, the aim of this study is to investigate the effcacy of tumor markers in diagnostic staging, therapeu-tic evaluation and prediction of disease relapsing.Methods Patients with SCLC who receiving the ifrst line chemotherapy in Cancer Hospital, Chinese Academy of Medical Sciences were enrolled and retrospectively analyzed. Clinical characteristic (includes NSE, ProGRP and LDH level before and atfer 2 cycles chemotherapy), effcacy evaluation, progression-free survival (PFS) were analyzed.Results Before treatment, Serum NSE, ProGRP and LDH in patients with extensive disease (ED) were signiifcantly higher than those with limited disease (LD)(allP4 cycles chemotherapy and with obvious decrease of ProGRP than those who accepted ≤4 cycles chemotherapy and with less obvious decrease of ProGRP in LD group; ED patients with no more than 2 distant metastasis, normal LDH level before treat-ment and obvious decrease of ProGRP atfer chemotherapy had lower short term relapse risk. In addition, the types of relapse (sensitive relapse, drug resistance relapse and refractory relapse) were negatively correlated with decrease of ProGRP (P=0.044). By multivariate analysis, numbers of chemotherapy cycle was independent prognostic factor for PFS in LD SCLC; numbers of distant metastasis and decrease of ProGRP were independent prognostic factors for PFS in ED SCLC.Conclusion Increase level of serum tumor markers is related to tumor burden. Decrease level of ProGRP atfer treatment may prognose effcacy and relapse risk.%背景与目的小细胞肺癌(small cell lung cancer, SCLC)是一种生长迅速、具有神经内分泌

  17. Assessment of two malaria rapid diagnostic tests in children under five years of age, with follow-up of false-positive pLDH test results, in a hyperendemic falciparum malaria area, Sierra Leone

    Directory of Open Access Journals (Sweden)

    De Smet Martin

    2010-01-01

    Full Text Available Abstract Background Most malaria rapid diagnostic tests (RDTs use HRP2 detection, including Paracheck-Pf®, but their utility is limited by persistent false positivity after treatment. PLDH-based tests become negative more quickly, but sensitivity has been reported below the recommended standard of 90%. A new pLDH test, CareStart™ three-line P.f/PAN-pLDH, claims better sensitivity with continued rapid conversion to negative. The study aims were to 1 compare sensitivity and specificity of CareStart™ to Paracheck-Pf® to diagnose falciparum malaria in children under five years of age, 2 assess how quickly false-positive CareStart™ tests become negative and 3 evaluate ease of use and inter-reader agreement of both tests. Methods Participants were included if they were aged between two and 59 months, presenting to a Médecins Sans Frontières community health centre in eastern Sierra Leone with suspected malaria defined as fever (axillary temperature > 37.5°C and/or history of fever in the previous 72 hours and no signs of severe disease. The same capillary blood was used for the RDTs and the blood slide, the latter used as the gold standard reference. All positive participants were treated with supervised artesunate and amodiaquine treatment for three days. Participants with a persistent false-positive CareStart™, but a negative blood slide on Day 2, were followed with repeated CareStart™ and blood slide tests every seven days until CareStart™ became negative or a maximum of 28 days. Results Sensitivity of CareStart™ was 99.4% (CI 96.8-100.0, 168/169 and of Paracheck-Pf®, 98.8% (95% CI 95.8-99.8, 167/169. Specificity of CareStart™ was 96.0% (CI 91.9-98.4, 167/174 and of Paracheck-Pf®, 74.7% (CI 67.6-81.0, 130/174 (p ® with excellent inter-reader agreement. Conclusions Both RDTs were highly sensitive, met WHO standards for the detection of falciparum malaria monoinfections where parasitaemia was >100 parasites/μl and were easy

  18. 北京地区汉族人群ALT、AST、GGT和LDH的参考区间研究%Reference intervals for ALT, AST, GGT and LDH among the Han Chinese in Beijing area

    Institute of Scientific and Technical Information of China (English)

    曾洁; 闫颖; 张传宝; 朱玲; 潘洁; 赵艳燕; 高建平; 申子瑜

    2011-01-01

    Objective To establish the reference intervals for ALT,AST,GGT and LDH among the Han nationality in Beijing.Methods The document C28-P3 issued by CLSI was a guideline about how to define,establish,and verify reference intervals in the clinical laboratory.IFCC had established multicenter enzymes reference intervals based on the guideline.Exclusion criteria were designed for screening candidate reference individual according to the document C28-P3 and the multicenter study's experience.Blood specimens were collected from 315 healthy individuals aged 20 to 60 years old,including 132 males and 183 females.Reference materials were used to ensure the accuracy of the test results of the four liver enzymes.The methods which used to test the four liver enzymes could be traced to the IFCC enzymes reference measure procedure,the reagent of ALT and AST included pyridoxal phosphate.Results There was statistically difference between males and females of the reference ranges for ALT, AST and GGT.Therefore,gender-specific reference intervals were established as ALT:8.2 -50.8 U/L (F),12.7 -71.8 U/L (M) ; AST:15.0 -36.7 U/L ( F),16.6 -51.1 U/L (M) ;GGT:9.0 -37.3 U/L (F),12.0 -50.9 U/L (M).For LDH,the reference interval was 127 -224 U/L,as no significant gender difference was found.Conclusions The reference intervals for the four liver enzymes based on the population of the Han nationality in Beijing are established.The upper reference limit for ALT in Beijing Han population is higher than that from other similar studies.%目的 建立北京地区汉族人群的ALT、AST、GGT和LDH的参考区间.方法 按照CLSI文件C28-P3《医学实验室参考区间的定义、建立和确认》中的要求,借鉴IFCC多中心酶学参考区间研究模式及参考人群筛选标准,于2009年筛选卫生部北京医院20~60岁健康体检者315名,其中男132名,女183名,用于上述各种酶参考区间的研究.使用酶学参考物质保证本次研究结果的准确性.上述4种酶

  19. Effect of anti-parasite drugs on the recombinant lactate dehydrogenase from Clonorchis senensis%4种驱虫药对重组华支睾乳酸脱氢酶(CsLDH)作用研究

    Institute of Scientific and Technical Information of China (English)

    黄灿; 胡旭初; 王乐旬; 吕刚; 余新炳; 徐劲

    2009-01-01

    目的 检测吡喹酮以及苯并咪唑类药物对重组的华支睾吸虫乳酸脱氢酶(CsLDH)酶促功能的影响,探讨其药物作用机制.方法 将不同浓度的吡喹酮、阿苯达唑、芬苯达唑和甲苯咪唑加入CsLDH所催化的丙酮酸还原成乳酸(正反应)以及乳酸氧化成丙酮酸(逆反应)的标准反应体系当中,紫外分光光度法测定NADH在A340处吸光度,SPSS16.0统计软件分析试验数据.结果 相比于对照组,9mmol/L的吡喹酮对正、逆反应的抑制均可达到94%(P<0.01);0.2 mmol/L的阿苯达唑对正、逆反应的抑制分别为84%(P<0.01)和79%(P <0.01),0.06 mmol/L的芬苯达唑对正、逆反应的抑制分别为92%(P<0.01)和86%(P<0.01),0.1 mmol/L的甲苯咪唑对正、逆反应分别为98%(P<0.01)和87%(P<0.01).结论 吡喹酮、阿苯达唑、芬苯达唑和甲苯咪唑可能以CsLDH为作用靶点杀伤华支睾吸虫而发挥治疗作用.

  20. PLA/MgAlCu-LDH复合材料的制备、表征及性能研究%Fabrication, Characterization and Properties of PLA/MgAlCu-LDH Composites Materials

    Institute of Scientific and Technical Information of China (English)

    张红梅; 朱同贺

    2015-01-01

    用溶液插层法制备了PLA/MgAlCu-LDH共混溶液,通过涂膜法制备了不同MgAlCu-LDH含量的PLA/MgAlCu-LDH复合膜,采用扫描电镜(SEM)、红外光谱(FTIR)、X射线衍射(XRD)、力学性能测试仪器对制备的复合膜的形貌、结构及性能进行测试表征.结果表明:部分PLA分子链插入到MgAlCu-LDH片层间隙中,使得MgAlCu-LDH层间距变大,MgAlCu-LDH作为插层剂对整个共混聚合物的韧性和强度有明显的影响;PLA/MgAl-Cu-LDH复合材料的杨氏模量随MgAlCu-LDH含量的增加而增加,加入少量MgAlCu-LDH时PLA/MgAlCu-LDH复合材料的抗拉强度、屈服强度和断裂伸长率均增加,在室温下拉伸时,PLA/MgAlCu-LDH 3%(质量分数)复合材料的抗拉强度、屈服强度和断裂伸长率分别比PLA提高14.5%、11.5%和23.7%.

  1. Effect of a C298D Mutation in CaHydA [FeFe]-Hydrogenase: Insights into the Protein-Metal Cluster Interaction by EPR and FTIR Spectroscopic Investigation

    Energy Technology Data Exchange (ETDEWEB)

    Morra, Simone; Maurelli, Sara; Chiesa, Mario; Mulder, David W.; Ratzloff, Michael W.; Giamello, Elio; King, Paul W.; Gilardi, Gianfranco; Valettia, Francesca

    2016-01-01

    A conserved cysteine located in the signature motif of the catalytic center (H-cluster) of [FeFe]-hydrogenases functions in proton transfer. This residue corresponds to C298 in Clostridium acetobutylicum CaHydA. Despite the chemical and structural difference, the mutant C298D retains fast catalytic activity, while replacement with any other amino acid caused significant activity loss. Given the proximity of C298 to the H-cluster, the effect of the C298D mutation on the catalytic center was studied by continuous wave (CW) and pulse electron paramagnetic resonance (EPR) and by Fourier transform infrared (FTIR) spectroscopies. Comparison of the C298D mutant with the wild type CaHydA by CW and pulse EPR showed that the electronic structure of the center is not altered. FTIR spectroscopy confirmed that absorption peak values observed in the mutant are virtually identical to those observed in the wild type, indicating that the H-cluster is not generally affected by the mutation. Significant differences were observed only in the inhibited state Hox-CO: the vibrational modes assigned to the COexo and Fed-CO in this state are shifted to lower values in C298D, suggesting different interaction of these ligands with the protein moiety when C298 is changed to D298. More relevant to the catalytic cycle, the redox equilibrium between the Hox and Hred states is modified by the mutation, causing a prevalence of the oxidized state. This work highlights how the interactions between the protein environment and the H-cluster, a dynamic closely interconnected system, can be engineered and studied in the perspective of designing bio-inspired catalysts and mimics.

  2. Favorable Protonation of the (μ-edt)[Fe(2)(PMe(3))(4)(CO)(2)(H-terminal)](+) Hydrogenase Model Complex Over Its Bridging μ-H Counterpart: A Spectroscopic and DFT Study.

    Science.gov (United States)

    Galinato, Mary Grace I; Whaley, C Matthew; Roberts, Dean; Wang, Peng; Lehnert, Nicolai

    2011-03-01

    The mechanism of hydrogen production in [FeFe] hydrogenase remains elusive. However, a species featuring a terminal hydride bound to the distal Fe is thought to be the key intermediate leading to hydrogen production. In this study, density functional theory (DFT) calculations on the terminal (H-term) and bridging (μ-H) hydride isomers of (μ-edt)-[Fe(2)(PMe(3))(4)(CO)(2)H](+) are presented in order to understand the factors affecting their propensity for protonation. Relative to H-term, μ-H is 12.7 kcal/mol more stable, which contributes to its decreased reactivity towards an acid. Potential energy surface (PES) calculations for the reaction of the H-term isomer with 4-nitropyridinium, a proton source, further reveal a lower activation energy barrier (14.5 kcal/mol) for H-term than for μ-H (29 kcal/mol). Besides these energetic considerations, the H-term isomer displays a key molecular orbital (MO ) that has a relatively strong hydride (1s) contribution (23%), which is not present in the μ-H isomer. This indicates a potential orbital control of the reaction of the hydride complexes with acid. The lower activation energy barrier and this key MO together control the overall catalytic activity of (μ-edt)[Fe(2)(PMe(3))(4)(CO)(2)(H-term)](+). Lastly, Raman and IR spectroscopy were performed in order to probe the ν(Fe-H) stretching mode of the two isomers and their deuterated counterparts. A ν(Fe-H) stretching mode was observed for the μ-H complex at 1220 cm(-1). However, the corresponding mode is not observed for the less stable H-term isomer.

  3. Synthesis and vibrational spectroscopy of 57Fe-labeled models of [NiFe] hydrogenase: first direct observation of a nickel–iron interaction† †Electronic supplementary information (ESI) available: Experimental procedures, spectral data, computational chemistry details, animated vibrational modes as GIFs. See DOI: 10.1039/c4cc04572f Click here for additional data file. Click here for additional data file.

    Science.gov (United States)

    Pelmenschikov, Vladimir; Wang, Hongxin; Meier, Florian; Gee, Leland B.; Yoda, Yoshitaka; Kaupp, Martin; Rauchfuss, Thomas B.

    2014-01-01

    A new route to iron carbonyls has enabled synthesis of 57Fe-labeled [NiFe] hydrogenase mimic (OC)3 57Fe(pdt)Ni(dppe). Its study by nuclear resonance vibrational spectroscopy revealed Ni–57Fe vibrations, as confirmed by calculations. The modes are absent for [(OC)3 57Fe(pdt)Ni(dppe)]+, which lacks Ni–57Fe bonding, underscoring the utility of the analyses in identifying metal–metal interactions. PMID:25237680

  4. 胸水ADA、LDH诊断结核性胸膜炎的临床效能评价%Evaluation on the clinical diagnosis efficiency of pleural fluid ADA and LDH for tuberculous pleuritis

    Institute of Scientific and Technical Information of China (English)

    李多孚; 陈郁琳; 夏雨

    2015-01-01

    目的 探讨胸水中腺苷脱氨酶(ADA)、乳酸脱氢酶(LDH)对鉴别结核性、癌性和其它疾病胸腔积液的临床价值.方法 回顾性分析行胸腔积液常规检测的住院患者的资料,其中结核患者38例、肿瘤患者74例、其它疾病患者108例.应用受试者工作特征(ROC)曲线确定胸腔积液ADA、LDH诊断结核性胸膜炎的最佳临界值,并计算ADA、LDH诊断结核性胸膜炎的临床诊断效能.结果 结核组、肿瘤组、其它疾病组ADA活性{中位数(M)[四分位间距(Q)]}分别为47.30 (26.50)、8.15(6.50)、5.40(8.40) U/L,各组间差异均有统计学意义(Z值分别为6.981、6.978、2.302,P均<0.05);LDH活性[M(Q)]分别为453.68(242.07)、252.00(368.00)、101.50(192.00) U/L,各组间差异均有统计学意义(Z值分别为2.419、5.386、4.324,p均<0.05).ROC曲线确定胸腔积液ADA诊断结核性胸膜炎的最佳临界值为26.7 U/L,灵敏度为89.5%、特异性为89.6%;LDH诊断结核性胸膜炎的最佳临界值为173.5 U/L,灵敏度为92.1%、特异性为54.4%;ADA和LDH联合检测的灵敏度为89.5%、特异性为54.1%.结论 胸腔积液ADA是诊断与鉴别结核性胸膜炎重要的指标,LDH特异性相对较低,但也有一定的参考意义.两者联合检测的临床诊断效能并不升高.

  5. The differential diagnosis value of CEA, CYFRA21-1, NSE and LDH in the blood and pleural fluid of patients with benign or malignant pleural effusion%CEA、CYFRA21-1、NSE、LDH在良恶性胸腔积液中的鉴别诊断价值

    Institute of Scientific and Technical Information of China (English)

    巩强进; 吕志; 吴丹; 胡先纬; 胡杰贵

    2012-01-01

    Objective To determine the differential diagnosis value of the tumor markers Carcinoembryonic Antigen( CEA ), Cy-tokeratin 19 fragment (CYFRA21-1 ),Neuron Specific Enolase( NSE ) and LDH in the blood and pleural fluid of patients with benign or malignant pleural effusion. Methods We prospectively evaluated the value of CEA, CYFRA21-1, NSE and LDH in the blood and pleural fluid of 108 patients with pleural effusion. Of which, 55 were diagnosed with malignant pleural effusion, and 53 were diagnosed with benign effusion. The optimum cut-off points resulting from the best sensitivity-specificity balance in the ROC curves were constructed. Results There were significant differences between malignant and benign cases for CEA and CYFRA21-1, in blood,and for CYFRA21-1、NSE、 CEA、LDH in pleural fluid . In the pleural fluid, the sensitivity and specificity of CEA, CYFRA21-1, NSE and LDH was 90. 9、63. 6、 72.7、36.4% and 98. K83、67. 9、88.1% .respectively. In the blood, the sensitivity and specificity was 89. K87. 3、32. K25. 5% and 92. 5、79. 2、90. 6、92. 5% for CEA, CYFRA21-1, NSE and LDH, respectively. Conclusions The results suggest that these markers might be useful in the differentiation between malignant and benign pleural effusion.%目的 探讨血清及胸腔积液中癌胚抗原(CEA)、细胞角蛋白19片段(CYFRA21-1)、神经特异性烯醇化酶(NSE)、乳酸脱氢酶(LDH)在良恶性胸腔积液鉴别诊断价值.方法 分析我院55例肺癌患者和53例良性胸腔积液患者的血清及胸腔积液中CEA、CYFRA21-1、NSE、LDH检测水平,并根据受试者工作特性(ROC)曲线建立合理的临床判断临界值及检测的敏感性和特异性.结果 恶性患者胸水CYFRA21-1、NSE、CEA、LDH的水平高于良性患者(P<0.05).胸水CEA、CYFRA21-1、NSE、LDH的敏感性和特异性分别为90.9、63.6、72.7、36.4%和98.1、83、67.9、88.7%.血清CEA、CYFRA21-1、NSE、LDH的敏感性和特异性分别为89.1、87.3 、32.1

  6. Effect of Water Pollution onActivitiy of CAT and LDH of Carassius auratus in Yongcheng Coal Collapse Area%永煤塌陷区水污染对鲫鱼过氧化氢酶和乳酸脱氢酶活性的影响

    Institute of Scientific and Technical Information of China (English)

    闫永峰; 郑娜; 李壹

    2011-01-01

    To investigate the influence of low concentration mixed heavy metal pollution in Yongcheng coal collapse area towards the activitiy of blood catalase (CAT) and serum lactate dehydrogenase(LDH), Carassius auratus of Yongcheng coal collapse area was selected as the research object and that from a relatively pollution-free Tianmu lake of Shangqiu was used as the control. The results showed that compared to Tianmu lake,the activity of blood CAT and LDH of Yongcheng coal collapse area decreased significantly (P<0.01). The result suggested that the normal physiological process was inhibited by low concentration mixed heavy metal pollution.%2010年5月,选择永城煤矿塌陷区天然鱼塘的野生鲫鱼(Carassius auratus)为对象,以相对无煤矿污染的商丘市天沐湖为对照,研究了永城煤矿塌陷区低浓度混合重金属污染对鲫鱼血液过氧化氢酶(CAT)和血清乳酸脱氢酶(LDH)活性的影响.结果表明,永城煤矿塌陷区鲫鱼CAT的OD值(0.600)极显著高于天沐湖鲫鱼CAT的OD值(0.411)(P<0.01),塌陷区鲤鱼血清中LDH的OD值(0.310)极显著低于天沐湖鲫鱼CAT的OD值(0.421)(P<0.01),说明煤矿塌陷区低浓度混合重金属污染对鲫鱼的正常生理过程有明显的抑制.

  7. Role of Layered Double Hydroxide (LDH) in the Protection of Herring Testis DNA from Heavy Metals%阴离子黏土(层状双氢氧化物)对鲱鱼精DNA在重金属作用下的保护作用研究

    Institute of Scientific and Technical Information of China (English)

    唐旖旎; 吴平霄; 朱能武

    2012-01-01

    用X射线衍射(XRD)、傅立叶红外光谱(FTIR)、扫描电镜(SEM)、循环伏安法、紫外光谱法等方法,研究了在重金属Cd2+、Pb2+作用下,阴离子黏土(LDH)对鲱鱼精DNA的保护作用.实验表明,LDH层间距由0.76 nm增大到2.30 nm,DNA-LDH复合体上出现了DNA分子中骨架和碱基上的CO(1 534 cm-1和1 488 cm-1),C—O伸缩振动峰(1 228 cm-1),P—O对称伸缩振动(1 096 cm-1),说明DNA已通过离子交换插入LDH的层间.然而DNA分子并没有完全交换出LDH层中NO3-(DNA部分嵌入),未交换到层间的DNA分子通过吸附作用固定于LDH表面上.循环伏安曲线显示,DNA-LDH复合体中的DNA还原峰与原始DNA保持一致,两个还原峰出现在EP=-1.2 mV和EP=-2.4 mV位置,也没有产生阴极凹陷.这表明当DNA固定在LDH上时,Cd2+、Pb2+就不能插入DNA沟槽之中,与碱基或其它基团的发生缔合作用.研究结果表明,一方面,LDH吸附溶液中的Cd2+、Pb2+于外表面上从而固定Cd2+、Pb2+,另一方面,LDH层板为DNA提供了保护空间,阻止Cd2+、Pb2+进入层间域与DNA作用从而保护DNA分子.%The role of layered double hydroxide(LDH) in the protection of herring testis DNA from heavy metals Cd2+and Pb2+ was studied by X-ray diffraction(XRD)spectra,Fourier transform infrared(FTIR)spectra,Scanning Electron Microscopy(SEM),Cyclic Voltammetry and Ultraviolet Spectrometry.Size expansion of the basal spacing(003) from 0.76 nm in LDH to 2.30 nm was observed in the resulting DNA-LDH nanohybrids and it gave peaks corresponding to CO(1 534 cm-1 and 1 488 cm-1) in skeleton and bases,C—O stretching vibration(1 228 cm-1),and P—O symmetrical stretching vibration(1 096 cm-1) in functional groups of DNA,indicating that DNA were intercalated into the LDH by the ion exchange.However,the displacement of NO-3 was not fully complete(partial intercalation of DNA).The DNA outside LDH interlayers was absorbed on the surface of

  8. Bi2MoO6/Ni-Fe LDH复合材料的制备及可见光催化性能%Preparation and Visible Light Responsive Photocatalytic Activity of Bi2MoO6/Ni-Fe LDH Composites

    Institute of Scientific and Technical Information of China (English)

    曲婷; 黄强; 赵振波

    2015-01-01

    采用水热法和共沉淀法结合制备Bi2MoO6/Ni-Fe LDH复合材料,通过XRD、FT-IR、SEM、TEM、XPS和N2物理吸附等对样品的结构和形貌进行表征。以甲基橙、亚甲基蓝、罗丹明 B和苯酚为目标降解物,在可见光下进行复合材料的光催化性能测试,以降解甲基橙溶液为例研究复合材料的光催化反应机理。结果表明,复合材料的BET比表面积随着Ni-Fe LDH含量的增加而增大,光催化活性明显提高。Bi2MoO6/Ni-Fe LDH复合材料中Ni-Fe LDH的含量为4.5%时具有最好的光催化效果,可见光照射60 min,甲基橙的降解率达91%,较Bi2MoO6和Ni-Fe LDH分别提高52%和16%。Bi2MoO6/Ni-Fe LDH复合材料具有良好的稳定性,循环使用5次,甲基橙(MO)的降解率为88%。复合材料光催化降解甲基橙反应遵循一级反应动力学。%Bi2MoO6/Ni-Fe LDH composites were prepared by hydrothermal method and co-precipitation. The morphology and structure of the sample were characterized by XRD, FT-IR, SEM, TEM, XPS and N2-physisorption. Photocatalytic degradation activity and mechanism of the samples were investigated by the photocatalytic degrada-tion of methyl orange (MO), methylene blue, butyl rhodamine B and phenol under visible light irradiation. The re-sults showed that BET specific surface area of the composites increased with the LDH content increase. Photocata-lytic degradation activity of MO under visible irradiation exhibited significant enhancement. After visible light ir-radiation for 60 min, the Bi2MoO6/Ni-Fe LDH composites with LDH content of 4.5wt% showed the highest degra-dation rate of 91%, higher than that of Bi2MoO6and Ni-Fe LDH by 52% and 16%, respectively. And the composites photocatalytic degradation followed first-order reaction kinetics. The composites decolorizing rate still remained 88% after 5 times recycle, showing high catalytic stability.

  9. 间歇低氧干预不影响实验性糖尿病大鼠骨骼肌SDH、LDH活性%Intermittent hypoxia did not have effect on the SDH and LDH activities of skeletal muscle in experimental diabetic rats

    Institute of Scientific and Technical Information of China (English)

    周燚; 方彩华; 李良鸣; 石家瑾

    2012-01-01

    目的:探讨间歇低氧干预对糖尿病大鼠骨骼肌糖代谢酶活性的影响。方法:采用长期高糖高脂膳食加小剂量链脲佐菌素(STZ)的方法建立糖尿病大鼠模型,将复制成功的模型大鼠随机分为模型对照组与低氧干预组。模型对照组在常氧环境下正常生活,低氧干预组大鼠每天在低氧帐篷内低氧暴露1h(O2浓度15.4±0.2%)、每周休息1d、共干预28d后36h取股四头肌测试琥珀酸脱氢酶(SDH)、乳酸脱氢酶(LDH)活性。结果:模型对照组大鼠股四头肌SDH活性显著低于正常对照组(P〈0.05),LDH活性高于正常对照组(P〉0.05),低氧干预组SDH、LDH活性与模型对照组比较均无明显差异(P〉0.05)。结论:实验性糖尿病大鼠骨骼肌SDH活性降低,有氧氧化供能能力下降,而LDH活性增强,无氧酵解能力提高;本实验所设计的间歇低氧干预模式对实验性糖尿病大鼠骨骼肌SDH、LDH活性均无明显积极作用。%Objective The purpose of this study was to investigate the effect of intermittent hypoxia on the SDH and LDH activities in skeletal muscle of diabetic rats. Methods The diabetic rat model was established by long-term high sugar and fat diet with low dose (30mg/kg body weight) of the streptozocin (STZ) by intraperitoneal injection only once. The diabetic rats were randomly divided into model control (MC) group and hypoxia interfering (HI) group. The MC rats were living in normal environment while the HI rats were exposed to hypoxia for one hour per day in hypoxic tent (oxygen concentration 15.4 ±0. 2 % ), six days a week for four weeks. The activities of SDH and LDH in the quadriceps femoris rats were measured. Results Compared with the rats in normal control group, the SDH activity reduced significantly ( P 〈 0. 05 ) and LDH activity increased only slightly in model control group. No significant differences of the

  10. Models of the iron-only hydrogenase: a comparison of chelate and bridge isomers of Fe2(CO)4{Ph2PN(R)PPh2}(μ-pdt) as proton-reduction catalysts.

    Science.gov (United States)

    Ghosh, Shishir; Hogarth, Graeme; Hollingsworth, Nathan; Holt, Katherine B; Richards, Idris; Richmond, Michael G; Sanchez, Ben E; Unwin, David

    2013-05-21

    the two metal centres based on the DFT calculations. The LUMO in the isomeric model compounds is similar in nature and is best described as an antibonding Fe-Fe interaction that contains differing amounts of aryl π* contributions from the ancillary PNP ligand. The proton reduction catalysis observed under electrochemical conditions at ca. -1.55 V is discussed as a function of the initial isomer and a mechanism that involves an initial protonation step involving the iron-iron bond. The measured CV currents were higher at this potential for the chelating complex, indicating faster turnover. Digital simulations showed that the faster rate of catalysis of the chelating complex can be traced to its greater propensity for protonation. This supports the theory that asymmetric distribution of electron density along the iron-iron bond leads to faster catalysis for models of the Fe-Fe hydrogenase active site.

  11. RNAi knock-down of LHCBM1, 2 and 3 increases photosynthetic H2 production efficiency of the green alga Chlamydomonas reinhardtii.

    Directory of Open Access Journals (Sweden)

    Melanie Oey

    Full Text Available Single cell green algae (microalgae are rapidly emerging as a platform for the production of sustainable fuels. Solar-driven H2 production from H2O theoretically provides the highest-efficiency route to fuel production in microalgae. This is because the H2-producing hydrogenase (HYDA is directly coupled to the photosynthetic electron transport chain, thereby eliminating downstream energetic losses associated with the synthesis of carbohydrate and oils (feedstocks for methane, ethanol and oil-based fuels. Here we report the simultaneous knock-down of three light-harvesting complex proteins (LHCMB1, 2 and 3 in the high H2-producing Chlamydomonas reinhardtii mutant Stm6Glc4 using an RNAi triple knock-down strategy. The resultant Stm6Glc4L01 mutant exhibited a light green phenotype, reduced expression of LHCBM1 (20.6% ±0.27%, LHCBM2 (81.2% ±0.037% and LHCBM3 (41.4% ±0.05% compared to 100% control levels, and improved light to H2 (180% and biomass (165% conversion efficiencies. The improved H2 production efficiency was achieved at increased solar flux densities (450 instead of ∼100 µE m(-2 s(-1 and high cell densities which are best suited for microalgae production as light is ideally the limiting factor. Our data suggests that the overall improved photon-to-H2 conversion efficiency is due to: 1 reduced loss of absorbed energy by non-photochemical quenching (fluorescence and heat losses near the photobioreactor surface; 2 improved light distribution in the reactor; 3 reduced photoinhibition; 4 early onset of HYDA expression and 5 reduction of O2-induced inhibition of HYDA. The Stm6Glc4L01 phenotype therefore provides important insights for the development of high-efficiency photobiological H2 production systems.

  12. The Comparison Analysis of the Albumin LDH,CEA in Pleural Effusion and Serum with Tuberculous Pleuritis%结核性胸膜炎胸液与血清的蛋白、LDH、CEA的含量对比分析

    Institute of Scientific and Technical Information of China (English)

    王枫

    2003-01-01

    目的:探讨结核性胸膜炎确诊的可靠率,精选确切的诊断指标.方法:对32例结核性胸膜炎患者通过首次胸液的蛋白、LDH、CEA及血清的蛋白、LDH、CEA的含量进行对比分析.结果:胸液与血清蛋白、LDH、CEA平均数比值分别为0.70(>0.5)、3.30(>0.6),0.75(血清中CEA高于胸液).胸液蛋白32例均>30(g/L);LDH 31例>200(u/L);均CEA14-0.1(μg/L)<15.结论:上述三项指标对于结核性胸膜炎的诊断有价值.

  13. Precipitation of Zn2Al LDH by urease enzyme.

    Science.gov (United States)

    Vial, Stephanie; Ghanbaja, Jaafar; Forano, Claude

    2006-01-21

    A biomineralization process based on the promotion of precipit