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Sample records for cis-trans isomerase involved

  1. Cellular peptidyl-prolyl cis/trans isomerase Pin1 facilitates replication of feline coronavirus.

    Science.gov (United States)

    Tanaka, Yoshikazu; Amano, Arisa; Morisaki, Masateru; Sato, Yuka; Sasaki, Takashi

    2016-02-01

    Although feline coronavirus (FCoV) causes feline infectious peritonitis (FIP), which is a fatal infectious disease, there are no effective therapeutic medicines or vaccines. Previously, in vitro studies have shown that cyclosporin (CsA) and FK506 inhibit virus replication in diverse coronaviruses. CsA and FK506 are targets of clinically relevant immunosuppressive drugs and bind to cellular cyclophilins (Cyps) or FK506 binding proteins (FKBPs), respectively. Both Cyp and FKBP have peptidyl-prolyl cis-trans isomerase (PPIase) activity. However, protein interacting with NIMA (Pin1), a member of the parvulin subfamily of PPIases that differs from Cyps and FKBPs, is essential for various signaling pathways. Here we demonstrated that genetic silencing or knockout of Pin1 resulted in decreased FCoV replication in vitro. Dipentamethylene thiuram monosulfide, a specific inhibitor of Pin1, inhibited FCoV replication. These data indicate that Pin1 modulates FCoV propagation.

  2. Identification and comparative analysis of sixteen fungal peptidyl-prolyl cis/trans isomerase repertoires

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    Pemberton Trevor J

    2006-09-01

    Full Text Available Abstract Background The peptidyl-prolyl cis/trans isomerase (PPIase class of proteins is present in all known eukaryotes, prokaryotes, and archaea, and it is comprised of three member families that share the ability to catalyze the cis/trans isomerisation of a prolyl bond. Some fungi have been used as model systems to investigate the role of PPIases within the cell, however how representative these repertoires are of other fungi or humans has not been fully investigated. Results PPIase numbers within these fungal repertoires appears associated with genome size and orthology between repertoires was found to be low. Phylogenetic analysis showed the single-domain FKBPs to evolve prior to the multi-domain FKBPs, whereas the multi-domain cyclophilins appear to evolve throughout cyclophilin evolution. A comparison of their known functions has identified, besides a common role within protein folding, multiple roles for the cyclophilins within pre-mRNA splicing and cellular signalling, and within transcription and cell cycle regulation for the parvulins. However, no such commonality was found with the FKBPs. Twelve of the 17 human cyclophilins and both human parvulins, but only one of the 13 human FKBPs, identified orthologues within these fungi. hPar14 orthologues were restricted to the Pezizomycotina fungi, and R. oryzae is unique in the known fungi in possessing an hCyp33 orthologue and a TPR-containing FKBP. The repertoires of Cryptococcus neoformans, Aspergillus fumigatus, and Aspergillus nidulans were found to exhibit the highest orthology to the human repertoire, and Saccharomyces cerevisiae one of the lowest. Conclusion Given this data, we would hypothesize that: (i the evolution of the fungal PPIases is driven, at least in part, by the size of the proteome, (ii evolutionary pressures differ both between the different PPIase families and the different fungi, and (iii whilst the cyclophilins and parvulins have evolved to perform conserved

  3. NMR assignments of the peptidyl-prolyl cis-trans isomerase domain of trigger factor from E. coli.

    Science.gov (United States)

    Huang, Chih-Ting; Hsu, Shang-Te Danny

    2016-04-01

    Trigger factor (TF) is a highly conserved multi-domain molecular chaperone in bacteria. It binds via its ribosome binding domain (RBD) to the ribosomal tunnel exit and facilitates co-translational folding of a broad range of protein substrates primarily through interactions with the substrate binding domain (SBD) adjacent to the RBD. Within the SBD, a peptidyl-prolyl cis-trans isomerase (PPIase) domain is inserted leading to an unusual domain insertion, which may provide stabilizing effect to the highly plastic SBD. Here we report the near complete NMR assignments of TF PPIase providing the basis for subsequent structural and folding in the context of the chaperone activity of TF.

  4. Penicillin-binding protein folding is dependent on the PrsA peptidyl-prolyl cis-trans isomerase in Bacillus subtilis

    NARCIS (Netherlands)

    Hyyrylainen, Hanne-Leena; Marciniak, Bogumila C.; Dahncke, Kathleen; Pietiainen, Milla; Courtin, Pascal; Vitikainen, Marika; Seppala, Raili; Otto, Andreas; Becher, Doerte; Chapot-Chartier, Marie-Pierre; Kuipers, Oscar P.; Kontinen, Vesa P.; Hyyryläinen, Hanne-Leena; Pietiäinen, Milla

    2010-01-01

    P>The PrsA protein is a membrane-anchored peptidyl-prolyl cis-trans isomerase in Bacillus subtilis and most other Gram-positive bacteria. It catalyses the post-translocational folding of exported proteins and is essential for normal growth of B. subtilis. We studied the mechanism behind this indispe

  5. Penicillin-binding protein folding is dependent on the PrsA peptidyl-prolyl cis-trans isomerase in Bacillus subtilis

    OpenAIRE

    Hyyryläinen, Hanne-Leena; Marciniak, Bogumila C.; Dahncke, Kathleen; Pietiäinen, Milla; Courtin, Pascal; Vitikainen, Marika; Seppala, Raili; Otto, Andreas; Becher, Dörte; Chapot-Chartier, Marie-Pierre; Oscar P. Kuipers; Kontinen, Vesa Pekka

    2010-01-01

    Abstract The PrsA protein is a membrane-anchored peptidyl-prolyl cis-trans isomerase in Bacillus subtilis and most other gram-positive bacteria. It catalyzes the post-translocational folding of exported proteins and is essential for normal growth of B. subtilis. We studied the mechanism behind this indispensability. We could construct a viable prsA null mutant in the presence of a high concentration of magnesium. Various changes in cell morphology in the absence of PrsA suggested t...

  6. Single-Domain Peptidyl-Prolyl cis/trans Isomerase FkpA from Corynebacterium glutamicum Improves the Biomass Yield at Increased Growth Temperatures.

    Science.gov (United States)

    Kallscheuer, Nicolai; Bott, Michael; van Ooyen, Jan; Polen, Tino

    2015-11-01

    Peptidyl-prolyl cis/trans isomerases (PPIases) catalyze the rate-limiting protein folding step at peptidyl bonds preceding proline residues and were found to be involved in several biological processes, including gene expression, signal transduction, and protein secretion. Representative enzymes were found in almost all sequenced genomes, including Corynebacterium glutamicum, a facultative anaerobic Gram-positive and industrial workhorse for the production of amino acids. In C. glutamicum, a predicted single-domain FK-506 (tacrolimus) binding protein (FKBP)-type PPIase (FkpA) is encoded directly downstream of gltA, which encodes citrate synthase (CS). This gene cluster is also present in other Actinobacteria. Here we carried out in vitro and in vivo experiments to study the function and influence of predicted FkpA in C. glutamicum. In vitro, FkpA indeed shows typical PPIase activity with artificial substrates and is inhibited by FK-506. Furthermore, FkpA delays the aggregation of CS, which is also inhibited by FK-506. Surprisingly, FkpA has a positive effect on the activity and temperature range of CS in vitro. Deletion of fkpA causes a 50% reduced biomass yield compared to that of the wild type when grown at 37°C, whereas there is only a 10% reduced biomass yield at the optimal growth temperature of 30°C accompanied by accumulation of 7 mM l-glutamate and 22 mM 2-oxoglutarate. Thus, FkpA may be exploited for improved product formation in biotechnical processes. Comparative transcriptome analysis revealed 69 genes which exhibit ≥2-fold mRNA level changes in C. glutamicum ΔfkpA, giving insight into the transcriptional response upon mild heat stress when FkpA is absent.

  7. Role of the cysteine residues in Arabidopsis thaliana cyclophilin CYP20-3 in peptidyl-prolyl cis-trans isomerase and redox-related functions.

    Science.gov (United States)

    Laxa, Miriam; König, Janine; Dietz, Karl-Josef; Kandlbinder, Andrea

    2007-01-01

    Cyps (cyclophilins) are ubiquitous proteins of the immunophilin superfamily with proposed functions in protein folding, protein degradation, stress response and signal transduction. Conserved cysteine residues further suggest a role in redox regulation. In order to get insight into the conformational change mechanism and functional properties of the chloroplast-located CYP20-3, site-directed mutagenized cysteine-->serine variants were generated and analysed for enzymatic and conformational properties under reducing and oxidizing conditions. Compared with the wild-type form, elimination of three out of the four cysteine residues decreased the catalytic efficiency of PPI (peptidyl-prolyl cis-trans isomerase) activity of the reduced CYP20-3, indicating a regulatory role of dithiol-disulfide transitions in protein function. Oxidation was accompanied by conformational changes with a predominant role in the structural rearrangement of the disulfide bridge formed between Cys(54) and Cys(171). The rather negative E(m) (midpoint redox potential) of -319 mV places CYP20-3 into the redox hierarchy of the chloroplast, suggesting the activation of CYP20-3 in the light under conditions of limited acceptor availability for photosynthesis as realized under environmental stress. Chloroplast Prx (peroxiredoxins) were identified as interacting partners of CYP20-3 in a DNA-protection assay. A catalytic role in the reduction of 2-Cys PrxA and 2-Cys PrxB was assigned to Cys(129) and Cys(171). In addition, it was shown that the isomerization and disulfide-reduction activities are two independent functions of CYP20-3 that both are regulated by the redox state of its active centre.

  8. An Intracellular Peptidyl-Prolyl cis/trans Isomerase Is Required for Folding and Activity of the Staphylococcus aureus Secreted Virulence Factor Nuclease.

    Science.gov (United States)

    Wiemels, Richard E; Cech, Stephanie M; Meyer, Nikki M; Burke, Caleb A; Weiss, Andy; Parks, Anastacia R; Shaw, Lindsey N; Carroll, Ronan K

    2017-01-01

    Staphylococcus aureus is an important human pathogen that relies on a large repertoire of secreted and cell wall-associated proteins for pathogenesis. Consequently, the ability of the organism to cause disease is absolutely dependent on its ability to synthesize and successfully secrete these proteins. In this study, we investigate the role of peptidyl-prolyl cis/trans isomerases (PPIases) on the activity of the S. aureus secreted virulence factor nuclease (Nuc). We identify a staphylococcal cyclophilin-type PPIase (PpiB) that is required for optimal activity of Nuc. Disruption of ppiB results in decreased nuclease activity in culture supernatants; however, the levels of Nuc protein are not altered, suggesting that the decrease in activity results from misfolding of Nuc in the absence of PpiB. We go on to demonstrate that PpiB exhibits PPIase activity in vitro, is localized to the bacterial cytosol, and directly interacts with Nuc in vitro to accelerate the rate of Nuc refolding. Finally, we demonstrate an additional role for PpiB in S. aureus hemolysis and demonstrate that the S. aureus parvulin-type PPIase PrsA also plays a role in the activity of secreted virulence factors. The deletion of prsA leads to a decrease in secreted protease and phospholipase activity, similar to that observed in other Gram-positive pathogens. Together, these results demonstrate, for the first time to our knowledge, that PPIases play an important role in the secretion of virulence factors in S. aureus IMPORTANCE: Staphylococcus aureus is a highly dangerous bacterial pathogen capable of causing a variety of infections throughout the human body. The ability of S. aureus to cause disease is largely due to an extensive repertoire of secreted and cell wall-associated proteins, including adhesins, toxins, exoenzymes, and superantigens. These virulence factors, once produced, are typically transported across the cell membrane by the secretory (Sec) system in a denatured state. Consequently

  9. Aluminum(III) interferes with the structure and the activity of the peptidyl-prolyl cis-trans isomerase (Pin1): a new mechanism contributing to the pathogenesis of Alzheimer's disease and cancers?

    Science.gov (United States)

    Wang, Jing-Zhang; Liu, Ji; Lin, Tao; Han, Yong-Guang; Luo, Yue; Xi, Lei; Du, Lin-Fang

    2013-09-01

    The enzyme peptidyl-prolyl cis-trans isomerase (Pin1) may play an important role in preventing the development of Alzheimer's disease (AD). The structural and functional stability of Pin1 is extremely important. Previously, we have determined the stability of Pin1 under stressed conditions, such as thermal treatment and acidic-pH. Considering that aluminum (Al(III)) is well known for its potential neurotoxicity in the pathogenesis of AD, we examined whether Al(III) affects the structure and function of Pin1, by means of a PPIase activity assay, intrinsic fluorescence, circular dichroism (CD) spectroscopy, FTIR, and differential scanning calorimetry (DSC). The intrinsic tryptophan fluorescence measurements mainly show that Al(III) may bind to the clusters nearby W11 and W34 in the WW domain of Pin1, quenching the intrinsic fluorescence of the two tryptophan residues, which possibly results in the decreased binding affinity of Pin1 to substrates. The secondary structural analysis as revealed by FTIR and CD measurements indicate that Al(III) induces the increase in β-sheet and the decrease in α-helix in Pin1. Furthermore, the changes of the thermodynamic parameters for Pin1 as monitored by DSC confirm that the thermal stability of Pin1 significantly increases in the presence of Al(III). The Al(III)-induced structural changes of Pin1 result in a sharp decrease of the PPIase activity of Pin1. To some extent, our research is suggestive that Al(III) may inhibit the isomerization activity of Pin1 in vivo, which may contribute to the pathogenesis of AD.

  10. The intriguing Cyclophilin A-HIV-1 Vpr interaction: prolyl cis/trans isomerisation catalysis and specific binding

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    Henklein Petra

    2010-10-01

    Full Text Available Abstract Background Cyclophilin A (CypA represents a potential target for antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1 replication, although the mechanism through which CypA modulates HIV-1 infectivity still remains unclear. The interaction of HIV-1 viral protein R (Vpr with the human peptidyl prolyl isomerase CypA is known to occur in vitro and in vivo. However, the nature of the interaction of CypA with Pro-35 of N-terminal Vpr has remained undefined. Results Characterization of the interactions of human CypA with N-terminal peptides of HIV-1 Vpr has been achieved using a combination of nuclear magnetic resonace (NMR exchange spectroscopy and surface plasmon resonance spectroscopy (SPR. NMR data at atomic resolution indicate prolyl cis/trans isomerisation of the highly conserved proline residues Pro-5, -10, -14 and -35 of Vpr are catalyzed by human CypA and require only very low concentrations of the isomerase relative to that of the peptide substrates. Of the N-terminal peptides of Vpr only those containing Pro-35 bind to CypA in a biosensor assay. SPR studies of specific N-terminal peptides with decreasing numbers of residues revealed that a seven-residue motif centred at Pro-35 consisting of RHFPRIW, which under membrane-like solution conditions comprises the loop region connecting helix 1 and 2 of Vpr and the two terminal residues of helix 1, is sufficient to maintain strong specific binding. Conclusions Only N-terminal peptides of Vpr containing Pro-35, which appears to be vital for manifold functions of Vpr, bind to CypA in a biosensor assay. This indicates that Pro-35 is essential for a specific CypA-Vpr binding interaction, in contrast to the general prolyl cis/trans isomerisation observed for all proline residues of Vpr, which only involve transient enzyme-substrate interactions. Previously suggested models depicting CypA as a chaperone that plays a role in HIV-1 virulence are

  11. Structural characterization of the HIV-1 Vpr N terminus: evidence of cis/trans-proline isomerism.

    Science.gov (United States)

    Bruns, Karsten; Fossen, Torgils; Wray, Victor; Henklein, Peter; Tessmer, Uwe; Schubert, Ulrich

    2003-10-31

    The 96-residue human immunodeficiency virus (HIV) accessory protein Vpr serves manifold functions in the retroviral life cycle including augmentation of viral replication in non-dividing host cells, induction of G2 cell cycle arrest, and modulation of HIV-induced apoptosis. Using a combination of dynamic light scattering, circular dichroism, and NMR spectroscopy the N terminus of Vpr is shown to be a unique domain of the molecule that behaves differently from the C-terminal domain in terms of self-association and secondary structure folding. Interestingly, the four highly conserved proline residues in the N terminus are predicted to have a high propensity for cis/trans isomerism. Thus the high resolution structure and folding of a synthetic N-terminal peptide (Vpr1-40) and smaller fragments thereof have been investigated. 1H NMR data indicate Vpr1-40 possesses helical structure between residues 17-32, and for the first time, this helix, which is bound by proline residues, was observed even in aqueous solution devoid of any detergent supplements. In addition, NMR data revealed that all of the proline residues undergo a cis/ trans isomerism to such an extent that approximately 40% of all Vpr molecules possess at least one proline in a cis conformation. This phenomenon of cis/trans isomerism, which is unprecedented for HIV-1 Vpr, not only provides an explanation for the molecular heterogeneity observed in the full-length molecule but also indicates that in vivo the folding and function of Vpr should depend on a cis/trans-proline isomerase activity, particularly as two of the proline residues in positions 14 and 35 show considerable amounts of cis isomers. This prediction correlates well with our recent observation (Zander, K., Sherman, M. P., Tessmer, U., Bruns, K., Wray, V., Prechtel, A. T., Schubert, E., Henklein, P., Luban, J., Neidleman, J., Greene, W. C., and Schubert, U. (2003) J. Biol. Chem. 278, 43170-43181) of a functional interaction between the major

  12. Semiconductor photocatalysis. Cis-trans photoisomerization of simple alkenes induced by trapped holes at surface states

    Energy Technology Data Exchange (ETDEWEB)

    Yanagida, S.; Mizumoto, K.; Pac, C.

    1986-02-19

    The use of ZnS or CdS as photocatalysts induces an efficient cis-trans photoisomerization of simple alkenes, e.g., the 2-octenes, 3-hexen-1-ols, and methyl 9-octadecenoates in photostationary cis-trans ratios almost identical with the thermodynamic equilibrium ratios achieved by the phenylthio radical. Quantum yields for the cis-trans photoisomerization, phi/sub c-t/, exceed largely over unity. Mechanistic studies involving Stern-Volmer analyses, quenching effect of oxygen, and ESR analyses under band-gap irradiation of ZnS in methanol demonstrate that the photoisomerizations take place with high turnover numbers at active sites where trapped holes at surface states, i.e., sulfur radicals arising from Zn vacancies and/or interstitial sulfur on sulfide semiconductors, play decisive roles. A highly efficient catalysis occurs with ZnS sols prepared from polysulfide-containing Na/sub 2/S solution. The trapped-hole mechanism is further supported by the enhanced effect of water acting as a good electron acceptor as well as the quenching effect of diethylamine acting as an electron donor.

  13. Graphene oxide catalyzed cis-trans isomerization of azobenzene

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    Dongha Shin

    2014-09-01

    Full Text Available We report the fast cis-trans isomerization of an amine-substituted azobenzene catalyzed by graphene oxide (GO, where the amine functionality facilitates the charge transfer from azobenzene to graphene oxide in contrast to non-substituted azobenzene. This catalytic effect was not observed in stilbene analogues, which strongly supports the existence of different isomerization pathways between azobenzene and stilbene. The graphene oxide catalyzed isomerization is expected to be useful as a new photoisomerization based sensing platform complementary to GO-based fluorescence quenching methods.

  14. Synthesis of novel biscrown ethers with rigid cis/trans ethylene bridge

    Institute of Scientific and Technical Information of China (English)

    Jian Quan Fan; La Sheng Jiang; Min Zhang; Na Wei; Ya Hui Feng; Shu Juan Han; Hui Wang

    2008-01-01

    Two novel biscrown ethers with rigid cisltrans ethylene linker were synthesized v/a Wittig reaction in high yield (about 80%).Their pure cis/trans-isomers were obtained by column chromatography separation. And their structure/configuration was confirmedby 1H NMR, 13C NMR, ESI mass spectrum, elemental analysis and UV-vis spectra.? 2008 La Sheng Jiang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

  15. Photosensitized cis/trans isomerization of 1-(1-propenyl)cycloalkenes

    Energy Technology Data Exchange (ETDEWEB)

    Inman, W.D.; Sanchez, K.A.J.; Chaidez, M.A.; Paulson, D.R. (California State Univ., Los Angeles (USA))

    1989-09-29

    The photosensitized cis/trans isomerization of a series of 1-(1-propenyl)cycloalkenes is reported. A plot of the photostationary state trans/cis ratio vs the sensitizer triplet energy for 1-(1-propenyl)cyclopentene shows a constant trans/cis ratio of {approx} 1.0 with high-energy sensitizers (E{sub T} > 61 kcal/mol). The plot shows one maxima at E{sub T} {approx} 55 kcal/mol with low-energy sensitizers (E{sub T} < 61 kcal/mol). This type of plot is very similar to those obtained with acyclic dienes such as piperylene. The 1-(1-propenyl)cyclohexene system shows a similar plot with high-energy sensitizers, but with low-energy sensitizers this system shows two maxima occurring at 56 and 47 kcal/mol, respectively. This double-maxima plot is rationalized by an unusually low trans/cis decay ratio for the s-cis relaxed triplet state of the 1-(1-propenyl)cyclohexene system. This double maxima is not observed in other diene systems due to a high trans/cis decay ratio for their s-cis relaxed triplet states. The photosensitized cis/trans isomerization of 2-ethylidene-10-methyl-1(9)-octalin was also studied as a model for a conformationally locked s-trans system. The 1-(1-propenyl)cycloheptene system undergoes photosensitized cis/trans isomerization, but photostationary cis/trans isomerization data could not be obtained due to a very efficient photosensitized dimerization of this diene system.

  16. Discrimination of cis-trans sex pheromone components in two sympatric Lepidopteran species.

    Science.gov (United States)

    Zhang, Sufang; Kong, Xiangbo; Ze, Sangzi; Wang, Hongbin; Lin, Aizhu; Liu, Fu; Zhang, Zhen

    2016-06-01

    Pheromone-binding proteins (PBPs) play an important role in the recognition of pheromones by insects. However, the abilities of these PBPs to discriminate pheromone components and recognize the isomers are unclear. Dendrolimus houi and Dendrolimus kikuchii are two sympatric coniferous pests whose pheromones have cis-trans isomers. We used these insect species to detect the precise recognition abilities of PBPs. The four PBPs examined showed male-biased antenna-intensive expression patterns, whereas PBP1 showed higher expression than PBP2 in the antenna. DhouPBP1 only bound to a minor interspecific pheromone component, whereas DhouPBP2 bound to all three intraspecific components and another minor interspecific component. DkikPBP1 and DkikPBP2 could recognize all three intraspecific components with affinities negatively correlated with their ratios, and they bound to interspecific pheromones with affinity that was positively correlated with the ratios. The four PBPs have different cis-trans isomer discrimination abilities, i.e., DhouPBP1 and DkikPBP1 could not discriminate the two cis-trans isomer pairs of pheromones from the two species, whereas DhouPBP2 could discriminate between both pairs, and DkikPBP2 could only discriminate one pair. Overall, PBPs from D. houi and D. kikuchii use different strategies to help the moths to discriminate the intra- and interspecific pheromone components. Our work will contribute to better understanding of the sex pheromone recognition mechanism in these two sister species of moths and provide insights into more effective management practices of these pest species.

  17. Robust trans-amide helical structure of oligomers of bicyclic mimics of β-proline: impact of positional switching of bridgehead substituent on amide cis-trans equilibrium.

    Science.gov (United States)

    Wang, Siyuan; Otani, Yuko; Liu, Xin; Kawahata, Masatoshi; Yamaguchi, Kentaro; Ohwada, Tomohiko

    2014-06-06

    Because homooligomers of 7-azabicyclo[2.2.1]heptane-2-endo-carboxylic acid, a bridged β-proline analogue with a substituent installed at the remote C4-bridgehead position, completely biased the amide cis-trans equilibrium to the cis-amide structure, we expected that introduction of a substituent at the C1-bridgehead position adjacent to the carboxylic acid moiety, rather than the remote C4-bridgehead position, would tip the cis-trans amide equilibrium toward trans-amide structure without the aid of hydrogen bonding. Thus, in this work we established an efficient synthetic route to an optically active bicyclic analogue of 1,1-disubstituted β-proline, bearing a substituent at the C1-bridgehead position. Crystallographic, spectroscopic, and computational studies showed that indeed oligomers of this analogue take a consistent helical structure involving all-trans-amide linkages, independently of the number of residues, from the dimer up to the octamer. Oligomers composed of (R)-β-amino acid units form an extended left-handed helix with about 2.7 residues per turn and an approximately 4.0 Å rise per residue, characterized by complete lack of main-chain hydrogen bonding. This unique helical structure shows some similarity in shape to the trans-amide-based polyproline II (PPII) helix. The present helix was stable in various kinds of solvents such as alcohols. The present work provided a fundamental structural basis for future applications.

  18. Cis-trans isomerisation of azobenzenes studied by laser-coupled NMR spectroscopy and DFT calculations.

    Science.gov (United States)

    Wazzan, Nuha A; Richardson, Patricia R; Jones, Anita C

    2010-07-30

    In a combined experimental and computational study of a group of para-substituted azobenzenes, the effects of substituents and solvent on the kinetics of thermal cis-to-trans isomerisation have been examined and the success of DFT calculations in predicting kinetic parameters assessed. Mono-substituted species are predicted to isomerise by inversion in both non-polar and polar solvent, whereas for push-pull azobenzenes the mechanism is predicted to change from inversion to rotation on going from non-polar to polar solvent. Computed free energies of activation qualitatively reproduce experimental trends but do not quantitatively predict the kinetics of cis-trans isomerisation. The polarisable continuum model of solvation fails to predict the experimentally observed influence of solvent on the entropy of activation.

  19. How Is cis-trans Isomerization Controlled in Dronpa Mutants? A Replica Exchange Molecular Dynamics Study.

    Science.gov (United States)

    Moors, Samuel L C; Michielssens, Servaas; Flors, Cristina; Dedecker, Peter; Hofkens, Johan; Ceulemans, Arnout

    2008-06-01

    The reversibly photoactivatable green fluorescent protein analog Dronpa holds great promise as a marker for various new cellular imaging applications. Using a replica exchange method which combines both Hamiltonian and temperature exchanges, the ground-state dynamics of Dronpa and two mutants with increased switching kinetics, Val157Gly and Met159Thr, were compared. The dominant chromophore state was found to be the cis isomer in all three proteins. The simulation data suggest that both mutations strongly increase the chromophore flexibility and cis-trans isomerization rate. We identify three key amino acids, Val157, Met159, and Phe173, which are able to impede the bottom hula-twist transition path, depending on their position and rotameric state. We believe our insights will help to understand the switching process and provide useful information for the design of new variants with improved fluorescence properties.

  20. A mixed-valent cyclodiphosphazane: Transition metal chemistry and cis/trans isomerisation

    Indian Academy of Sciences (India)

    Guddekoppa S Ananthnag; Joel T Mague; Maravanji S Balakrishna

    2015-06-01

    The hydrolysis of cis-{ClP(-NBu)2P(NHBu)} (1) produced a mixed PIII/PV derivative of cyclodiphosphazane, cis-{(BuNH)P(-NBu)2P(O)H} (2). The treatment of 2 with elemental selenium resulted in the formation of the monoselenide, trans-{(BuNH)P(Se)(-NBu)2P(O)H} (3) in good yield. The reactions of two equivalent of 2 with [Pd(-Cl)(3-C3H5)]2 or [Ru(6--cymene)(-Cl)Cl]2 in dichloromethane afforded corresponding mononuclear complexes, [(3-C3H5)PdCl{(BuNH)P(-NBu)2P(O)H}] (4) and [((6--cymene)RuCl2){(BuNH)P(-NBu)2P(O)H}] (5). The treatment of 2 with M(COD)Cl2 (M = Pd and Pt) in dichloromethane at room temperature gave [MCl2{(BuNH)P(-NBu)2P(O)H}2] (6 M = Pd; 7 M = Pt) in good yield. Owing to the cis/trans isomerisation of the cyclodiphosphazane rings, the complexes 6 and 7 exist as a mixture of two isomers. Various NMR spectroscopic techniques were employed for structural elucidation. The molecular structures of 5 and 7 were established by single crystal X-ray crystallographic studies.

  1. Quantum Yields from Stationary States: Cis-Trans Isomerization of Model Retinal

    CERN Document Server

    Tscherbul, T V

    2014-01-01

    Cis-trans isomerization in retinal, the first step in vision, is often computationally studied from a time dependent viewpoint. Motivation for such studies lies in coherent pulsed laser experiments that explore the isomerization dynamics. However, such biological processes take place naturally in the presence of incoherent light, which excites a non-evolving mixture of stationary states. Here the isomerization problem is considered from the latter viewpoint and applied to a standard two-state, two-mode linear vibronic coupling model of retinal that explicitly includes a conical intersection between the ground and first excited electronic states. The calculated quantum yield at 500 nm agrees well with both the previous time-dependent calculations of Hahn and Stock (0.63) and with experiment ($0.65\\pm0.01$), as does its wavelength dependence. Significantly, the effects of environmental relaxation on the quantum yield in this well-established model are found to be negligible. The results make clear the connectio...

  2. Separation of cis/trans fatty acid isomers on gas chromatography compared to the Ag-TLC method

    Energy Technology Data Exchange (ETDEWEB)

    Ravi Kiran, C.; Reshma, M. V.; Sundaresan, A.

    2013-05-01

    The present study investigates the separation of the cis/ trans isomers of fatty acids on the 75 m SP2560 column under different gas chromatographic (GC) conditions including an isothermal program and a time-temperature program. The time-temperature program showed improved separation of cis/trans isomers of C{sub 1}4:1, C{sub 1}6:1, C{sub 1}8:1, C{sub 1}8:2 and C{sub 1}8:3 fatty acids along with short chain fatty acids compared to the isothermal program. The separation of trans/trans isomers of C{sub 1}8:1 fatty acids including elaidic acid (C{sub 1}8:1 .9t) and vaccenic acid (C{sub 1}8:1 {Delta}11t) was difficult with the time-temperature program. The thin layer chromatography impregnated with silver nitrate (Ag- TLC) method was performed to separate cis/trans fractions and GC analysis was carried out with the trans fraction. But GC analysis showed a co-elution of trans isomers of C{sub 1}8:1 fatty acid. Thus the study shows that a time-temperature programmed GC method with the highly polar cyanopropyl column is sufficient to resolve trans fatty acids along with short chain fatty acids when a large number of samples has to be analyzed. (Author) 33 refs.

  3. Peptidyl prolyl isomerase Pin1-inhibitory activity of D-glutamic and D-aspartic acid derivatives bearing a cyclic aliphatic amine moiety.

    Science.gov (United States)

    Nakagawa, Hidehiko; Seike, Suguru; Sugimoto, Masatoshi; Ieda, Naoya; Kawaguchi, Mitsuyasu; Suzuki, Takayoshi; Miyata, Naoki

    2015-12-01

    Pin1 is a peptidyl prolyl isomerase that specifically catalyzes cis-trans isomerization of phosphorylated Thr/Ser-Pro peptide bonds in substrate proteins and peptides. Pin1 is involved in many important cellular processes, including cancer progression, so it is a potential target of cancer therapy. We designed and synthesized a novel series of Pin1 inhibitors based on a glutamic acid or aspartic acid scaffold bearing an aromatic moiety to provide a hydrophobic surface and a cyclic aliphatic amine moiety with affinity for the proline-binding site of Pin1. Glutamic acid derivatives bearing cycloalkylamino and phenylthiazole groups showed potent Pin1-inhibitory activity comparable with that of known inhibitor VER-1. The results indicate that steric interaction of the cyclic alkyl amine moiety with binding site residues plays a key role in enhancing Pin1-inhibitory activity.

  4. Red- and blue-shifted hydrogen bonds in the cis-trans noncyclic formic acid dimer.

    Science.gov (United States)

    Zhou, Pan-Pan; Qiu, Wen-Yuan

    2009-08-01

    The cis-trans noncyclic formic acid dimer was studied by means of MP2 method with 6-31G(d,p), 6-31+G(d,p) and 6-311+G(d,p) basis sets. It exhibits simultaneously red-shifted O-H...O and blue-shifted C-H...O hydrogen bonds. AIM and NBO analyses are performed at the MP2/6-31+G(d,p) level to explore their properties and origins. AIM analysis provides the evidence that the O-H bond becomes weaker and the C-H bond becomes stronger upon the hydrogen bond formations. Intermolecular and intramolecular hyperconjugations have important influence on the electron densities in the X-H (X = O, C) sigma bonding orbital and its sigma* antibonding orbital. The electron densities in the two orbitals are closely connected with the X-H (X = O, C) bond length, and they are used to quantitatively estimate the bond length variation. The larger amount of charge transfer in the red-shifted O-H...O hydrogen bond is due to its favorable H...O electron channel, whereas the H...O electron channel in the blue-shifted C-H...O hydrogen bond is weaker. Structural reorganization effects shorten the C-H bond by approximately 30% when compared to the C-H bond contraction upon the dimerization. Strikingly, it leads to a small elongation and a slight red shift of the O-H bond. Both rehybridization and repolarization result in the X-H (X = O, C) bond contraction, but their effects on the O-H bond do not hold a dominant position. The hydrogen-bonding processes go through the electrostatic attractions, van der Waals interactions, charge-transfer interactions, hydrogen-bonding interactions and electrostatic repulsions. Electrostatic attractions are of great importance on the origin of the red-shifted O-H...O hydrogen bond, especially the strong H(delta+)...O(delta-) attraction. For the blue-shifted C-H...O hydrogen bond, the considerable nucleus-nucleus repulsion between H and O atoms caused by the strong electrostatic attraction between C and O atoms is a possible reason for the C-H bond contraction and

  5. Separation of cis/trans fatty acid isomers on gas chromatography compared to the Ag-TLC method

    Directory of Open Access Journals (Sweden)

    Sundaresan, A.

    2013-03-01

    Full Text Available The present study investigates the separation of the cis/trans isomers of fatty acids on the 75 m SP2560 column under different gas chromatographic (GC conditions including an isothermal program and a time-temperature program. The time-temperature program showed improved separation of cis/trans isomers of C14:1, C16:1, C18:1, C18:2 and C18:3 fatty acids along with short chain fatty acids compared to the isothermal program. The separation of trans/trans isomers of C18:1 fatty acids including elaidic acid (C18:1 ∆9t and vaccenic acid (C18:1 ∆11t was difficult with the time-temperature program. The thin layer chromatography impregnated with silver nitrate (Ag- TLC method was performed to separate cis/trans fractions and GC analysis was carried out with the trans fraction. But GC analysis showed a co-elution of trans isomers of C18:1 fatty acid. Thus the study shows that a time-temperature programmed GC method with the highly polar cyanopropyl column is sufficient to resolve trans fatty acids along with short chain fatty acids when a large number of samples has to be analyzed.El presente estudio investiga la separación de isómeros cis/trans de ácidos grasos mediante cromatografía de gases (GC utilizando una columna de SP2560 de 75 m y diferentes condiciones que incluyen un programa isotérmico y una programación temperatura-tiempo. La programación temperatura- tiempo mostró una mejor separación de isómeros cis / trans de los ácidos grasos C14:1, C16:1, C18:1, C18:2 y C18:3 con los ácidos grasos de cadena corta en comparación con el programa isotermo. La separación de los isómeros trans/trans de los ácidos grasos C18:1 incluyendo ácido elaídico (C18:1 Δ9t y ácido vaccénico (C18:1Δ11t fué difícil mediante programación temperatura-tiempo. La cromatografía en capa fina impregnada con nitrato de plata (Ag-TLC se realizó para separar fracciones cis/trans y el análisis de la fracciones se llevó a cabo mediante GC. El an

  6. Study on the thermal decomposition kinetics of cis/trans-[Cu(gly)2]·H2O

    Institute of Scientific and Technical Information of China (English)

    ZHANG Fengxing; LI Jun; LI Hengxin; DU Guixiang; SONG Shuqing

    2005-01-01

    The thermal decomposition reactions of the complexes cis/trans-[Cu(gly)2]· H2Owere studied by TG-DSC methods. The results showed that they have similar decomposition process, which occur in two steps. The first step is the loss of water and the second step is the decomposition of anhydrous complexes.But for cis-[Cu(gly)2]· H2O,the tempoerature of losing water is higher than that of trans-isomer. Their reaction mechanisms of the two-step decomposition were also proposed.

  7. Mass-spectrometry-directed analysis and purification of pyrrolizidine alkaloid cis/trans isomers in Gynura japonica.

    Science.gov (United States)

    Fang, Lianxiang; Xiong, Aizhen; Yang, Xiao; Cheng, Wenzhi; Yang, Li; Wang, Zhengtao

    2014-08-01

    Pyrrolizidine alkaloids are highly hepatotoxic natural chemicals that produce irreversible chronic and acute hepatotoxic effects on human beings. Purification of large amounts of pyrrolizidine alkaloids is necessary for toxicity studies. In this study, an efficient method for targeted analysis and purification of pyrrolizidine alkaloid cis/trans isomers from herbal materials was developed for the first time. Targeted analysis of the hepatotoxic pyrrolizidine alkaloids was performed by liquid chromatography with tandem mass spectrometry (precursor ion scan and daughter ion scan), and the purification of pyrrolizidine alkaloids was achieved with a mass-directed auto purification system. The extraction and preparative liquid chromatography conditions were optimized. The developed method was applied to analysis of Gynura japonica (Thunb.) Juel., a herbal medicine traditionally used for detumescence and relieving pain but is potentially hepatotoxic as it contains pyrrolizidine alkaloids. Twelve pyrrolizidine alkaloids (six cis/trans isomer pairs) were identified with reference compounds or characterized by liquid chromatography with tandem mass spectrometry, and five individual pyrrolizidine alkaloids, including (E)-seneciphylline, seneciphylline, integerrimine, senecionine, and seneciphyllinine, were prepared from G. japonica roots with high efficiency. The results of this work provide a new technique for the preparation of large amounts of pyrrolizidine alkaloid reference substances, which will also benefit toxicological studies of pyrrolizidine alkaloids and treatments for pyrrolizidine alkaloid-induced toxicity.

  8. DEB025 (Alisporivir inhibits hepatitis C virus replication by preventing a cyclophilin A induced cis-trans isomerisation in domain II of NS5A.

    Directory of Open Access Journals (Sweden)

    Lotte Coelmont

    Full Text Available DEB025/Debio 025 (Alisporivir is a cyclophilin (Cyp-binding molecule with potent anti-hepatitis C virus (HCV activity both in vitro and in vivo. It is currently being evaluated in phase II clinical trials. DEB025 binds to CypA, a peptidyl-prolyl cis-trans isomerase which is a crucial cofactor for HCV replication. Here we report that it was very difficult to select resistant replicons (genotype 1b to DEB025, requiring an average of 20 weeks (four independent experiments, compared to the typically <2 weeks with protease or polymerase inhibitors. This indicates a high genetic barrier to resistance for DEB025. Mutation D320E in NS5A was the only mutation consistently selected in the replicon genome. This mutation alone conferred a low-level (3.9-fold resistance. Replacing the NS5A gene (but not the NS5B gene from the wild type (WT genome with the corresponding sequence from the DEB025(res replicon resulted in transfer of resistance. Cross-resistance with cyclosporine A (CsA was observed, whereas NS3 protease and NS5B polymerase inhibitors retained WT-activity against DEB025(res replicons. Unlike WT, DEB025(res replicon replicated efficiently in CypA knock down cells. However, DEB025 disrupted the interaction between CypA and NS5A regardless of whether the NS5A protein was derived from WT or DEB025(res replicon. NMR titration experiments with peptides derived from the WT or the DEB025(res domain II of NS5A corroborated this observation in a quantitative manner. Interestingly, comparative NMR studies on two 20-mer NS5A peptides that contain D320 or E320 revealed a shift in population between the major and minor conformers. These data suggest that D320E conferred low-level resistance to DEB025 probably by reducing the need for CypA-dependent isomerisation of NS5A. Prolonged DEB025 treatment and multiple genotypic changes may be necessary to generate significant resistance to DEB025, underlying the high barrier to resistance.

  9. The 2 1Ag state of isolated cis-trans-1,3,5,7-octatetraene: two-color resonance enhanced two-photon ionization studies

    NARCIS (Netherlands)

    B.E. Kohler; T. Shaler; W.J. Buma

    1992-01-01

    Vibrationally resolved 1 1Ag2 1Ag excitation spectra and decay times for cis,trans-1,3,5,7-octatetraene seeded in a supersonic He expansion have been measured by two-color resonance enhanced two-photon ionization spectroscopy. The excitation energy of the 1 1Ag2 1Ag 0-0 band (29 035 cm-1 ) is ~6500

  10. Transient-Absorption Spectroscopy of Cis-Trans Isomerization of N,N-dimethyl-4,4'-Azodianiline with 3D-Printed Temperature-Controlled Sample Holder

    Science.gov (United States)

    Kosenkov, Dmytro; Shaw, James; Zuczek, Jennifer; Kholod, Yana

    2016-01-01

    The laboratory unit demonstrates a project based approach to teaching physical chemistry laboratory where upper-division undergraduates carry out a transient-absorption experiment investigating the kinetics of cis-trans isomerization of N,N-dimethyl-4,4'-azodianiline. Students participate in modification of a standard flash-photolysis spectrometer…

  11. Asymmetric epoxidation of cis/trans-β-methylstyrene catalysed by immobilised Mn(salen) with different linkages: heterogenisation of homogeneous asymmetric catalysis.

    Science.gov (United States)

    Zhang, Haidong; Zou, Yu; Wang, Yi-Meng; Shen, Yu; Zheng, Xuxu

    2014-06-16

    Immobilised Mn(salen) catalysts with two different linkages were studied in the asymmetric epoxidation of cis/trans-β-methylstyrene using NaClO as oxidant. The immobilised Mn(salen) complexes inside nanopores can lead to different catalytic behaviour compared with that of homogeneous Jacobsen catalyst. The rigidity of the linkage was found to be a key factor affecting the catalytic performance of immobilised catalysts. The immobilised catalyst with a rigid linkage exhibited comparable chemical selectivity, enantioselectivity and cis/trans ratio of product formation to that obtained with homogeneous Jacobsen catalysts. In contrast, the immobilised catalyst with a flexible linkage gave remarkably lower chemical selectivity, enantioselectivity and inverted cis/trans ratio compared with the results obtained with the homogeneous Jacobsen catalyst and the immobilised catalyst with rigid linkage. Thus, for immobilised Mn(salen) catalysts, a rigid linkage connecting active centres to the support is essential to obtain activity and enantioselectivity as high as those obtained in homogeneous systems.

  12. Role of the cysteinyl residues in Arabidopsis thaliana cyclophilin CYP20-3 in peptidyl-prolyl cis-trans isomerase and redox-related functions

    OpenAIRE

    Laxa, Miriam; König, Janine; Dietz, Karl-Josef; Kandlbinder, Andrea

    2006-01-01

    Abstract Cyclophilins (Cyps) are ubiquitous proteins of the immunophilin superfamily with proposed functions in protein folding, protein degradation, stress response and signal transduction. Conserved Cys-residues further suggest a role in redox regulation. In order to get insight into the conformational change mechanism and functional properties of the chloroplast located CYP20-3, site-directed mutagenised Cys->Ser-variants were generated and analyzed for enzymatic and conformatio...

  13. Echinococcus multilocularis phosphoglucose isomerase (EmPGI): a glycolytic enzyme involved in metacestode growth and parasite-host cell interactions.

    Science.gov (United States)

    Stadelmann, Britta; Spiliotis, Markus; Müller, Joachim; Scholl, Sabrina; Müller, Norbert; Gottstein, Bruno; Hemphill, Andrew

    2010-11-01

    In Echinococcus multilocularis metacestodes, the surface-associated and highly glycosylated laminated layer, and molecules associated with this structure, is believed to be involved in modulating the host-parasite interface. We report on the molecular and functional characterisation of E. multilocularis phosphoglucose isomerase (EmPGI), which is a component of this laminated layer. The EmPGI amino acid sequence is virtually identical to that of its homologue in Echinococcus granulosus, and shares 64% identity and 86% similarity with human PGI. Mammalian PGI is a multi-functional protein which, besides its glycolytic function, can also act as a cytokine, growth factor and inducer of angiogenesis, and plays a role in tumour growth, development and metastasis formation. Recombinant EmPGI (recEmPGI) is also functionally active as a glycolytic enzyme and was found to be present, besides the laminated layer, in vesicle fluid and in germinal layer cell extracts. EmPGI is released from metacestodes and induces a humoral immune response in experimentally infected mice, and vaccination of mice with recEmPGI renders these mice more resistant towards secondary challenge infection, indicating that EmPGI plays an important role in parasite development and/or in modulating the host-parasite relationship. We show that recEmPGI stimulates the growth of isolated E. multilocularis germinal layer cells in vitro and selectively stimulates the proliferation of bovine adrenal cortex endothelial cells but not of human fibroblasts and rat hepatocytes. Thus, besides its role in glycolysis, EmPGI could also act as a factor that stimulates parasite growth and potentially induces the formation of novel blood vessels around the developing metacestode in vivo.

  14. Cis-trans isomerization in the S1 state of acetylene: identification of cis-well vibrational levels.

    Science.gov (United States)

    Merer, Anthony J; Steeves, Adam H; Baraban, Joshua H; Bechtel, Hans A; Field, Robert W

    2011-06-28

    acetylene that was previously thought to be unobservable, as well as the first high resolution spectroscopic results describing cis-trans isomerization.

  15. Amide cis-trans isomerization in aqueous solutions of methyl N-formyl-D-glucosaminides and methyl N-acetyl-D-glucosaminides: chemical equilibria and exchange kinetics.

    Science.gov (United States)

    Hu, Xiaosong; Zhang, Wenhui; Carmichael, Ian; Serianni, Anthony S

    2010-04-07

    Amide cis-trans isomerization (CTI) in methyl 2-deoxy-2-acylamido-d-glucopyranosides was investigated by (1)H and (13)C NMR spectroscopy. Singly (13)C-labeled methyl 2-deoxy-2-formamido-d-glucopyranoside (MeGlcNFm) anomers provided standard (1)H and (13)C chemical shifts and (1)H-(1)H and (13)C-(13)C spin-coupling constants for cis and trans amides that are detected readily in aqueous solution. Equipped with this information, doubly (13)C-labeled methyl 2-deoxy-2-acetamido-d-glucopyranoside (MeGlcNAc) anomers were investigated, leading to the detection and quantification of cis and trans amides in this biologically important aminosugar. In comparison to MeGlcNFm anomers, the percentage of cis amide in aqueous solutions of MeGlcNAc anomers is small ( approximately 23% for MeGlcNFm versus approximately 1.8% for MeGlcNAc at 42 degrees C) but nevertheless observable with assistance from (13)C-labeling. Temperature studies gave thermodynamic parameters DeltaG degrees , DeltaH degrees , and DeltaS degrees for cis-trans interconversion in MeGlcNFm and MeGlcNAc anomers. Cis/trans equilibria depended on anomeric configuration, with solutions of alpha-anomers containing less cis amide than those of beta-anomers. Confirmation of the presence of cis amide in MeGlcNAc solutions derived from quantitative (13)C saturation transfer measurements of CTI rate constants as a function of solution temperature, yielding activation parameters E(act), DeltaG degrees (), DeltaH degrees (), and DeltaS degrees () for saccharide CTI. Rate constants for the conversion of trans to cis amide in MeGlcNFm and MeGlcNAc anomers ranged from 0.02 to 3.59 s(-1) over 31-85 degrees C, compared to 0.24-80 s(-1) for the conversion of cis to trans amide over the same temperature range. Energies of activation ranged from 16-19 and 19-20 kcal/mol for the cis --> trans and trans --> cis processes, respectively. Complementary DFT calculations on MeGlcNFm and MeGlcNAc model structures were conducted to evaluate

  16. Positron annihilation and relaxation dynamics from dielectric spectroscopy and nuclear magnetic resonance: Cis-trans-1,4-poly(butadiene)

    Science.gov (United States)

    Bartoš, J.; Šauša, O.; Schwartz, G. A.; Alegría, A.; Alberdi, J. M.; Arbe, A.; Krištiak, J.; Colmenero, J.

    2011-04-01

    We report a joint analysis of positron annihilation lifetime spectroscopy (PALS), dielectric spectroscopy (BDS), and nuclear magnetic resonance (NMR) on cis-trans-1,4-poly(butadiene) (c-t-1,4-PBD). Phenomenological analysis of the orthopositronium lifetime τ3 - T dependence by linear fitting reveals four characteristic PALS temperatures: T_{b1} ^G = 0{.63}T_g^{PALS}, T_g^{PALS}, T_{b1} ^L = 1.22T_g^{PALS}, and T_{b2} ^L = 1.52T_g^{PALS}. Slight bend effects in the glassy and supercooled liquid states are related to the fast or slow secondary β process, from neutron scattering, respectively, the latter being connected with the trans-isomers. In addition, the first bend effect in the supercooled liquid coincides with a deviation of the slow effective secondary βeff relaxation related to the cis-isomers from low-T Arrhenius behavior to non-Arrhenius one and correlates with the onset of the primary α process from BDS. The second plateau effect in the liquid state occurs when τ3 becomes commensurable with the structural relaxation time τα(Tb2). It is also approximately related to its crossover from non-Arrhenius to Arrhenius regime in the combined BDS and NMR data. Finally, the combined BDS and NMR structural relaxation data, when analyzed in terms of the two-order parameter (TOP) model, suggest the influence of solidlike domains on both the annihilation behavior and the local and segmental chain mobility in the supercooled liquid. All these findings indicate the influence of the dynamic heterogeneity in both the primary and secondary relaxations due to the cis-trans isomerism in c-t-1,4-PBD and their impact into the PALS response.

  17. Fatty acids attached to all-trans-astaxanthin alter its cis-trans equilibrium, and consequently its stability, upon light-accelerated autoxidation.

    Science.gov (United States)

    de Bruijn, Wouter J C; Weesepoel, Yannick; Vincken, Jean-Paul; Gruppen, Harry

    2016-03-01

    Fatty acid esterification, common in naturally occurring astaxanthin, has been suggested to influence both colour stability and degradation of all-trans-astaxanthin. Therefore, astaxanthin stability was studied as influenced by monoesterification and diesterification with palmitate. Increased esterification decelerated degradation of all-trans-astaxanthin (RP-UHPLC-PDA), whereas, it had no influence on colour loss over time (spectrophotometry). This difference might be explained by the observation that palmitate esterification influenced the cis-trans equilibrium. Free astaxanthin produced larger amounts of 9-cis isomer whereas monopalmitate esterification resulted in increased 13-cis isomerization. The molar ratios of 9-cis:13-cis after 60min were 1:1.7 (free), 1:4.8 (monopalmitate) and 1:2.6 (dipalmitate). The formation of 9-cis astaxanthin, with its higher molar extinction coefficient than that of all-trans-astaxanthin, might compensate for colour loss induced by conjugated double bond cleavage. As such, it was concluded that spectrophotometry is not an accurate measure of the degradation of the all-trans-astaxanthin molecule.

  18. Exploración estocástica de las superficies de energía potencial de dímeros cis-trans y trans-trans del ácido fórmico

    Directory of Open Access Journals (Sweden)

    Said F. Figueredo

    2014-01-01

    Full Text Available Potential energy surface (PES of cis-trans and trans-trans formic acid dimers were sampled using a stochastic method, and the geometries, energies, and vibrational frequencies were computed at B3LYP/6-311++G(3df,2p level of theory. The results show that molar free energy of dimerization deviated up to 108.4% when basis set superposition error (BSSE and zero-point energy (ZPE were not considered. For cis-trans dimers, C=O and O - H bond weakened, whereas C - O bonds strengthened due to dimerization. Also, trans-trans FA dimers did not show a trend regarding strengthening or weakening of the C=O, O - H and C - O bonds.

  19. High-performance liquid chromatography separation of cis-trans anthocyanin isomers from wild Lycium ruthenicum Murr. employing a mixed-mode reversed-phase/strong anion-exchange stationary phase.

    Science.gov (United States)

    Jin, Hongli; Liu, Yanfang; Guo, Zhimou; Yang, Fan; Wang, Jixia; Li, Xiaolong; Peng, Xiaojun; Liang, Xinmiao

    2015-01-21

    The cis-trans isomerism is a common phenomenon for acylated anthocyanins. Nevertheless, few studies reported effective methods for the preparation of isomeric anthocyanins from natural products. In this work, a high-performance liquid chromatography (HPLC) method was developed to efficiently purify anthocyanin isomers from Lycium ruthenicum Murr. based on a mixed-mode reversed-phase/strong anion-exchange column (named XCharge C8SAX). Four commercially available columns were evaluated with a pair of isomeric anthocyanins, and the results demonstrated that the XCharge C8SAX column exhibited improved selectivity and column efficiency for the isomers. The chromatographic parameters, including pH, organic content, and ionic strength, were investigated. Optimal separation quality for the anthocyanin isomers was achieved on the XCharge C8SAX column. Six pure anthocyanins, including two pairs of cis-trans isomeric anthocyanins with one new anthocyanin, were purified from L. ruthenicum and identified. All of the results indicated that this method is an effective way to separate anthocyanins, especially for cis-trans isomers.

  20. Chiral Cyclobutane β-Amino Acid-Based Amphiphiles: Influence of Cis/Trans Stereochemistry on Condensed Phase and Monolayer Structure.

    Science.gov (United States)

    Sorrenti, Alessandro; Illa, Ona; Ortuño, Rosa M; Pons, Ramon

    2016-07-12

    New diastereomeric nonionic amphiphiles, cis- and trans-1, based on an optically pure cyclobutane β-amino ester moiety have been investigated to gain insight into the influence exerted by cis/trans stereochemistry and stereochemical constraints on the physicochemical behavior, molecular organization, and morphology of their Langmuir monolayers and dry solid states. All these features are relevant to the rational design of functional materials. trans-1 showed a higher thermal stability than cis-1. For the latter, a higher fluidity of its monolayers was observed when compared with the films formed by trans-1 whose BAM images revealed the formation of condensed phase domains with a dendritic shape, which are chiral, and all of them feature the same chiral sign. Although the formation of LC phase domains was not observed by BAM for cis-1, compact dendritic crystals floating on a fluid subphase were observed beyond the collapse, which are attributable to multilayered 3D structures. These differences can be explained by the formation of hydrogen bonds between the amide groups of consecutive molecules allowing the formation of extended chains for trans-1 giving ordered arrangements. However, for cis-1, this alignment coexists with another one that allows the simultaneous formation of two hydrogen bonds between the amide and the ester groups of adjacent molecules. In addition, the propensity to form intramolecular hydrogen bonds must be considered to justify the formation of different patterns of hydrogen bonding and, consequently, the formation of less ordered phases. Those characteristics are congruent also with the results obtained from SAXS-WAXS experiments which suggest a more bent configuration for cis-1 than for trans-1.

  1. Chiral Cyclobutane β-Amino Acid-Based Amphiphiles: Influence of Cis/Trans Stereochemistry on Solution Self-Aggregation and Recognition.

    Science.gov (United States)

    Sorrenti, Alessandro; Illa, Ona; Pons, Ramon; Ortuño, Rosa M

    2015-09-08

    Novel diastereomeric anionic amphiphiles based on the rigid cyclobutane β-amino acid scaffold have been synthesized and deeply investigated with the aim of generating new functional supramolecular architectures on the basis of the rational design of original amphiphilic molecules and the control of their self-assembly. The main interest has been focused on the effect that cis/trans stereochemistry exerts on their molecular organization and recognition. In diluted solutions, the relative stereochemistry mainly influences the headgroup solvation and anionic-charge stabilization, i.e., better stabilized in the cis diastereoisomer due to intramolecular hydrogen-bonding and/or charge-dipole interactions. This provokes differences in their physicochemical behavior (pKa, cmc, conductivity) as well as in the structural parameters of the spherical micelles formed. Although both diastereoisomers form fibers that evolve with time from the spherical micelles, they display markedly different morphology and kinetics of formation. In the lyotropic liquid crystal domain, the greatest differences are observed at the highest concentrations and can be ascribed to different hydrogen-bonding and molecular packing imposed by the stereochemical constraints. Remarkably, the spherical micelles of the two anionic surfactants show dramatically diverse enantioselection ability for bilirubin enantiomers. In addition, both the surfactants form heteroaggregates with bilirubin at submicellar concentrations but with a different expression of supramolecular chirality. This points out that the unlike relative configuration of the two surfactants influences their chiral recognition ability as well as the fashion in which chirality is expressed at the supramolecular level by controlling the molecular organization in both micellar aggregates and surfactant/bilirubin heteroaggregates. All these differential features can be appropriate and useful for the design and development of new soft materials with

  2. Synthesis and characterization of fac-[M(CO)3(P)(OO)] and cis-trans-[M(CO)2(P)2(OO)] complexes (M = Re, (99m)Tc) with acetylacetone and curcumin as OO donor bidentate ligands.

    Science.gov (United States)

    Triantis, Charalampos; Tsotakos, Theodoros; Tsoukalas, Charalampos; Sagnou, Marina; Raptopoulou, Catherine; Terzis, Aris; Psycharis, Vassilis; Pelecanou, Maria; Pirmettis, Ioannis; Papadopoulos, Minas

    2013-11-18

    The synthesis and characterization of neutral mixed ligand complexes fac-[M(CO)3(P)(OO)] and cis-trans-[M(CO)2(P)2(OO)] (M = Re, (99m)Tc), with deprotonated acetylacetone or curcumin as the OO donor bidentate ligands and a phosphine (triphenylphosphine or methyldiphenylphosphine) as the monodentate P ligand, is described. The complexes were synthesized through the corresponding fac-[M(CO)3(H2O)(OO)] (M = Re, (99m)Tc) intermediate aqua complex. In the presence of phosphine, replacement of the H2O molecule of the intermediate complex at room temperature generates the neutral tricarbonyl monophosphine fac-[Re(CO)3(P)(OO)] complex, while under reflux conditions further replacement of the trans to the phosphine carbonyl generates the new stable dicarbonyl bisphosphine complex cis-trans-[Re(CO)2(P)2(OO)]. The Re complexes were fully characterized by elemental analysis, spectroscopic methods, and X-ray crystallography showing a distorted octahedral geometry around Re. Both the monophosphine and the bisphosphine complexes of curcumin show selective binding to β-amyloid plaques of Alzheimer's disease. At the (99m)Tc tracer level, the same type of complexes, fac-[(99m)Tc(CO)3(P)(OO)] and cis-trans-[(99m)Tc(CO)2(P)2(OO)], are formed introducing new donor combinations for (99m)Tc(I). Overall, β-diketonate and phosphine constitute a versatile ligand combination for Re(I) and (99m)Tc(I), and the successful employment of the multipotent curcumin as β-diketone provides a solid example of the pharmacological potential of this system.

  3. Discovery of Evolutionary Divergence of Biological Nitrogen Fixation and Photosynthesis: Fine Tuning of Biogenesis of the NifH and the ChlL by a Peptidyl-Prolyl Cis/Trans Isomerase

    Directory of Open Access Journals (Sweden)

    Nara Gavini

    2011-01-01

    Full Text Available Problem statement: Despite the structural and functional similarities between the nitrogenase that performs biological nitrogen fixation reaction and the Dark Protochlorphyllide Oxidoreductase (DPOR that performs chlorophyll-biosynthesis, attempts to substitute nitrogenase-components with DPOR-components have hitherto failed. This investigation was undertaken to test if Chlamydomonas reinhardtii protochlorophyllide (Pchlide reductase (ChlL that shares some structural similarity with Nitrogenase Reductase (NifH could complement the functions of NifH in biological nitrogen fixation of Azotobacter vinelandii. Approach: Genetic complementation studies were performed to test if the chlL gene and its mutants cloned under transcriptional control of nifH promoter (nifHp in a broad-host range low copy plasmid pBG1380 could render a Nif+ phenotype to NifH-deficient A. vinelandii strains. Results: Expression of ChlL could render Nif+ phenotype to NifH-deficient A. vinelandii only in the absence of NifM, a nif-specific PPIase essential for biogenesis of NifH. The ChlL mutants Cys95Thr and Cys129Thr were unable to substitute for NifH. Thus, the conserved cysteine ligands of [4Fe-4S] cluster in ChlL are essential for successful substitution of NifH by ChlL. Since C-termini of NifH and ChlL demonstrated the least similarity and Pro258, a substrate for the PPIase activity of NifM, is located in the C-terminus of NifH, we posited that replacing the C-terminus of NifH with that of ChlL would render NifM-independence to NifH. The NifH-ChlL chimera could support the growth of NifH- and NifM-deficient A. vinelandii in nitrogen limiting conditions implying that it has acquired NifM-independence. Conclusion/Recommendations: Collectively, these observations suggest that NifM, an evolutionarily conserved nif-specific PPIase, could have contributed to the functional divergence of biological nitrogen fixation and photosynthesis during evolution by virtue of its ability to exert opposing effects on structurally similar substrates, ChlL and NifH.

  4. Discovery of Evolutionary Divergence of Biological Nitrogen Fixation and Photosynthesis: Fine Tuning of Biogenesis of the NifH and the ChlL by a Peptidyl-Prolyl Cis/Trans Isomerase

    OpenAIRE

    Nara Gavini; Sinny Delacroix; Kelvin Harris Jr.; Lakshmi Pulakat

    2011-01-01

    Problem statement: Despite the structural and functional similarities between the nitrogenase that performs biological nitrogen fixation reaction and the Dark Protochlorphyllide Oxidoreductase (DPOR) that performs chlorophyll-biosynthesis, attempts to substitute nitrogenase-components with DPOR-components have hitherto failed. This investigation was undertaken to test if Chlamydomonas reinhardtii protochlorophyllide (Pchlide) reductase (ChlL) that shares some structural similarity with Nitrog...

  5. Water-stable helical structure of tertiary amides of bicyclic β-amino acid bearing 7-azabicyclo[2.2.1]heptane. Full control of amide cis-trans equilibrium by bridgehead substitution.

    Science.gov (United States)

    Hosoya, Masahiro; Otani, Yuko; Kawahata, Masatoshi; Yamaguchi, Kentaro; Ohwada, Tomohiko

    2010-10-27

    Helical structures of oligomers of non-natural β-amino acids are significantly stabilized by intramolecular hydrogen bonding between main-chain amide moieties in many cases, but the structures are generally susceptible to the environment; that is, helices may unfold in protic solvents such as water. For the generation of non-hydrogen-bonded ordered structures of amides (tertiary amides in most cases), control of cis-trans isomerization is crucial, even though there is only a small sterical difference with respect to cis and trans orientations. We have established methods for synthesis of conformationally constrained β-proline mimics, that is, bridgehead-substituted 7-azabicyclo[2.2.1]heptane-2-endo-carboxylic acids. Our crystallographic, 1D- and 2D-NMR, and CD spectroscopic studies in solution revealed that a bridgehead methoxymethyl substituent completely biased the cis-trans equilibrium to the cis-amide structure along the main chain, and helical structures based on the cis-amide linkage were generated independently of the number of residues, from the minimalist dimer through the tetramer, hexamer, and up to the octamer, and irrespective of the solvent (e.g., water, alcohol, halogenated solvents, and cyclohexane). Generality of the control of the amide equilibrium by bridgehead substitution was also examined.

  6. Development of a mariner-Based Transposon and Identification of Listeria monocytogenes Determinants, Including the Peptidyl-Prolyl Isomerase PrsA2, That Contribute to Its Hemolytic Phenotype▿

    Science.gov (United States)

    Zemansky, Jason; Kline, Benjamin C.; Woodward, Joshua J.; Leber, Jess H.; Marquis, Hélène; Portnoy, Daniel A.

    2009-01-01

    Listeriolysin O (LLO) is a pore-forming toxin that mediates phagosomal escape and cell-to-cell spread of the intracellular pathogen Listeria monocytogenes. In order to identify factors that control the production, activity, or secretion of this essential virulence factor, we constructed a Himar1 mariner transposon delivery system and screened 50,000 mutants for a hypohemolytic phenotype on blood agar plates. Approximately 200 hypohemolytic mutants were identified, and the 51 most prominent mutants were screened ex vivo for intracellular growth defects. Eight mutants with a phenotype were identified, and they contained insertions in the following genes: lmo0964 (similar to yjbH), lmo1268 (clpX), lmo1401 (similar to ymdB), lmo1575 (similar to ytqI), lmo1695 (mprF), lmo1821 (similar to prpC), lmo2219 (prsA2), and lmo2460 (similar to cggR). Some of these genes are involved in previously unexplored areas of research with L. monocytogenes: the genes yjbH and clpX regulate the disulfide stress response in Bacillus subtilis, and the prpC phosphatase has been implicated in virulence in other gram-positive pathogens. Here we demonstrate that prsA2, an extracytoplasmic peptidyl-prolyl cis/trans isomerase, is critical for virulence and contributes to the folding of LLO and to the activity of another virulence factor, the broad-range phospholipase C (PC-PLC). Furthermore, although it has been shown that prsA2 expression is linked to PrfA, the master virulence transcription factor in L. monocytogenes pathogenesis, we demonstrate that prsA2 is not directly controlled by PrfA. Finally, we show that PrsA2 is involved in flagellum-based motility, indicating that this factor likely serves a broad physiological role. PMID:19376879

  7. Development of a mariner-based transposon and identification of Listeria monocytogenes determinants, including the peptidyl-prolyl isomerase PrsA2, that contribute to its hemolytic phenotype.

    Science.gov (United States)

    Zemansky, Jason; Kline, Benjamin C; Woodward, Joshua J; Leber, Jess H; Marquis, Hélène; Portnoy, Daniel A

    2009-06-01

    Listeriolysin O (LLO) is a pore-forming toxin that mediates phagosomal escape and cell-to-cell spread of the intracellular pathogen Listeria monocytogenes. In order to identify factors that control the production, activity, or secretion of this essential virulence factor, we constructed a Himar1 mariner transposon delivery system and screened 50,000 mutants for a hypohemolytic phenotype on blood agar plates. Approximately 200 hypohemolytic mutants were identified, and the 51 most prominent mutants were screened ex vivo for intracellular growth defects. Eight mutants with a phenotype were identified, and they contained insertions in the following genes: lmo0964 (similar to yjbH), lmo1268 (clpX), lmo1401 (similar to ymdB), lmo1575 (similar to ytqI), lmo1695 (mprF), lmo1821 (similar to prpC), lmo2219 (prsA2), and lmo2460 (similar to cggR). Some of these genes are involved in previously unexplored areas of research with L. monocytogenes: the genes yjbH and clpX regulate the disulfide stress response in Bacillus subtilis, and the prpC phosphatase has been implicated in virulence in other gram-positive pathogens. Here we demonstrate that prsA2, an extracytoplasmic peptidyl-prolyl cis/trans isomerase, is critical for virulence and contributes to the folding of LLO and to the activity of another virulence factor, the broad-range phospholipase C (PC-PLC). Furthermore, although it has been shown that prsA2 expression is linked to PrfA, the master virulence transcription factor in L. monocytogenes pathogenesis, we demonstrate that prsA2 is not directly controlled by PrfA. Finally, we show that PrsA2 is involved in flagellum-based motility, indicating that this factor likely serves a broad physiological role.

  8. Epistasis in tomato color mutations involves regulation of phytoene synthase 1 expression by cis-carotenoids.

    Science.gov (United States)

    Kachanovsky, David E; Filler, Shdema; Isaacson, Tal; Hirschberg, Joseph

    2012-11-13

    Tomato (Solanum lycopersicum) fruit accumulate the red carotenoid pigment lycopene. The recessive mutation yellow-flesh (locus r) in tomato eliminates fruit carotenoids by disrupting the activity of the fruit-specific phytoene synthase (PSY1), the first committed step in the carotenoid biosynthesis pathway. Fruits of the recessive mutation tangerine (t) appear orange due to accumulation of 7,9,7',9'-tetra-cis-lycopene (prolycopene) as a result of a mutation in the carotenoid cis-trans isomerase. It was established 60 y ago that tangerine is epistatic to yellow-flesh. This uncharacteristic epistasis interaction defies a paradigm in biochemical genetics arguing that mutations that disrupt enzymes acting early in a biosynthetic pathway are epistatic to other mutations that block downstream steps in the same pathway. To explain this conundrum, we have investigated the interaction between tangerine and yellow-flesh at the molecular level. Results presented here indicate that allele r(2997) of yellow-flesh eliminates transcription of PSY1 in fruits. In a genetic background of tangerine, transcription of PSY1 is partially restored to a level sufficient for producing phytoene and downstream carotenoids. Our results revealed the molecular mechanism underlying the epistasis of t over r and suggest the involvement of cis-carotenoid metabolites in a feedback regulation of PSY1 gene expression.

  9. Determination of the cis-trans isomerization barriers of L-alanyl-L-proline in aqueous solutions and at water/hydrophobic interfaces by on-line temperature-jump relaxation HPLC and dynamic on-column reaction HPLC.

    Science.gov (United States)

    Shibukawa, Masami; Miyake, Ayaka; Eda, Sayaka; Saito, Shingo

    2015-09-15

    Proline cis-trans isomerization is known to play a key role in the rate-determining steps of protein folding. It is thus very important to understand the influence of environments, not only bulk solutions but also microenvironments such as interfaces, on the isomerization reaction of proline peptides. Here we present two HPLC methods for measurements of kinetic and equilibrium parameters for the isomerization reactions in bulk solutions and at liquid/solid interfaces. On-line temperature-jump relaxation HPLC (T-jump HPLC) allows the determination of forward and reverse rate constants of the isomerization in a bulk solution by monitoring the whole time course of conversion of pure isomers from both sides of the reaction, in contrast to other HPLC and capillary zone electrophoresis as well as spectrometric and calorimetric methods, which use a mixture of the isomers. We can then determine cis-trans isomerization barriers of the peptide at liquid/solid interfaces from the kinetic data obtained by dynamic on-column reaction HPLC and T-jump HPLC. We observed that the interconversion around the peptide bond for l-alanyl-l-proline (Ala-Pro) in water is accelerated at the surfaces of an alkyl-bonded silica and a poly(styrene-divinylbenzene) copolymer resin, and this is caused by a remarkable decrease in the enthalpy of activation. The molecular structures of the cis and trans forms of Ala-Pro estimated by quantum mechanics calculation reveal that an equilibrium shift toward the cis form as well as the rapid isomerization of Ala-Pro at the water/hydrophobic interfaces can be attributed to the lower polarity of the interfacial water at the surfaces of the hydrophobic materials compared to that of bulk water.

  10. A high-throughput screen for inhibitors of the prolyl isomerase, Pin1, identifies a seaweed polyphenol that reduces adipose cell differentiation.

    Science.gov (United States)

    Mori, Tadashi; Hidaka, Masafumi; Ikuji, Hiroko; Yoshizawa, Ibuki; Toyohara, Haruhiko; Okuda, Toru; Uchida, Chiyoko; Asano, Tomoichiro; Yotsu-Yamashita, Mari; Uchida, Takafumi

    2014-01-01

    The peptidyl prolyl cis/trans isomerase Pin1 enhances the uptake of triglycerides and the differentiation of fibroblasts into adipose cells in response to insulin stimulation. Pin1 downregulation could be a potential approach to prevent and treat obesity-related disorders. In order to identify an inhibitor of Pin1 that exhibited minimal cytotoxicity, we established a high-throughput screen for Pin1 inhibitors and used this method to identify an inhibitor from 1,056 crude fractions of two natural product libraries. The candidate, a phlorotannin called 974-B, was isolated from the seaweed, Ecklonia kurome. 974-B inhibited the differentiation of mouse embryonic fibroblasts and 3T3-L1 cells into adipose cells without inducing cytotoxicity. We discovered the Pin1 inhibitor, 974-B, from the seaweed, E. kurome, and showed that it blocks the differentiation of fibroblasts into adipose cells, suggesting that 974-B could be a lead drug candidate for obesity-related disorders.

  11. Synergistic Interaction of Light Alcohol Administration in the Presence of Mild Iron Overload in a Mouse Model of Liver Injury: Involvement of Triosephosphate Isomerase Nitration and Inactivation

    Science.gov (United States)

    Gao, Wanxia; Zhao, Jie; Gao, Zhonghong

    2017-01-01

    It is well known that iron overload promotes alcoholic liver injury, but the doses of iron or alcohol used in studies are usually able to induce liver injury independently. Little attention has been paid to the coexistence of low alcohol consumption and mild iron overload when either of them is insufficient to cause obvious liver damage, although this situation is very common among some people. We studied the interactive effects and the underlining mechanism of mild doses of iron and alcohol on liver injury in a mouse model. Forty eight male Kunming mice were randomly divided into four groups: control, iron (300 mg/kg iron dextran, i.p.), alcohol (2 g/kg/day ethanol for four weeks i.g.), and iron plus alcohol group. After 4 weeks of treatment, mice were sacrificed and blood and livers were collected for biochemical analysis. Protein nitration level in liver tissue was determined by immunoprecipitation and Western blot analysis. Although neither iron overload nor alcohol consumption at our tested doses can cause severe liver injury, it was found that co-administration of the same doses of alcohol and iron resulted in liver injury and hepatic dysfunction, accompanied with elevated ratio of NADH/NAD+, reduced antioxidant ability, increased oxidative stress, and subsequent elevated protein nitration level. Further study revealed that triosephosphate isomerase, an important glycolytic enzyme, was one of the targets to be oxidized and nitrated, which was responsible for its inactivation. These data indicate that even under low alcohol intake, a certain amount of iron overload can cause significant liver oxidative damage, and the modification of triosephosphate isomerasemight be the important underlining mechanism of hepatic dysfunction. PMID:28103293

  12. NMR-based metabolomics reveals that conjugated double bond content and lipid storage efficiency in HepG2 cells are affected by fatty acid cis/trans configuration and chain length

    DEFF Research Database (Denmark)

    Najbjerg, Heidi; Young, Jette F; Bertram, Hanne Christine S.

    2011-01-01

    -11), linoleic acid (C18:2), or palmitic acid (C16:0), and multivariate data analysis revealed a strong effect of fatty acid on the lipophilic metabolite fraction. Inspection of the spectra revealed that the difference between the observed responses could be ascribed to the appearance of resonances......:0), myristic acid (C14:0), or palmitic acid (C16:0), an effect of fatty acid length was also evident, and data indicated that short-chain fatty acids (C4C6) are immediately converted, whereas mediumlong-chain fatty acids (C1216) are incorporated into triglycerides and deposited in the cells. In conclusion......In the present study the metabolic response to various fatty acids was investigated in HepG2 cells by using a 1HNMRbased approach. To elucidate the effect of cis/trans configuration, the cells were exposed to either oleic acid (C18:1 cis-9), elaidic acid (C18:1 trans-9), vaccenic acid (C18:1 trans...

  13. Zero-point Energy is Needed in Molecular Dynamics Calculations to Access the Saddle Point for H+HCN→H2CN* and cis/trans-HCNH* on a New Potential Energy Surface.

    Science.gov (United States)

    Wang, Xiaohong; Bowman, Joel M

    2013-02-12

    We calculate the probabilities for the association reactions H+HCN→H2CN* and cis/trans-HCNH*, using quasiclassical trajectory (QCT) and classical trajectory (CT) calculations, on a new global ab initio potential energy surface (PES) for H2CN including the reaction channels. The surface is a linear least-squares fit of roughly 60 000 CCSD(T)-F12b/aug-cc-pVDZ electronic energies, using a permutationally invariant basis with Morse-type variables. The reaction probabilities are obtained at a variety of collision energies and impact parameters. Large differences in the threshold energies in the two types of dynamics calculations are traced to the absence of zero-point energy in the CT calculations. We argue that the QCT threshold energy is the realistic one. In addition, trajectories find a direct pathway to trans-HCNH, even though there is no obvious transition state (TS) for this pathway. Instead the saddle point (SP) for the addition to cis-HCNH is evidently also the TS for direct formation of trans-HCNH.

  14. Probing cis-trans isomerization in the S{sub 1} state of C{sub 2}H{sub 2} via H-atom action and hot band-pumped IR-UV double resonance spectroscopies

    Energy Technology Data Exchange (ETDEWEB)

    Changala, P. Bryan; Baraban, Joshua H.; Field, Robert W., E-mail: rwfield@mit.edu [Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 (United States); Merer, Anthony J. [Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 10617, Taiwan (China); Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1 (Canada)

    2015-08-28

    We report novel experimental strategies that should prove instrumental in extending the vibrational and rotational assignments of the S{sub 1} state of acetylene, C{sub 2}H{sub 2}, in the region of the cis-trans isomerization barrier. At present, the assignments are essentially complete up to ∼500 cm{sup −1} below the barrier. Two difficulties arise when the assignments are continued to higher energies. One is that predissociation into C{sub 2}H + H sets in roughly 1100 cm{sup −1} below the barrier; the resulting quenching of laser-induced fluorescence (LIF) reduces its value for recording spectra in this region. The other difficulty is that tunneling through the barrier causes a staggering in the K-rotational structure of isomerizing vibrational levels. The assignment of these levels requires data for K values up to at least 3. Given the rotational selection rule K′ − ℓ{sup ′′} = ± 1, such data must be obtained via excited vibrational levels of the ground state with ℓ{sup ′′} > 0. In this paper, high resolution H-atom resonance-enhanced multiphoton ionization spectra are demonstrated to contain predissociated bands which are almost invisible in LIF spectra, while preliminary data using a hyperthermal pulsed nozzle show that ℓ{sup ′′} = 2 states can be selectively populated in a jet, giving access to K′ = 3 states in IR-UV double resonance.

  15. AcEST: DK947818 [AcEST

    Lifescience Database Archive (English)

    Full Text Available eptidyl-prolyl cis-trans isomerase CYP19-... 223 6e-58 sp|Q5R8S7|PPIA_PONPY Peptidyl-prolyl cis-trans isomerase A OS=Po....lyl cis-trans isomerase OS=Po... 276 9e-73 tr|Q6TMX3|Q6TMX3_THEHA Peptidyl-prolyl cis-trans isomerase OS=Th...... 274 3e-72 tr|A9PIY0|A9PIY0_POPJC Peptidyl-prolyl cis-trans isomerase OS=Po... ...TR Peptidyl-prolyl cis-trans isomerase OS=Po... 272 1e-71 tr|A9PJ47|A9PJ47_POPJC Peptidyl-prolyl cis-trans isomerase OS=Po....|A9P9C8|A9P9C8_POPTR Peptidyl-prolyl cis-trans isomerase OS=Po... 271 2e-71 tr|Q8

  16. Separation of cis -, trans - isomer of methyl oleate by urea inclusion%尿素包合法分离油酸甲酯顺反异构体的研究

    Institute of Scientific and Technical Information of China (English)

    郝建秀; 贾时宇; 齐永琴; 侯相林

    2011-01-01

    Cis -, trans - isomer of methyl oleate were separated from methyl oleate by urea inclusion method.The effect of mass ratio of methyl oleate to urea to methanol, inclusion temperature, cooling rate, inclusion time on separating was reviewed.By single - factor experiments, the best conditions were found as follows: mass ratio of methyl oleate to urea 1∶ 2, inclusion temperature 30 ℃, cooling rate 30℃/h, mass ratio of methyl oleate to methanol 1∶10 and inclusion time 5 h.Under the above conditions, the purity of cis - methyl oleate was 96.52%, and its recovery was 47.99%.In addition, the product of cis -methyl oleate did not contain trans -methyl oleate.%以顺反混合的油酸甲酯异构体为原料,采用尿素包合法对其顺、反式异构体进行分离.分别考察了油酸甲酯、尿素与甲醇质量比,包合温度,降温速率,包合时间等因素对分离效果的影响.确定最优分离条件为:油酸甲酯、尿素和甲醇质量比1:2:10,包合温度30℃,降温速率30℃/h,包合时间5 h.在此条件下,顺式油酸甲酯纯度可达到96.52%,回收率为47.99%,同时顺式油酸甲酯产品中不含反式油酸甲酯.

  17. FK506 binding protein 8 peptidylprolyl isomerase activity manages a late stage of cystic fibrosis transmembrane conductance regulator (CFTR) folding and stability.

    Science.gov (United States)

    Hutt, Darren M; Roth, Daniela Martino; Chalfant, Monica A; Youker, Robert T; Matteson, Jeanne; Brodsky, Jeffrey L; Balch, William E

    2012-06-22

    Cystic fibrosis (CF) is caused by mutations in the apical chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) with 90% of patients carrying at least one deletion of the F508 (ΔF508) allele. This mutant form of CFTR is characterized by a folding and trafficking defect that prevents exit from the endoplasmic reticulum. We previously reported that ΔF508 CFTR can be recovered in a complex with Hsp90 and its co-chaperones as an on-pathway folding intermediate, suggesting that Δ508 CF disease arises due to a failure of the proteostasis network (PN), which manages protein folding and degradation in the cell. We have now examined the role of FK506-binding protein 8 (FKBP8), a component of the CFTR interactome, during the biogenesis of wild-type and ΔF508 CFTR. FKBP8 is a member of the peptidylprolyl isomerase family that mediates the cis/trans interconversion of peptidyl prolyl bonds. Our results suggest that FKBP8 is a key PN factor required at a post-Hsp90 step in CFTR biogenesis. In addition, changes in its expression level or alteration of its activity by a peptidylprolyl isomerase inhibitor alter CFTR stability and transport. We propose that CF is caused by the sequential failure of the prevailing PN pathway to stabilize ΔF508-CFTR for endoplasmic reticulum export, a pathway that can be therapeutically managed.

  18. Prolyl isomerase Pin1 regulates doxorubicin-inducible P-glycoprotein level by reducing Foxo3 stability.

    Science.gov (United States)

    Shimizu, Taiki; Bamba, Yoshimasa; Kawabe, Yosuke; Fukuda, Tomokazu; Fujimori, Fumihiro; Takahashi, Katsuhiko; Uchida, Chiyoko; Uchida, Takafumi

    2016-03-01

    It has been known that the phosphoSer/Thr-Pro-specific peptidyl prolyl cis/trans isomerase Pin1 regulates a variety of intracellular signaling pathways, including the response to the genotoxic drug doxorubicin. Pin1 binds phosphorylated p53 and stabilizes p53 to cause cell cycle arrest and apoptosis quickly in response to doxorubicin. Here we show another mechanism of Pin1 to maintain cell sensitivity to genotoxic stress, irrespective of whether p53 is present or not. In response to the genotoxic drug, Pin1 binds and decreases levels of the phosphorylated Foxo3, the positive transcription factor of P-glycoprotein (P-gp) gene. Through this mechanism of action, Pin1 decreases the level of P-gp and signals the cell to pump the genotoxic drugs out. This shows that Pin1 is implemented in maintaining the susceptibility to the genotoxic drugs by controlling P-gp level as well as p53-dependent apoptosis and cell cycle signaling pathways.

  19. Brown Algae Polyphenol, a Prolyl Isomerase Pin1 Inhibitor, Prevents Obesity by Inhibiting the Differentiation of Stem Cells into Adipocytes

    Science.gov (United States)

    Suzuki, Atsuko; Saeki, Toshiyuki; Ikuji, Hiroko; Uchida, Chiyoko; Uchida, Takafumi

    2016-01-01

    Background While screening for an inhibitor of the peptidyl prolyl cis/trans isomerase, Pin1, we came across a brown algae polyphenol that blocks the differentiation of fibroblasts into adipocytes. However, its effectiveness on the accumulation of fat in the body has never been studied. Methodology/Principal Findings Oral administration of brown algae polyphenol to mice fed with a high fat diet, suppressed the increase in fat volume to a level observed in mice fed with a normal diet. We speculate that Pin1 might be required for the differentiation of stem cell to adipocytes. We established wild type (WT) and Pin1-/- (Pin1-KO) adipose-derived mesenchymal stem cell (ASC) lines and found that WT ASCs differentiate to adipocytes but Pin1-KO ASCs do not. Conclusion and Significance Oral administration of brown algae polyphenol, a Pin1 inhibitor, reduced fat buildup in mice. We showed that Pin1 is required for the differentiation of stem cells into adipocytes. We propose that oral intake of brown algae polyphenol is useful for the treatment of obesity. PMID:28036348

  20. Fine-tuning the extent and dynamics of binding cleft opening as a potential general regulatory mechanism in parvulin-type peptidyl prolyl isomerases

    Science.gov (United States)

    Czajlik, András; Kovács, Bertalan; Permi, Perttu; Gáspári, Zoltán

    2017-03-01

    Parvulins or rotamases form a distinct group within peptidyl prolyl cis-trans isomerases. Their exact mode of action as well as the role of conserved residues in the family are still not unambiguously resolved. Using backbone S2 order parameters and NOEs as restraints, we have generated dynamic structural ensembles of three distinct parvulins, SaPrsA, TbPin1 and CsPinA. The resulting ensembles are in good agreement with the experimental data but reveal important differences between the three enzymes. The largest difference can be attributed to the extent of the opening of the substrate binding cleft, along which motional mode the three molecules occupy distinct regions. Comparison with a wide range of other available parvulin structures highlights structural divergence along the bottom of the binding cleft acting as a hinge during the opening-closing motion. In the prototype WW-domain containing parvulin, Pin1, this region is also important in forming contacts with the WW domain known to modulate enzymatic activity of the catalytic domain. We hypothesize that modulation of the extent and dynamics of the identified ‘breathing motion’ might be one of the factors responsible for functional differences in the distinct parvulin subfamilies.

  1. Ribose 5-phosphate isomerase B knockdown compromises Trypanosoma brucei bloodstream form infectivity.

    Science.gov (United States)

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

    2015-01-01

    Ribose 5-phosphate isomerase is an enzyme involved in the non-oxidative branch of the pentose phosphate pathway, and catalyzes the inter-conversion of D-ribose 5-phosphate and D-ribulose 5-phosphate. Trypanosomatids, including the agent of African sleeping sickness namely Trypanosoma brucei, have a type B ribose-5-phosphate isomerase. This enzyme is absent from humans, which have a structurally unrelated ribose 5-phosphate isomerase type A, and therefore has been proposed as an attractive drug target waiting further characterization. In this study, Trypanosoma brucei ribose 5-phosphate isomerase B showed in vitro isomerase activity. RNAi against this enzyme reduced parasites' in vitro growth, and more importantly, bloodstream forms infectivity. Mice infected with induced RNAi clones exhibited lower parasitaemia and a prolonged survival compared to control mice. Phenotypic reversion was achieved by complementing induced RNAi clones with an ectopic copy of Trypanosoma cruzi gene. Our results present the first functional characterization of Trypanosoma brucei ribose 5-phosphate isomerase B, and show the relevance of an enzyme belonging to the non-oxidative branch of the pentose phosphate pathway in the context of Trypanosoma brucei infection.

  2. Nerve growth factor stimulates interaction of Cayman ataxia protein BNIP-H/Caytaxin with peptidyl-prolyl isomerase Pin1 in differentiating neurons.

    Directory of Open Access Journals (Sweden)

    Jan Paul Buschdorf

    Full Text Available Mutations in ATCAY that encodes the brain-specific protein BNIP-H (or Caytaxin lead to Cayman cerebellar ataxia. BNIP-H binds to glutaminase, a neurotransmitter-producing enzyme, and affects its activity and intracellular localization. Here we describe the identification and characterization of the binding between BNIP-H and Pin1, a peptidyl-prolyl cis/trans isomerase. BNIP-H interacted with Pin1 after nerve growth factor-stimulation and they co-localized in the neurites and cytosol of differentiating pheochromocytoma PC12 cells and the embryonic carcinoma P19 cells. Deletional mutagenesis revealed two cryptic binding sites within the C-terminus of BNIP-H such that single point mutants affecting the WW domain of Pin1 completely abolished their binding. Although these two sites do not contain any of the canonical Pin1-binding motifs they showed differential binding profiles to Pin1 WW domain mutants S16E, S16A and W34A, and the catalytically inert C113A of its isomerase domain. Furthermore, their direct interaction would occur only upon disrupting the ability of BNIP-H to form an intramolecular interaction by two similar regions. Furthermore, expression of Pin1 disrupted the BNIP-H/glutaminase complex formation in PC12 cells under nerve growth factor-stimulation. These results indicate that nerve growth factor may stimulate the interaction of BNIP-H with Pin1 by releasing its intramolecular inhibition. Such a mechanism could provide a post-translational regulation on the cellular activity of BNIP-H during neuronal differentiation.

  3. The cloning, characterization, and functional analysis of a gene encoding an isopentenyl diphosphate isomerase involved in triterpene biosynthesis in the Lingzhi or reishi medicinal mushroom Ganoderma lucidum (higher Basidiomycetes).

    Science.gov (United States)

    Wu, Feng-Li; Shi, Liang; Yao, Jian; Ren, Ang; Zhou, Chao; Mu, Da-Shuai; Zhao, Ming-Wen

    2013-01-01

    An isopentenyl diphosphate isomerase (IDI) gene, GlIDI, was isolated from Ganoderma lucidum, which produces triterpenes through the mevalonate pathway. The open reading frame of GlIDI encodes a 252 amino acid polypeptide with a theoretical molecular mass of 28.71 kDa and a theoretical isoelectric point of 5.36. GlIDI is highly homologous to other fungal IDIs and contains conserved active residues and nudix motifs shared by the IDI protein family. The color complementation assay indicated that GlIDI can accelerate the accumulation of β-carotene and confirmed that the cloned complementary DNA encoded a functional GlIDI protein. Gene expression analysis showed that the GlIDI transcription level was relatively low in the mycelia and reached a relatively high level in the mushroom primordia. In addition, its expression level could be up-regulated by 254 µM methyl jasmonate. Our results suggest that this enzyme may play an important role in triterpene biosynthesis.

  4. 紫花苜蓿肽基脯氨酰顺反异构酶基因全长cDNA的电子克隆及同源建模%Full-length cDNA in Silico Cloning and Homology Modeling of Gene Encoding Peptidyl Prolyl Cis-trans Isomerase from Medicago sativa L.

    Institute of Scientific and Technical Information of China (English)

    王海波

    2010-01-01

    [目的]研究对紫花苜蓿肽基脯氨酰顺反异构酶基因全长cDNA进行电子克隆及同源建模的方法.[方法]根据物种间同源基因相对保守的特点,利用NCBI数据库,对公共EST数据库进行同源检索筛选和重叠群装配,获得拼接cDNA序列,经过ORF Finder分析后获得相应蛋白质的全长序列,并利用Swiss-Mode服务器预测各原子坐标,进而利用Swiss-PdbViewer生成三维空间模型.[结果]采用该方法成功获得紫花苜蓿肽基脯氨酰顺反异构酶基因的完整cDNA序列,全长为1 880 bp,mPPI蛋白质编码68AA,分子量约为7 829.9 Da,理论等电点pI=8.99,原子组成为C_(338)H_(547)N_(103)O_(103)S_4,摩尔消光系数280 nm处为4 595,不稳定系数为42.16,脂肪系数为83.24,总平均亲水性为-0.456.[结论]根据物种间同源基因序列对跨物种间EST数据库进行同源检索筛选和拼接,是基因克隆的一条有效途径.

  5. Thermoinactivation Mechanism of Glucose Isomerase

    Science.gov (United States)

    Lim, Leng Hong; Saville, Bradley A.

    In this article, the mechanisms of thermoinactivation of glucose isomerase (GI) from Streptomyces rubiginosus (in soluble and immobilized forms) were investigated, particularly the contributions of thiol oxidation of the enzyme's cysteine residue and a "Maillard-like" reaction between the enzyme and sugars in high fructose corn syrup (HFCS). Soluble GI (SGI) was successfully immobilized on silica gel (13.5 μm particle size), with an activity yield between 20 and 40%. The immobilized GI (IGI) has high enzyme retention on the support during the glucose isomerization process. In batch reactors, SGI (half-life =145 h) was more stable than IGI (half-life=27 h) at 60°C in HFCS, whereas at 80°C, IGI (half-life=12 h) was more stable than SGI (half-life=5.2 h). IGI was subject to thiol oxidation at 60°C, which contributed to the enzyme's deactivation. IGI was subject to thiol oxidation at 80°C, but this did not contribute to the deactivation of the enzyme. SGI did not undergo thiol oxidation at 60°C, but at 80°C SGI underwent severe precipitation and thiol oxidation, which caused the enzyme to deactivate. Experimental results show that immobilization suppresses the destablizing effect of thiol oxidation on GI. A "Maillard-like" reaction between SGI and the sugars also caused SGI thermoinactivation at 60, 70, and 80°C, but had minimal effect on IGI. At 60 and 80°C, IGI had higher thermostability in continuous reactors than in batch reactors, possibily because of reduced contact with deleterious compounds in HFCS.

  6. Evidence supporting a cis-enediol-based mechanism for Pyrococcus furiosus phosphoglucose isomerase

    NARCIS (Netherlands)

    Berrisford, J.M.; Hounslow, A.M.; Akerboom, A.P.; Hagen, W.R.; Brouns, S.J.J.; Oost, van der J.; Murray, I.A.; Blackburn, G.M.; Waltho, J.P.; Rice, D.W.; Baker, P.J.

    2006-01-01

    The enzymatic aldose ketose isomerisation of glucose and fructose sugars involves the transfer of a hydrogen between their C1 and C2 carbon atoms and, in principle, can proceed through either a direct hydride shift or via a cis-enediol intermediate. Pyrococcus furiosus phosphoglucose isomerase (PfPG

  7. Genetics Home Reference: triosephosphate isomerase deficiency

    Science.gov (United States)

    ... Oláh J, Ovádi J. Triosephosphate isomerase deficiency: new insights into an enigmatic disease. Biochim Biophys Acta. 2009 ... healthcare professional . About Genetics Home Reference Site Map Customer Support Selection Criteria for Links USA.gov Copyright ...

  8. Functional differences in yeast protein disulfide isomerases

    DEFF Research Database (Denmark)

    Nørgaard, P; Westphal, V; Tachibana, C;

    2001-01-01

    PDI1 is the essential gene encoding protein disulfide isomerase in yeast. The Saccharomyces cerevisiae genome, however, contains four other nonessential genes with homology to PDI1: MPD1, MPD2, EUG1, and EPS1. We have investigated the effects of simultaneous deletions of these genes. In several...

  9. The Periplasmic Chaperone Network of Campylobacter jejuni: Evidence that SalC (Cj1289) and PpiD (Cj0694) Are Involved in Maintaining Outer Membrane Integrity

    Science.gov (United States)

    Taylor, Aidan J.; Zakai, Shadi A. I.; Kelly, David J.

    2017-01-01

    The outer membrane (OM) of Gram-negative pathogenic bacteria is a key structure in host–pathogen interactions that contains a plethora of proteins, performing a range of functions including adhesion, nutrient uptake, export of effectors and interaction with innate and adaptive components of the immune system. In addition, the OM can exclude drugs and thus contribute to antimicrobial resistance. The OM of the food-borne pathogen Campylobacter jejuni contains porins, adhesins and other virulence factors that must be specifically localized to this membrane, but the protein sorting mechanisms involved are only partially understood. In particular, chaperones are required to ferry OM proteins across the periplasm after they emerge from the Sec translocation system. The SurA-related chaperone PEB4 (Cj0596) is the only protein with a proven role in OM biogenesis and integrity in C. jejuni. In this work, we have constructed a set of isogenic deletion mutants in genes encoding both known and predicted chaperones (cj0596, cj0694, cj1069, cj1228c, and cj1289) using NCTC 11168H as the parental strain. These mutants were characterized using a range of assays to determine effects on growth, agglutination, biofilm formation, membrane permeability and hydrophobicity. We focused on Cj1289 and Cj0694, which our previous work suggested possessed both chaperone and peptidyl-proyl cis/trans isomerase (PPIase) domains. Mutants in either cj1289 or cj0694 showed growth defects, increased motility, agglutination and biofilm formation and severe OM permeability defects as measured by a lysozyme accessibility assay, that were comparable to those exhibited by the isogenic peb4 mutant. 2D-gel comparisons showed a general decrease in OM proteins in these mutants. We heterologously overproduced and purified Cj0694 and obtained evidence that this protein was an active PPIase, as judged by its acceleration of the refolding rate of reduced and alkylated ribonuclease T1 and that it also possessed

  10. 21 CFR 184.1372 - Insoluble glucose isomerase enzyme preparations.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Insoluble glucose isomerase enzyme preparations... enzyme preparations. (a) Insoluble glucose isomerase enzyme preparations are used in the production of... additional requirements for enzyme preparations in the Food Chemicals Codex, 3d Ed. (1981), p. 107, which...

  11. A novel chloroplastic isopentenyl diphosphate isomerase gene from Jatropha curcas: Cloning, characterization and subcellular localization

    OpenAIRE

    Wei, Lei; Yin, Li; Hu,Xiaole; Xu, Ying; Chen,Fang

    2014-01-01

    Background Jatropha curcas is a rich reservoir of pharmaceutically active terpenoids. More than 25 terpenoids have been isolated from this plant, and their activities are anti-bacterial, anti-fungal, anti-cancer, insecticidal, rodenticidal, cytotoxic and molluscicidal. But not much is known about the pathway involved in the biosynthesis of terpenoids. The present investigation describes the cloning, characterization and subcellular localization of isopentenyl diphosphate isomerase (IPI) gene ...

  12. AcEST: DK945695 [AcEST

    Lifescience Database Archive (English)

    Full Text Available TH Peptidyl-prolyl cis-trans isomerase CYP19-... 223 6e-58 sp|Q5R8S7|PPIA_PONPY Peptidyl-prolyl cis-trans isomerase A OS=Po....-prolyl cis-trans isomerase OS=Po... 276 9e-73 tr|Q6TMX3|Q6TMX3_THEHA Peptidyl-prolyl cis-trans isomerase OS...=Th... 274 3e-72 tr|A9PIY0|A9PIY0_POPJC Peptidyl-prolyl cis-trans isomerase OS=Po...._POPTR Peptidyl-prolyl cis-trans isomerase OS=Po... 272 1e-71 tr|A9PJ47|A9PJ47_POPJC Peptidyl-prolyl cis-trans isomerase OS=Po....1 tr|A9P9C8|A9P9C8_POPTR Peptidyl-prolyl cis-trans isomerase OS=Po... 271 2e-71 t

  13. AcEST: DK957169 [AcEST

    Lifescience Database Archive (English)

    Full Text Available prolyl cis-trans isomerase OS=Po... 243 7e-63 tr|Q6TMX3|Q6TMX3_THEHA Peptidyl-prolyl cis-trans isomerase OS=...Th... 241 2e-62 tr|A9PJ47|A9PJ47_POPJC Peptidyl-prolyl cis-trans isomerase OS=Po...... 241 2e-62 tr|A9P9C8|A9P9C8_POPTR Peptidyl-prolyl cis-trans isomerase OS=Po... 241 3e-62 tr|A5BQN6|A5BQN6_...VITVI Peptidyl-prolyl cis-trans isomerase OS=Vi... 241 3e-62 tr|A9PIY0|A9PIY0_POPJC Peptidyl-prolyl cis-trans isomerase OS=Po....prolyl cis-trans isomerase OS=Po... 239 1e-61 tr|A0MTQ0|A0MTQ0_SOLSG Peptidyl-prolyl cis-trans isomerase OS=

  14. AcEST: DK959102 [AcEST

    Lifescience Database Archive (English)

    Full Text Available ptidyl-prolyl cis-trans isomerase CYP19-... 223 9e-58 sp|Q5R8S7|PPIA_PONPY Peptidyl-prolyl cis-trans isomerase A OS=Po....|A9P8B6_POPTR Peptidyl-prolyl cis-trans isomerase OS=Po... 276 1e-72 tr|Q6TMX3|Q6TMX3_THEHA Peptidyl-prolyl ...cis-trans isomerase OS=Th... 274 6e-72 tr|A9PIY0|A9PIY0_POPJC Peptidyl-prolyl cis-trans isomerase OS=Po... 2...R Peptidyl-prolyl cis-trans isomerase OS=Po... 272 2e-71 tr|A9PJ47|A9PJ47_POPJC P...eptidyl-prolyl cis-trans isomerase OS=Po... 271 3e-71 tr|Q52UN0|Q52UN0_CUCSA Peptidyl-prolyl cis-trans isome

  15. Physiological and Pathogenic Roles of Prolyl Isomerase Pin1 in Metabolic Regulations via Multiple Signal Transduction Pathway Modulations

    Directory of Open Access Journals (Sweden)

    Yusuke Nakatsu

    2016-09-01

    Full Text Available Prolyl isomerases are divided into three groups, the FKBP family, Cyclophilin and the Parvulin family (Pin1 and Par14. Among these isomerases, Pin1 is a unique prolyl isomerase binding to the motif including pSer/pThr-Pro that is phosphorylated by kinases. Once bound, Pin1 modulates the enzymatic activity, protein stability or subcellular localization of target proteins by changing the cis- and trans-formations of proline. Several studies have examined the roles of Pin1 in the pathogenesis of cancers and Alzheimer’s disease. On the other hand, recent studies have newly demonstrated Pin1 to be involved in regulating glucose and lipid metabolism. Interestingly, while Pin1 expression is markedly increased by high-fat diet feeding, Pin1 KO mice are resistant to diet-induced obesity, non-alcoholic steatohepatitis and diabetic vascular dysfunction. These phenomena result from the binding of Pin1 to several key factors regulating metabolic functions, which include insulin receptor substrate-1, AMPK, Crtc2 and NF-κB p65. In this review, we focus on recent advances in elucidating the physiological roles of Pin1 as well as the pathogenesis of disorders involving this isomerase, from the viewpoint of the relationships between signal transductions and metabolic functions.

  16. Physiological and Pathogenic Roles of Prolyl Isomerase Pin1 in Metabolic Regulations via Multiple Signal Transduction Pathway Modulations.

    Science.gov (United States)

    Nakatsu, Yusuke; Matsunaga, Yasuka; Yamamotoya, Takeshi; Ueda, Koji; Inoue, Yuki; Mori, Keiichi; Sakoda, Hideyuki; Fujishiro, Midori; Ono, Hiraku; Kushiyama, Akifumi; Asano, Tomoichiro

    2016-09-07

    Prolyl isomerases are divided into three groups, the FKBP family, Cyclophilin and the Parvulin family (Pin1 and Par14). Among these isomerases, Pin1 is a unique prolyl isomerase binding to the motif including pSer/pThr-Pro that is phosphorylated by kinases. Once bound, Pin1 modulates the enzymatic activity, protein stability or subcellular localization of target proteins by changing the cis- and trans-formations of proline. Several studies have examined the roles of Pin1 in the pathogenesis of cancers and Alzheimer's disease. On the other hand, recent studies have newly demonstrated Pin1 to be involved in regulating glucose and lipid metabolism. Interestingly, while Pin1 expression is markedly increased by high-fat diet feeding, Pin1 KO mice are resistant to diet-induced obesity, non-alcoholic steatohepatitis and diabetic vascular dysfunction. These phenomena result from the binding of Pin1 to several key factors regulating metabolic functions, which include insulin receptor substrate-1, AMPK, Crtc2 and NF-κB p65. In this review, we focus on recent advances in elucidating the physiological roles of Pin1 as well as the pathogenesis of disorders involving this isomerase, from the viewpoint of the relationships between signal transductions and metabolic functions.

  17. AcEST: DK952836 [AcEST

    Lifescience Database Archive (English)

    Full Text Available Peptidyl-prolyl cis-trans isomerase A OS=Po... 223 9e-58 sp|P62941|PPIA_PAPAN Peptidyl-prolyl cis-trans isom...A9P8B6|A9P8B6_POPTR Peptidyl-prolyl cis-trans isomerase OS=Po... 276 1e-72 tr|Q6TMX3|Q6TMX3_THEHA Peptidyl-p...yl cis-trans isomerase OS=Po... 273 5e-72 tr|A8CYN7|A8CYN7_GERHY Peptidyl-prolyl cis-trans isomerase OS=Ge...... 273 7e-72 tr|A9P8L4|A9P8L4_POPTR Peptidyl-prolyl cis-trans isomerase OS=Po... 272 1e-71 tr|A9PJ47|A9PJ47_P...OPJC Peptidyl-prolyl cis-trans isomerase OS=Po... 271 2e-71 tr|Q52UN0|Q52UN0_CUCSA Peptidyl-prolyl cis-trans

  18. Dynamical role of phosphorylation on serine/threonine-proline Pin1 substrates from constant force molecular dynamics simulations.

    Science.gov (United States)

    Velazquez, Hector A; Hamelberg, Donald

    2015-02-21

    Cis-trans isomerization of peptidyl-prolyl bonds of the protein backbone plays an important role in numerous biological processes. Cis-trans isomerization can be the rate-limiting step due its extremely slow dynamics, compared to the millisecond time scale of many processes, and is catalyzed by a widely studied family of peptidyl-prolyl cis-trans isomerase enzymes. Also, mechanical forces along the peptide chain can speed up the rate of isomerization, resulting in "mechanical catalysis," and have been used to study peptidyl-prolyl cis-trans isomerization and other mechanical properties of proteins. Here, we use constant force molecular dynamics simulations to study the dynamical effects of phosphorylation on serine/threonine-proline protein motifs that are involved in the function of many proteins and have been implicated in many aberrant biological processes. We show that the rate of cis-trans isomerization is slowed down by phosphorylation, in excellent agreement with experiments. We use a well-grounded theory to describe the force dependent rate of isomerization. The calculated rates at zero force are also in excellent agreement with experimentally measured rates, providing additional validation of the models and force field parameters. Our results suggest that the slowdown in the rate upon phosphorylation is mainly due to an increase in the friction along the peptidyl-prolyl bond angle during isomerization. Our results provide a microscopic description of the dynamical effects of post-translational phosphorylation on cis-trans isomerization and insights into the properties of proteins under tension.

  19. AcEST: DK961519 [AcEST

    Lifescience Database Archive (English)

    Full Text Available ptidyl-prolyl cis-trans isomerase A OS=Po... 38 0.030 sp|P62936|PPIA_PIG Peptidyl-prolyl cis-trans isomerase...is-trans isomerase OS=Vi... 49 1e-04 tr|Q68PF9|Q68PF9_POPTR Peptidyl-prolyl cis-trans isomerase OS=Po... 48 ...ptidyl-prolyl cis-trans isomerase OS=Po... 47 7e-04 tr|A9P7Y6|A9P7Y6_POPTR Peptid...yl-prolyl cis-trans isomerase OS=Po... 47 0.001 tr|Q8VX73|Q8VX73_RICCO Peptidyl-prolyl cis-trans isomerase O...somerase OS=Gl... 45 0.002 tr|A9PJ47|A9PJ47_POPJC Peptidyl-prolyl cis-trans isomerase OS=Po... 45 0.002 tr|A

  20. Compact conformations of human protein disulfide isomerase.

    Directory of Open Access Journals (Sweden)

    Shang Yang

    Full Text Available Protein disulfide isomerase (PDI composed of four thioredoxin-like domains a, b, b', and a', is a key enzyme catalyzing oxidative protein folding in the endoplasmic reticulum. Large scale molecular dynamics simulations starting from the crystal structures of human PDI (hPDI in the oxidized and reduced states were performed. The results indicate that hPDI adopts more compact conformations in solution than in the crystal structures, which are stabilized primarily by inter-domain interactions, including the salt bridges between domains a and b' observed for the first time. A prominent feature of the compact conformations is that the two catalytic domains a and a' can locate close enough for intra-molecular electron transfer, which was confirmed by the characterization of an intermediate with a disulfide between the two domains. Mutations, which disrupt the inter-domain interactions, lead to decreased reductase activity of hPDI. Our molecular dynamics simulations and biochemical experiments reveal the intrinsic conformational dynamics of hPDI and its biological impact.

  1. The human protein disulfide isomerase gene family

    Directory of Open Access Journals (Sweden)

    Galligan James J

    2012-07-01

    Full Text Available Abstract Enzyme-mediated disulfide bond formation is a highly conserved process affecting over one-third of all eukaryotic proteins. The enzymes primarily responsible for facilitating thiol-disulfide exchange are members of an expanding family of proteins known as protein disulfide isomerases (PDIs. These proteins are part of a larger superfamily of proteins known as the thioredoxin protein family (TRX. As members of the PDI family of proteins, all proteins contain a TRX-like structural domain and are predominantly expressed in the endoplasmic reticulum. Subcellular localization and the presence of a TRX domain, however, comprise the short list of distinguishing features required for gene family classification. To date, the PDI gene family contains 21 members, varying in domain composition, molecular weight, tissue expression, and cellular processing. Given their vital role in protein-folding, loss of PDI activity has been associated with the pathogenesis of numerous disease states, most commonly related to the unfolded protein response (UPR. Over the past decade, UPR has become a very attractive therapeutic target for multiple pathologies including Alzheimer disease, Parkinson disease, alcoholic and non-alcoholic liver disease, and type-2 diabetes. Understanding the mechanisms of protein-folding, specifically thiol-disulfide exchange, may lead to development of a novel class of therapeutics that would help alleviate a wide range of diseases by targeting the UPR.

  2. AcEST: DK944198 [AcEST

    Lifescience Database Archive (English)

    Full Text Available -prolyl cis-trans isomerase CYP19-... 223 6e-58 sp|Q5R8S7|PPIA_PONPY Peptidyl-prolyl cis-trans isomerase A OS=Po....3FNQ1_HEVBR Cyclophilin OS=Hevea brasiliensis PE=2 SV=1 276 9e-73 tr|A9P8B6|A9P8B6_POPTR Peptidyl-prolyl cis-trans isomerase OS=Po....3e-72 tr|A9PIY0|A9PIY0_POPJC Peptidyl-prolyl cis-trans isomerase OS=Po... 273 4e-...idyl-prolyl cis-trans isomerase OS=Po... 272 1e-71 tr|A9PJ47|A9PJ47_POPJC Peptidyl-prolyl cis-trans isomerase OS=Po....|A9P9C8_POPTR Peptidyl-prolyl cis-trans isomerase OS=Po... 271 2e-71 tr|Q8L5T1|Q8

  3. AcEST: DK947760 [AcEST

    Lifescience Database Archive (English)

    Full Text Available mitoch... 139 5e-33 sp|Q5R8S7|PPIA_PONPY Peptidyl-prolyl cis-trans isomerase A OS=Po... 139 5e-33 sp|P62941...Peptidyl-prolyl cis-trans isomerase OS=Ae... 147 2e-34 tr|A9PIY0|A9PIY0_POPJC Peptidyl-prolyl cis-trans isomerase OS=Po....P8L4|A9P8L4_POPTR Peptidyl-prolyl cis-trans isomerase OS=Po... 146 6e-34 tr|A9P8B6|A9P8B6_POPTR Peptidyl-pro...lyl cis-trans isomerase OS=Po... 146 6e-34 tr|Q9M530|Q9M530_EUPES Peptidyl-prolyl... cis-trans isomerase (Frag... 144 3e-33 tr|A9PJ47|A9PJ47_POPJC Peptidyl-prolyl cis-trans isomerase OS=Po...

  4. AcEST: DK958384 [AcEST

    Lifescience Database Archive (English)

    Full Text Available l-prolyl cis-trans isomerase A OS=Po... 223 5e-58 sp|P62941|PPIA_PAPAN Peptidyl-prolyl cis-trans isomerase A...POPTR Peptidyl-prolyl cis-trans isomerase OS=Po... 276 6e-73 tr|A9PIY0|A9PIY0_POPJC Peptidyl-prolyl cis-trans isomerase OS=Po....|A9P8L4|A9P8L4_POPTR Peptidyl-prolyl cis-trans isomerase OS=Po... 272 9e-72 tr|A9PJ47|A9PJ47_POPJC Peptidyl-...prolyl cis-trans isomerase OS=Po... 272 1e-71 tr|A9P9C8|A9P9C8_POPTR Peptidyl-prolyl cis-trans isomerase OS=Po....S=Sorghum bicolor GN=Cyp PE=... 265 1e-69 tr|A9P7Y6|A9P7Y6_POPTR Peptidyl-prolyl

  5. NMR studies on mechanism of isomerisation of fructose 6-phosphate to glucose 6-phosphate catalysed by phosphoglucose isomerase from Thermococcus kodakarensis.

    Science.gov (United States)

    Abbas, Shahzada Nadeem; Mok, Kenneth Hun; Rashid, Naeem; Xie, Yongjing; Ruether, Manuel; O'Brien, John; Akhtar, Muhammad

    2016-06-01

    The fate of hydrogen atoms at C-2 of glucose 6-phosphate (G6P) and C-1 of fructose 6-phosphate (F6P) was studied in the reaction catalysed by phosphoglucose isomerase from Thermococcus kodakarensis (TkPGI) through 1D and 2D NMR methods. When the reaction was performed in (2)H2O the hydrogen atoms in the aforementioned positions were exchanged with deuterons indicating that the isomerization occurred by a cis-enediol intermediate involving C-1 pro-R hydrogen of F6P. These features are similar to those described for phosphoglucose isomerases from rabbit muscle and Pyrococcus furiosus.

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

    Science.gov (United States)

    Jewett, Kathy; Sandwick, Roger K.

    2011-01-01

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

  7. Main: 1J6Y [RPSD[Archive

    Lifescience Database Archive (English)

    Full Text Available 1J6Y シロイヌナズナ Arabidopsis Arabidopsis thaliana (L.) Heynh. Peptidyl-Prolyl Cis-Trans Isomerase 1 Name=Pin...dyl-Prolyl Cis-Trans Isomerase; Chain: A; Engineered: Yes Isomerase I.Landrieu, J.M.Wieruszeski, R.Wintjens,... D.Inze, G.Lippens I.Landrieu, J.M.Wieruszeski, R.Wintjens, D.Inze, G.Lippens Sol...ution Structure Of The Single-Domain Prolyl Cis/Trans Isomerase Pin1at From Arabidopsis Thaliana J.Mol.Biol.... V. 320 321 2002 Prolyl Cis/Trans Isomerase, Parvulin, Pin1, Phosphorylation GB:AAD20122,4406814|EMBL; AC006

  8. AcEST: DK956467 [AcEST

    Lifescience Database Archive (English)

    Full Text Available PY Peptidyl-prolyl cis-trans isomerase A OS=Po... 223 8e-58 sp|P62941|PPIA_PAPAN Peptidyl-prolyl cis-trans i...tr|A9P8B6|A9P8B6_POPTR Peptidyl-prolyl cis-trans isomerase OS=Po... 276 9e-73 tr|Q6TMX3|Q6TMX3_THEHA Peptidy...rolyl cis-trans isomerase OS=Po... 273 5e-72 tr|A8CYN7|A8CYN7_GERHY Peptidyl-prolyl cis-trans isomerase OS=G...e... 273 6e-72 tr|A9P8L4|A9P8L4_POPTR Peptidyl-prolyl cis-trans isomerase OS=Po... 272 1e-71 tr|A9PJ47|A9PJ4...7_POPJC Peptidyl-prolyl cis-trans isomerase OS=Po... 271 2e-71 tr|Q52UN0|Q52UN0_CUCSA Peptidyl-prolyl cis-tr

  9. L-Ribose isomerase and mannose-6-phosphate isomerase: properties and applications for L-ribose production.

    Science.gov (United States)

    Xu, Zheng; Sha, Yuanyuan; Liu, Chao; Li, Sha; Liang, Jinfeng; Zhou, Jiahai; Xu, Hong

    2016-11-01

    L-Ribose is a synthetic L-form monosaccharide. It is a building block of many novel nucleotide analog anti-viral drugs. Bio-production of L-ribose relies on a two-step reaction: (i) conversion of L-arabinose to L-ribulose by the catalytic action of L-arabinose isomerase (L-AI) and (ii) conversion of L-ribulose to L-ribose by the catalytic action of L-ribose isomerase (L-RI, EC 5.3.1.B3) or mannose-6-phosphate isomerase (MPI, EC 5.3.1.8, alternately named as phosphomannose isomerase). Between the two enzymes, L-RI is a rare enzyme that was discovered in 1996 by Professor Izumori's group, whereas MPI is an essential enzyme in metabolic pathways in humans and microorganisms. Recent studies have focused on their potentials for industrial production of L-ribose. This review summarizes the applications of L-RI and MPI for L-ribose production.

  10. AcEST: BP913577 [AcEST

    Lifescience Database Archive (English)

    Full Text Available ans isomerase slr1251... 179 1e-44 sp|Q5R8S7|PPIA_PONPY Peptidyl-prolyl cis-trans isomerase A OS=Po... 178 2...rolyl cis-trans isomerase OS=Po... 221 3e-56 tr|A9P8L4|A9P8L4_POPTR Peptidyl-prolyl cis-trans isomerase OS=Po...... 220 4e-56 tr|A9P8B6|A9P8B6_POPTR Peptidyl-prolyl cis-trans isomerase OS=Po.....isomerase OS=Vi... 213 9e-54 tr|A9PJ47|A9PJ47_POPJC Peptidyl-prolyl cis-trans isomerase OS=Po...... 212 1e-53 tr|A9P9C8|A9P9C8_POPTR Peptidyl-prolyl cis-trans isomerase OS=Po... 212 2e-53 tr|Q

  11. Erythritol feeds the pentose phosphate pathway via three new isomerases leading to D-erythrose-4-phosphate in Brucella.

    Science.gov (United States)

    Barbier, Thibault; Collard, François; Zúñiga-Ripa, Amaia; Moriyón, Ignacio; Godard, Thibault; Becker, Judith; Wittmann, Christoph; Van Schaftingen, Emile; Letesson, Jean-Jacques

    2014-12-16

    Erythritol is an important nutrient for several α-2 Proteobacteria, including N2-fixing plant endosymbionts and Brucella, a worldwide pathogen that finds this four-carbon polyol in genital tissues. Erythritol metabolism involves phosphorylation to L-erythritol-4-phosphate by the kinase EryA and oxidation of the latter to L-3-tetrulose 4-phosphate by the dehydrogenase EryB. It is accepted that further steps involve oxidation by the putative dehydrogenase EryC and subsequent decarboxylation to yield triose-phosphates. Accordingly, growth on erythritol as the sole C source should require aldolase and fructose-1,6-bisphosphatase to produce essential hexose-6-monophosphate. However, we observed that a mutant devoid of fructose-1,6-bisphosphatases grew normally on erythritol and that EryC, which was assumed to be a dehydrogenase, actually belongs to the xylose isomerase superfamily. Moreover, we found that TpiA2 and RpiB, distant homologs of triose phosphate isomerase and ribose 5-phosphate isomerase B, were necessary, as previously shown for Rhizobium. By using purified recombinant enzymes, we demonstrated that L-3-tetrulose-4-phosphate was converted to D-erythrose 4-phosphate through three previously unknown isomerization reactions catalyzed by EryC (tetrulose-4-phosphate racemase), TpiA2 (D-3-tetrulose-4-phosphate isomerase; renamed EryH), and RpiB (D-erythrose-4-phosphate isomerase; renamed EryI), a pathway fully consistent with the isotopomer distribution of the erythrose-4-phosphate-derived amino acids phenylalanine and tyrosine obtained from bacteria grown on (13)C-labeled erythritol. D-erythrose-4-phosphate is then converted by enzymes of the pentose phosphate pathway to glyceraldehyde 3-phosphate and fructose 6-phosphate, thus bypassing fructose-1,6-bisphosphatase. This is the first description to our knowledge of a route feeding carbohydrate metabolism exclusively via D-erythrose 4-phosphate, a pathway that may provide clues to the preferential metabolism of

  12. The fermentation of lignocellulose hydrolysates with xylose isomerases and yeasts

    Energy Technology Data Exchange (ETDEWEB)

    Linden, T.

    1992-01-01

    Untreated spent sulphite liquor (SSL) was fermented with Canida tropicalis, Pichia stipitis, Pachysolen tannophilus, Schizosaccharomyces pombe, Saccharomyces cerevisiae and a co-culture of P. Tannophilus and A. cerevisiae, in the presence of xylose isomerases and 4.6 mM azide. The highest yield of ethanol, 0.41 g/g total sugar was obtained with S. cerevisiae, C. tropicalis, and P. tannophilus produced considerble amounts of polyoles, mainly xylitol. With P. stipitis sugar uptake was rapidly inhibited in untreated SSL. The presence of azide contributed to the yield by about 0.04. The fermentation of hydrogen fluoride-pretreated and acid-hydrolysed wheat straw with S. cerevisiae, xylose isomerase, and azide gave a yield of 0.40 g ethanol/g total sugar. In this substrate the xylose utilisation was 84% compared with 51% in SSL. In the concentration range appropriate for enzymatic xylose isomerization, xylulose was measured in a lignocellulose hydrolysate using HPLC with two hydrogen loaded ion exchange columns in series. SSL was used as a model for lignocellulose hydrolysates. The enzymatic isomerization of xylose to xylulose was followed directly in SSL, providing a method for the direct determination of xylose isomerase activity in lignocellulose hydrolysates. Three different xylose isomerase preparations of L. brevis whole cells were compared with a commercial enzyme preparation Maxazyme GI-immob., with respect to activity and stability. From a continuous SSL fermentation plant, two species of yeasts were isolated, S. cerevisiae and Pichia membranaefaciens. One of the isolates of S. cerevisiae, no. 3 was heavily flocculating. Without acetic acid present, both bakers' yeast and isolate no. 3 showed catabolite repression and fermented glucose and galactose sequentially. Galactose fermentation with bakers' yeast was strongly inhibited by acetic acid at pH values below 6. Isolate no. 3 fermented galactose, glucose and mannose, in the presence of acetic acid

  13. The fermentation of lignocellulose hydrolysates with xylose isomerases and yeasts

    Energy Technology Data Exchange (ETDEWEB)

    Linden, T.

    1992-09-01

    Untreated spent sulphite liquor (SSL) was fermented with Canida tropicalis, Pichia stipitis, Pachysolen tannophilus, Schizosaccharomyces pombe, Saccharomyces cerevisiae and a co-culture of P. Tannophilus and A. cerevisiae, in the presence of xylose isomerases and 4.6 mM azide. The highest yield of ethanol, 0.41 g/g total sugar was obtained with S. cerevisiae, C. tropicalis, and P. tannophilus produced considerble amounts of polyoles, mainly xylitol. With P. stipitis sugar uptake was rapidly inhibited in untreated SSL. The presence of azide contributed to the yield by about 0.04. The fermentation of hydrogen fluoride-pretreated and acid-hydrolysed wheat straw with S. cerevisiae, xylose isomerase, and azide gave a yield of 0.40 g ethanol/g total sugar. In this substrate the xylose utilisation was 84% compared with 51% in SSL. In the concentration range appropriate for enzymatic xylose isomerization, xylulose was measured in a lignocellulose hydrolysate using HPLC with two hydrogen loaded ion exchange columns in series. SSL was used as a model for lignocellulose hydrolysates. The enzymatic isomerization of xylose to xylulose was followed directly in SSL, providing a method for the direct determination of xylose isomerase activity in lignocellulose hydrolysates. Three different xylose isomerase preparations of L. brevis whole cells were compared with a commercial enzyme preparation Maxazyme GI-immob., with respect to activity and stability. From a continuous SSL fermentation plant, two species of yeasts were isolated, S. cerevisiae and Pichia membranaefaciens. One of the isolates of S. cerevisiae, no. 3 was heavily flocculating. Without acetic acid present, both bakers` yeast and isolate no. 3 showed catabolite repression and fermented glucose and galactose sequentially. Galactose fermentation with bakers` yeast was strongly inhibited by acetic acid at pH values below 6. Isolate no. 3 fermented galactose, glucose and mannose, in the presence of acetic acid even at pH.

  14. Gene regulation in response to protein disulphide isomerase deficiency

    DEFF Research Database (Denmark)

    Nørgaard, Per; Tachibana, Christine; Bruun, Anette W

    2003-01-01

    We have examined the activities of promoters of a number of yeast genes encoding resident endoplasmic reticulum proteins, and found increased expression in a strain with severe protein disulphide isomerase deficiency. Serial deletion in the promoter of the MPD1 gene, which encodes a PDI1-homologu...... element. The sequence (GACACG) does not resemble the unfolded protein response element. It is present in the upstream regions of the MPD1, MPD2, KAR2, PDI1 and ERO1 genes....

  15. The small subunit 1 of the Arabidopsis isopropylmalate isomerase is required for normal growth and development and the early stages of glucosinolate formation.

    Science.gov (United States)

    Imhof, Janet; Huber, Florian; Reichelt, Michael; Gershenzon, Jonathan; Wiegreffe, Christoph; Lächler, Kurt; Binder, Stefan

    2014-01-01

    In Arabidopsis thaliana the evolutionary and functional relationship between Leu biosynthesis and the Met chain elongation pathway, the first part of glucosinolate formation, is well documented. Nevertheless the exact functions of some pathway components are still unclear. Isopropylmalate isomerase (IPMI), an enzyme usually involved in Leu biosynthesis, is a heterodimer consisting of a large and a small subunit. While the large protein is encoded by a single gene (isopropylmalate isomerase large subunit1), three genes encode small subunits (isopropylmalate isomerase small subunit1 to 3). We have now analyzed small subunit 1 (isopropylmalate isomerase small subunit1) employing artificial microRNA for a targeted knockdown of the encoding gene. Strong reduction of corresponding mRNA levels to less than 5% of wild-type levels resulted in a severe phenotype with stunted growth, narrow pale leaf blades with green vasculature and abnormal adaxial-abaxial patterning as well as anomalous flower morphology. Supplementation of the knockdown plants with leucine could only partially compensate for the morphological and developmental abnormalities. Detailed metabolite profiling of the knockdown plants revealed changes in the steady state levels of isopropylmalate and glucosinolates as well as their intermediates demonstrating a function of IPMI SSU1 in both leucine biosynthesis and the first cycle of Met chain elongation. Surprisingly the levels of free leucine slightly increased suggesting an imbalanced distribution of leucine within cells and/or within plant tissues.

  16. The small subunit 1 of the Arabidopsis isopropylmalate isomerase is required for normal growth and development and the early stages of glucosinolate formation.

    Directory of Open Access Journals (Sweden)

    Janet Imhof

    Full Text Available In Arabidopsis thaliana the evolutionary and functional relationship between Leu biosynthesis and the Met chain elongation pathway, the first part of glucosinolate formation, is well documented. Nevertheless the exact functions of some pathway components are still unclear. Isopropylmalate isomerase (IPMI, an enzyme usually involved in Leu biosynthesis, is a heterodimer consisting of a large and a small subunit. While the large protein is encoded by a single gene (isopropylmalate isomerase large subunit1, three genes encode small subunits (isopropylmalate isomerase small subunit1 to 3. We have now analyzed small subunit 1 (isopropylmalate isomerase small subunit1 employing artificial microRNA for a targeted knockdown of the encoding gene. Strong reduction of corresponding mRNA levels to less than 5% of wild-type levels resulted in a severe phenotype with stunted growth, narrow pale leaf blades with green vasculature and abnormal adaxial-abaxial patterning as well as anomalous flower morphology. Supplementation of the knockdown plants with leucine could only partially compensate for the morphological and developmental abnormalities. Detailed metabolite profiling of the knockdown plants revealed changes in the steady state levels of isopropylmalate and glucosinolates as well as their intermediates demonstrating a function of IPMI SSU1 in both leucine biosynthesis and the first cycle of Met chain elongation. Surprisingly the levels of free leucine slightly increased suggesting an imbalanced distribution of leucine within cells and/or within plant tissues.

  17. AcEST: DK959011 [AcEST

    Lifescience Database Archive (English)

    Full Text Available 8B6|A9P8B6_POPTR Peptidyl-prolyl cis-trans isomerase OS=Po... 250 3e-65 tr|B3FNQ1...is-trans isomerase OS=Vi... 249 5e-65 tr|A9PIY0|A9PIY0_POPJC Peptidyl-prolyl cis-trans isomerase OS=Po... 24...ptidyl-prolyl cis-trans isomerase OS=Po... 247 3e-64 tr|A9PJ47|A9PJ47_POPJC Peptidyl-prolyl cis-trans isomerase OS=Po...... 246 7e-64 tr|A9P9C8|A9P9C8_POPTR Peptidyl-prolyl cis-trans isomerase OS=Po... 246 7e-64 tr|Q8L5...Y6_POPTR Peptidyl-prolyl cis-trans isomerase OS=Po... 241 2e-62 >tr|Q8VX73|Q8VX73_RICCO Peptidyl-prolyl cis-

  18. Cyclophilin D Is Involved in the Regulation of Autophagy and Affects the Lifespan of P. anserina in Response to Mitochondrial Oxidative Stress

    Science.gov (United States)

    Kramer, Piet; Jung, Alexander T.; Hamann, Andrea; Osiewacz, Heinz D.

    2016-01-01

    The mitochondrial permeability transition pore plays a key role in programmed cell death and the induction of autophagy. Opening of the pore is regulated by the mitochondrial peptidyl prolyl-cis, trans-isomerase cyclophilin D (CYPD). Previously it was shown in the aging model organism Podospora anserina that PaCYPD abundance increases during aging and that PaCypD overexpressors are characterized by accelerated aging. Here, we describe a role of PaCYPD in the regulation of autophagy. We found that the accelerated aging phenotype observed in a strain overexpressing PaCypD is not metacaspase-dependent but is accompanied by an increase of general autophagy and mitophagy, the selective autophagic degradation of mitochondria. It thus is linked to what has been defined as “autophagic cell death” or “type II” programmed cell death. Moreover, we found that the previously demonstrated age-related induction of autophagy in wild-type aging depends on the presence of PaCYPD. Deletion of PaCypD leads to a decrease in autophagy in later stages of age and under paraquat-mediated oxidative stress. Finally, we report that PaCYPD is also required for mitohormesis, the beneficial effect of mild mitochondrial stress. Thus, PaCYPD plays a key role in the context-dependent regulation of pathways leading to pro-survival and pro-death effects of autophagy. PMID:27683587

  19. X-ray structure of linalool dehydratase/isomerase from Castellaniella defragrans reveals enzymatic alkene synthesis.

    Science.gov (United States)

    Weidenweber, Sina; Marmulla, Robert; Ermler, Ulrich; Harder, Jens

    2016-05-01

    Linalool dehydratase/isomerase (Ldi), an enzyme of terpene degradation in Castellaniella defragrans, isomerizes the primary monoterpene alcohol geraniol into the tertiary alcohol (S)-linalool and dehydrates (S)-linalool to the alkene β-myrcene. Here we report on the crystal structures of Ldi with and without terpene substrates, revealing a cofactor-free homopentameric enzyme. The substrates were embedded inside a hydrophobic channel between two monomers of the (α,α)6 barrel fold class and flanked by three clusters of polar residues involved in acid-base catalysis. The detailed view into the active site will guide future biotechnological applications of Ldi, in particular, for industrial butadiene and isoprene production from renewable sources.

  20. Thermodynamics of Enzyme-Catalyzed Reactions: Part 5. Isomerases and Ligases

    Science.gov (United States)

    Goldberg, Robert N.; Tewari, Yadu B.

    1995-11-01

    Equilibrium constants and enthalpy changes for reactions catalyzed by the isomerase and ligase classes of enzymes have been compiled. For each reaction the following information is given: the reference for the data; the reaction studied; the name of the enzyme used and its Enzyme Commission number; the method of measurement; the conditions of measurement (temperature, pH, ionic strength, and the buffer(s) and cofactor(s) used); the data and an evaluation of it; and, sometimes, commentary on the data and on any corrections which have been applied to it or any calculations for which the data have been used. The data from 176 references have been examined and evaluated. Chemical Abstract Service registry numbers are given for the substances involved in these various reactions. There is a cross reference between the substances and the Enzyme Commission numbers of the enzymes used to catalyze the reactions in which the substances participate.

  1. AcEST: DK957490 [AcEST

    Lifescience Database Archive (English)

    Full Text Available merase CYP19-... 223 7e-58 sp|Q5R8S7|PPIA_PONPY Peptidyl-prolyl cis-trans isomerase A OS=Po... 223 9e-58 sp|...in OS=Hevea brasiliensis PE=2 SV=1 276 1e-72 tr|A9P8B6|A9P8B6_POPTR Peptidyl-prolyl cis-trans isomerase OS=Po..... 274 4e-72 tr|A9PIY0|A9PIY0_POPJC Peptidyl-prolyl cis-trans isomerase OS=Po... 273 5e-72 tr|A8CYN7|A8CYN7_G...ERHY Peptidyl-prolyl cis-trans isomerase OS=Ge... 273 7e-72 tr|A9P8L4|A9P8L4_POPTR Peptidyl-prolyl cis-trans isomerase OS=Po...... 272 2e-71 tr|A9PJ47|A9PJ47_POPJC Peptidyl-prolyl cis-trans isomerase OS=Po... 271 2e-71

  2. AcEST: DK951271 [AcEST

    Lifescience Database Archive (English)

    Full Text Available R8S7|PPIA_PONPY Peptidyl-prolyl cis-trans isomerase A OS=Po... 220 5e-57 sp|P62941|PPIA_PAPAN Peptidyl-proly...5e-72 tr|A9P8B6|A9P8B6_POPTR Peptidyl-prolyl cis-trans isomerase OS=Po... 273 5e-72 tr|Q6TMX3|Q6TMX3_THEHA P...idyl-prolyl cis-trans isomerase OS=Po... 271 3e-71 tr|A8CYN7|A8CYN7_GERHY Peptidyl-prolyl cis-trans isomeras...e OS=Ge... 271 3e-71 tr|A9P8L4|A9P8L4_POPTR Peptidyl-prolyl cis-trans isomerase OS=Po... 270 8e-71 tr|A9PJ47...|A9PJ47_POPJC Peptidyl-prolyl cis-trans isomerase OS=Po... 269 1e-70 tr|Q52UN0|Q52UN0_CUCSA Peptidyl-prolyl

  3. AcEST: DK954860 [AcEST

    Lifescience Database Archive (English)

    Full Text Available CYPH_LUPLU Peptidyl-prolyl cis-trans isomerase OS=Lupi... 95 9e-20 sp|Q5R8S7|PPIA_PONPY Peptidyl-prolyl cis-trans isomerase A OS=Po....... 100 4e-20 tr|A9PIY0|A9PIY0_POPJC Peptidyl-prolyl cis-trans isomerase OS=Po... 100 5e-20 tr|A9P8L4|A9P8L4..._POPTR Peptidyl-prolyl cis-trans isomerase OS=Po... 100 6e-20 tr|A9P8B6|A9P8B6_POPTR Peptidyl-prolyl cis-trans isomerase OS=Po..... 97 3e-19 tr|A9PJ47|A9PJ47_POPJC Peptidyl-prolyl cis-trans isomerase OS=Po... 97 3e-19 >tr|B3FNQ1|B3FNQ1_HE

  4. AcEST: DK946707 [AcEST

    Lifescience Database Archive (English)

    Full Text Available tidyl-prolyl cis-trans isomerase OS=Po... 222 6e-57 tr|A5BQN6|A5BQN6_VITVI Peptidyl-prolyl cis-trans isomera...OS=Ge... 220 2e-56 tr|A9PIY0|A9PIY0_POPJC Peptidyl-prolyl cis-trans isomerase OS=Po... 220 3e-56 tr|Q8W171|Q...s-trans isomerase OS=Th... 219 5e-56 tr|A9PJ47|A9PJ47_POPJC Peptidyl-prolyl cis-trans isomerase OS=Po...... 219 7e-56 tr|A9P9C8|A9P9C8_POPTR Peptidyl-prolyl cis-trans isomerase OS=Po... 219 7e...tidyl-prolyl cis-trans isomerase OS=Po... 218 9e-56 tr|Q9XF12|Q9XF12_SOLTU Peptid

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

    OpenAIRE

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

    2015-01-01

    Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. It is involved in glycolysis and in the regeneration of glucose-6-P molecules in the oxidative pentose phosphate pathway (OPPP). In chloroplasts of illuminated mesophyll cells PGI also connects the Calvin-Benson cycle with the starch biosynthetic pathway. In this work we isolated pgi1-3, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of th...

  6. GPI Mount Scopus--a variant of glucosephosphate isomerase deficiency.

    Science.gov (United States)

    Shalev, O; Shalev, R S; Forman, L; Beutler, E

    1993-10-01

    Glucosephosphate isomerase (GPI) deficiency is an unusual cause of hereditary nonspherocytic hemolytic anemia. The disease, inherited as an autosomal recessive disorder, is most often manifested by symptoms and signs of chronic hemolysis, ameliorated by splenectomy. We recently diagnosed GPI deficiency in a 23-year-old Ashkenazi Jewish man who displayed the typical clinical course of this disorder. The biophysical characteristics of the GPI variant are slow electrophoretic mobility, presence of only one of the two bands normally present, and extreme thermolability. To the best of our knowledge, this is the first report of GPI deficiency in a patient of Jewish descent, and we propose to designate this enzyme variant "GPI Mount Scopus".

  7. Geometry matters: inverse cytotoxic relationship for cis/trans-Ru(ii) polypyridyl complexes from cis/trans-[PtCl2(NH3)2].

    Science.gov (United States)

    Wachter, Erin; Zamora, Ana; Heidary, David K; Ruiz, José; Glazer, Edith C

    2016-08-09

    Two thermally activated ruthenium(ii) polypyridyl complexes, cis-Ru(bpy)2Cl2 and trans-Ru(qpy)Cl2 were investigated to determine the impact of the geometric arrangement of the exchangable ligands on the potential of the compounds to act as chemotherapeutics. In contrast to the geometry requirements for cisplatin, trans-Ru(qpy)Cl2 was 7.1-9.5× more cytotoxic than cis-Ru(bpy)2Cl2. This discovery could open up a new area of metal-based chemotherapeutic research.

  8. Characterization of a mutant glucose isomerase from Thermoanaerobacterium saccharolyticum.

    Science.gov (United States)

    Xu, Heng; Shen, Dong; Wu, Xue-Qiang; Liu, Zhi-Wei; Yang, Qi-He

    2014-10-01

    A series of site-directed mutant glucose isomerase at tryptophan 139 from Thermoanaerobacterium saccharolyticum strain B6A were purified to gel electrophoretic homogeneity, and the biochemical properties were determined. W139F mutation is the most efficient mutant derivative with a tenfold increase in its catalytic efficiency toward glucose compared with the native GI. With a maximal activity at 80 °C of 59.58 U/mg on glucose, this mutant derivative is the most active type ever reported. The enzyme activity was maximal at 90 °C and like other glucose isomerase, this mutant enzyme required Co(2+) or Mg(2+) for enzyme activity and thermal stability (stable for 20 h at 80 °C in the absence of substrate). Its optimum pH was around 7.0, and it had 86 % of its maximum activity at pH 6.0 incubated for 12 h at 60 °C. This enzyme was determined as thermostable and weak-acid stable. These findings indicated that the mutant GI W139F from T. saccharolyticum strain B6A is appropriate for use as a potential candidate for high-fructose corn syrup producing enzyme.

  9. Preparation of fluorescence quenched libraries containing interchain disulphide bonds for studies of protein disulphide isomerases

    DEFF Research Database (Denmark)

    Spetzler, J C; Westphal, V; Winther, Jakob R.

    1998-01-01

    Protein disulphide isomerase is an enzyme that catalyses disulphide redox reactions in proteins. In this paper, fluorogenic and interchain disulphide bond containing peptide libraries and suitable substrates, useful in the study of protein disulphide isomerase, are described. In order to establish...... the quenching chromophore (Tyr(NO2)) and Cys(pNpys) activated for reaction with a second thiol. The formation and cleavage of the interchain disulphide bonds in the library were monitored under a fluorescence microscope. Substrates to investigate the properties of protein disulphide isomerase in solution were...

  10. Determination of the amino acid requirements for a protein hinge in triosephosphate isomerase.

    OpenAIRE

    Sun, J.; Sampson, N. S.

    1998-01-01

    We have determined the sequence requirements for a protein hinge in triosephosphate isomerase. The codons encoding the hinge at the C-terminus of the active-site lid of triosephosphate isomerase were replaced with a genetic library of all possible 8,000 amino acid combinations. The most active of these 8,000 mutants were selected using in vivo complementation of a triosephosphate isomerase deficient strain of E. coli, DF502. Approximately 3% of the mutants complement DF502 with an activity th...

  11. Solution structure of 3-oxo-delta5-steroid isomerase.

    Science.gov (United States)

    Wu, Z R; Ebrahimian, S; Zawrotny, M E; Thornburg, L D; Perez-Alvarado, G C; Brothers, P; Pollack, R M; Summers, M F

    1997-04-18

    The three-dimensional structure of the enzyme 3-oxo-delta5-steroid isomerase (E.C. 5.3.3.1), a 28-kilodalton symmetrical dimer, was solved by multidimensional heteronuclear magnetic resonance spectroscopy. The two independently folded monomers pack together by means of extensive hydrophobic and electrostatic interactions. Each monomer comprises three alpha helices and a six-strand mixed beta-pleated sheet arranged to form a deep hydrophobic cavity. Catalytically important residues Tyr14 (general acid) and Asp38 (general base) are located near the bottom of the cavity and positioned as expected from mechanistic hypotheses. An unexpected acid group (Asp99) is also located in the active site adjacent to Tyr14, and kinetic and binding studies of the Asp99 to Ala mutant demonstrate that Asp99 contributes to catalysis by stabilizing the intermediate.

  12. Three phenotypes of glucosephosphate isomerase in sheep: improved staining recipe.

    Science.gov (United States)

    Manwell, C; Baker, C M; Graydon, R J

    1985-01-01

    Contrary to results published recently, we observe three, rather than two, phenotypes for the enzyme glucosephosphate isomerase (EC 5.3.1.9) from sheep. The phenotypic electrophoretic patterns conform to the patterns observed for this dimeric enzyme in other species. Genotype frequencies in a flock of Southdowns do not deviate significantly from those predicted under the assumption of the Hardy-Weinberg equilibrium. A remarkable observation is that the electrophoretically distinct phenotypes of GPI are largely or entirely obliterated by the addition of 1-10 mmol/l MgCl2 to the electrophoretic buffers. Modification of the usual staining recipe for GPI result in greater resolution and shorter staining times.

  13. New role of flavin as a general acid-base catalyst with no redox function in type 2 isopentenyl-diphosphate isomerase.

    Science.gov (United States)

    Unno, Hideaki; Yamashita, Satoshi; Ikeda, Yosuke; Sekiguchi, Shin-Ya; Yoshida, Norie; Yoshimura, Tohru; Kusunoki, Masami; Nakayama, Toru; Nishino, Tokuzo; Hemmi, Hisashi

    2009-04-03

    Using FMN and a reducing agent such as NAD(P)H, type 2 isopentenyl-diphosphate isomerase catalyzes isomerization between isopentenyl diphosphate and dimethylallyl diphosphate, both of which are elemental units for the biosynthesis of highly diverse isoprenoid compounds. Although the flavin cofactor is expected to be integrally involved in catalysis, its exact role remains controversial. Here we report the crystal structures of the substrate-free and complex forms of type 2 isopentenyl-diphosphate isomerase from the thermoacidophilic archaeon Sulfolobus shibatae, not only in the oxidized state but also in the reduced state. Based on the active-site structures of the reduced FMN-substrate-enzyme ternary complexes, which are in the active state, and on the data from site-directed mutagenesis at highly conserved charged or polar amino acid residues around the active site, we demonstrate that only reduced FMN, not amino acid residues, can catalyze proton addition/elimination required for the isomerase reaction. This discovery is the first evidence for this long suspected, but previously unobserved, role of flavins just as a general acid-base catalyst without playing any redox roles, and thereby expands the known functions of these versatile coenzymes.

  14. Mammalian peptide isomerase: platypus-type activity is present in mouse heart.

    Science.gov (United States)

    Koh, Jennifer M S; Chow, Stephanie J P; Crossett, Ben; Kuchel, Philip W

    2010-06-01

    Male platypus (Ornithorhynchus anatinus) venom has a peptidyl aminoacyl L/D-isomerase (hereafter called peptide isomerase) that converts the second amino acid residue in from the N-terminus from the L- to the D-form, and vice versa. A reversed-phase high-performance liquid chromatography (RP-HPLC) assay has been developed to monitor the interconversion using synthetic hexapeptides derived from defensin-like peptide-2 (DLP-2) and DLP-4 as substrates. It was hypothesised that animals other than the platypus would have peptide isomerase with the same substrate specificity. Accordingly, eight mouse tissues were tested and heart was shown to have the activity. This is notable for being the first evidence of a peptide isomerase being present in a higher mammal and heralds finding the activity in man.

  15. Cumene peroxide and Fe(2+)-ascorbate-induced lipid peroxidation and effect of phosphoglucose isomerase.

    Science.gov (United States)

    Agadjanyan, Z S; Dugin, S F; Dmitriev, L F

    2006-09-01

    Malondialdehyde (MDA) is one of cytotoxic aldehydes produced in cells as a result of lipid peroxidation and further MDA metabolism in cytoplasm is not known. In our experiments the liver fraction 10,000 g containing phosphoglucose isomerase and enzymes of the glyoxalase system was used and obtained experimental data shows that in this fraction there is an aggregate of reactions taking place both in membranes (lipid peroxidation) and outside membranes. MDA accumulation is relatively slow because MDA is a substrate of aldehyde isomerase (MDA methylglyoxal). The well known enzyme phosphoglucose isomerase acts as an aldehyde isomerase (Michaelis constant for this enzyme Km = 133 +/- 8 microM). MDA conversion to methylglyoxal and further to neutral product D-lactate (with GSH as a cofactor) occurs in cytoplasm and D-lactate should be regarded as the end product of two different parametabolic reactions: lipid peroxidation or protein glycation.

  16. A single and two step isomerization process for d-tagatose and l-ribose bioproduction using l-arabinose isomerase and d-lyxose isomerase.

    Science.gov (United States)

    Patel, Manisha J; Akhani, Rekha C; Patel, Arti T; Dedania, Samir R; Patel, Darshan H

    2017-02-01

    l-ribose and d-tagatose are biochemically synthesized using sugar isomerases. The l-arabinose isomerase gene from Shigella flexneri (Sf-AI) was cloned and expressed in Escherichia coli BL-21. Sf-AI was applied for the bioproduction of d-tagatose from d-galactose. l-ribose synthesis was performed by two step isomerization using Sf-AI and d-lyxose/ribose isomerase from Cohnella laevoribosii. The overall 22.3% and 25% conversion rate were observed for d-tagatose and l-ribose production from d-galactose and l-arabinose respectively. In the present manuscript, synthesis of rare sugars from naturally available sugars is discussed along with the biochemical characterization of Sf-AI and its efficiency.

  17. Detection of platypus-type L/D-peptide isomerase activity in aqueous extracts of papaya fruit.

    Science.gov (United States)

    Arakawa, Kensuke; Koh, Jennifer M S; Crossett, Ben; Torres, Allan M; Kuchel, Philip W

    2012-09-01

    Peptide isomerase catalyses the post-translational isomerisation of the L: - to the D: -form of an amino acid residue around the N/C-termini of substrate peptides. To date, some peptide isomerases have been found in a limited number of animal secretions and cells. We show here that papaya extracts have weak peptide isomerase activity. The activity was detected in each 30-100 kDa fraction of the flesh and the seed extracts of unripe and ripe papaya fruit. The definitive activity was confirmed in the ripe papaya extracts, but even then it was much less active than that of the other peptide isomerases previously reported. The activity was markedly inhibited by methanol, and partly so by amastatin and diethyl pyrocarbonate. This is the first report of peptide isomerase activity in a plant and suggests that perhaps every living organism may have some peptide isomerase activity.

  18. The contributions of protein disulfide isomerase and its homologues to oxidative protein folding in the yeast endoplasmic reticulum

    DEFF Research Database (Denmark)

    Xiao, Ruoyu; Wilkinson, Bonney; Solovyov, Anton;

    2004-01-01

    and one or more of its ER homologues (Mpd1p, Mpd2p, Eug1p, Eps1p) are required for efficient carboxypeptidase Y maturation. Consistent with its function as a disulfide isomerase in vivo, the active sites of Pdi1p are partially reduced (32 +/- 8%) in vivo. These results suggest that PDI and its ER......In vitro, protein disulfide isomerase (Pdi1p) introduces disulfides into proteins (oxidase activity) and provides quality control by catalyzing the rearrangement of incorrect disulfides (isomerase activity). Protein disulfide isomerase (PDI) is an essential protein in Saccharomyces cerevisiae......, but the contributions of the catalytic activities of PDI to oxidative protein folding in the endoplasmic reticulum (ER) are unclear. Using variants of Pdi1p with impaired oxidase or isomerase activity, we show that isomerase-deficient mutants of PDI support wild-type growth even in a strain in which all of the PDI...

  19. Molecular phylogenetics and comparative modeling of HEN1, a methyltransferase involved in plant microRNA biogenesis

    Directory of Open Access Journals (Sweden)

    Obarska Agnieszka

    2006-01-01

    Full Text Available Abstract Background Recently, HEN1 protein from Arabidopsis thaliana was discovered as an essential enzyme in plant microRNA (miRNA biogenesis. HEN1 transfers a methyl group from S-adenosylmethionine to the 2'-OH or 3'-OH group of the last nucleotide of miRNA/miRNA* duplexes produced by the nuclease Dicer. Previously it was found that HEN1 possesses a Rossmann-fold methyltransferase (RFM domain and a long N-terminal extension including a putative double-stranded RNA-binding motif (DSRM. However, little is known about the details of the structure and the mechanism of action of this enzyme, and about its phylogenetic origin. Results Extensive database searches were carried out to identify orthologs and close paralogs of HEN1. Based on the multiple sequence alignment a phylogenetic tree of the HEN1 family was constructed. The fold-recognition approach was used to identify related methyltransferases with experimentally solved structures and to guide the homology modeling of the HEN1 catalytic domain. Additionally, we identified a La-like predicted RNA binding domain located C-terminally to the DSRM domain and a domain with a peptide prolyl cis/trans isomerase (PPIase fold, but without the conserved PPIase active site, located N-terminally to the catalytic domain. Conclusion The bioinformatics analysis revealed that the catalytic domain of HEN1 is not closely related to any known RNA:2'-OH methyltransferases (e.g. to the RrmJ/fibrillarin superfamily, but rather to small-molecule methyltransferases. The structural model was used as a platform to identify the putative active site and substrate-binding residues of HEN and to propose its mechanism of action.

  20. Glucose isomerization in simulated moving bed reactor by Glucose isomerase

    Directory of Open Access Journals (Sweden)

    Eduardo Alberto Borges da Silva

    2006-05-01

    Full Text Available Studies were carried out on the production of high-fructose syrup by Simulated Moving Bed (SMB technology. A mathematical model and numerical methodology were used to predict the behavior and performance of the simulated moving bed reactors and to verify some important aspects for application of this technology in the isomerization process. The developed algorithm used the strategy that considered equivalences between simulated moving bed reactors and true moving bed reactors. The kinetic parameters of the enzymatic reaction were obtained experimentally using discontinuous reactors by the Lineweaver-Burk technique. Mass transfer effects in the reaction conversion using the immobilized enzyme glucose isomerase were investigated. In the SMB reactive system, the operational variable flow rate of feed stream was evaluated to determine its influence on system performance. Results showed that there were some flow rate values at which greater purities could be obtained.Neste trabalho a tecnologia de Leito Móvel Simulado (LMS reativo é aplicada no processo de isomerização da glicose visando à produção de xarope concentrado de frutose. É apresentada a modelagem matemática e uma metodologia numérica para predizer o comportamento e o desempenho de unidades reativas de leito móvel simulado para verificar alguns aspectos importantes para o emprego desta tecnologia no processo de isomerização. O algoritmo desenvolvido utiliza a abordagem que considera as equivalências entre as unidades reativas de leito móvel simulado e leito móvel verdadeiro. Parâmetros cinéticos da reação enzimática são obtidos experimentalmente usando reatores em batelada pela técnica Lineweaver-Burk. Efeitos da transferência de massa na conversão de reação usando a enzima imobilizada glicose isomerase são verificados. No sistema reativo de LMS, a variável operacional vazão da corrente de alimentação é avaliada para conhecer o efeito de sua influência no

  1. Comparison of the Recombinant Glucosephosphate Isomerase from Different Zymodemes of Entamoeba histolytica with Their Natural Counterparts by Isoenzyme Electrophoresis

    Directory of Open Access Journals (Sweden)

    E Razmjou

    2005-09-01

    Full Text Available Entamoeba histolytica is the etiological agent of invasive amoebiasis, the third leading parasitic cause of mortality in the world. Our aim was to find a molecular correlation between a glucosephosphate isomerase zymodeme analyses in E. histolytica zymodemes. It was demonstrated that natural and recombinant glucosephosphate isomerase enzymes of E. histolytica comigrated in the starch gel electrophoresis, indicating that the isoenzyme pattern of E. histolytica glucosephosphate isomerase could be explained from the primary sequences alone and means that expression of the polypeptides of the described sequences in Escherichia coli are able to reproduce the classical glucosephosphate isomerase isoenzyme patterns.

  2. Diversifying selection underlies the origin of allozyme polymorphism at the phosphoglucose isomerase locus in Tigriopus californicus.

    Directory of Open Access Journals (Sweden)

    Sean D Schoville

    Full Text Available The marine copepod Tigriopus californicus lives in intertidal rock pools along the Pacific coast, where it exhibits strong, temporally stable population genetic structure. Previous allozyme surveys have found high frequency private alleles among neighboring subpopulations, indicating that there is limited genetic exchange between populations. Here we evaluate the factors responsible for the diversification and maintenance of alleles at the phosphoglucose isomerase (Pgi locus by evaluating patterns of nucleotide variation underlying previously identified allozyme polymorphism. Copepods were sampled from eleven sites throughout California and Baja California, revealing deep genetic structure among populations as well as genetic variability within populations. Evidence of recombination is limited to the sample from Pescadero and there is no support for linkage disequilibrium across the Pgi locus. Neutrality tests and codon-based models of substitution suggest the action of natural selection due to elevated non-synonymous substitutions at a small number of sites in Pgi. Two sites are identified as the charge-changing residues underlying allozyme polymorphisms in T. californicus. A reanalysis of allozyme variation at several focal populations, spanning a period of 26 years and over 200 generations, shows that Pgi alleles are maintained without notable frequency changes. Our data suggest that diversifying selection accounted for the origin of Pgi allozymes, while McDonald-Kreitman tests and the temporal stability of private allozyme alleles suggests that balancing selection may be involved in the maintenance of amino acid polymorphisms within populations.

  3. Diversifying selection underlies the origin of allozyme polymorphism at the phosphoglucose isomerase locus in Tigriopus californicus.

    Science.gov (United States)

    Schoville, Sean D; Flowers, Jonathan M; Burton, Ronald S

    2012-01-01

    The marine copepod Tigriopus californicus lives in intertidal rock pools along the Pacific coast, where it exhibits strong, temporally stable population genetic structure. Previous allozyme surveys have found high frequency private alleles among neighboring subpopulations, indicating that there is limited genetic exchange between populations. Here we evaluate the factors responsible for the diversification and maintenance of alleles at the phosphoglucose isomerase (Pgi) locus by evaluating patterns of nucleotide variation underlying previously identified allozyme polymorphism. Copepods were sampled from eleven sites throughout California and Baja California, revealing deep genetic structure among populations as well as genetic variability within populations. Evidence of recombination is limited to the sample from Pescadero and there is no support for linkage disequilibrium across the Pgi locus. Neutrality tests and codon-based models of substitution suggest the action of natural selection due to elevated non-synonymous substitutions at a small number of sites in Pgi. Two sites are identified as the charge-changing residues underlying allozyme polymorphisms in T. californicus. A reanalysis of allozyme variation at several focal populations, spanning a period of 26 years and over 200 generations, shows that Pgi alleles are maintained without notable frequency changes. Our data suggest that diversifying selection accounted for the origin of Pgi allozymes, while McDonald-Kreitman tests and the temporal stability of private allozyme alleles suggests that balancing selection may be involved in the maintenance of amino acid polymorphisms within populations.

  4. Mapping Soluble Guanylyl Cyclase and Protein Disulfide Isomerase Regions of Interaction.

    Directory of Open Access Journals (Sweden)

    Erin J Heckler

    Full Text Available Soluble guanylyl cyclase (sGC is a heterodimeric nitric oxide (NO receptor that produces cyclic GMP. This signaling mechanism is a key component in the cardiovascular system. NO binds to heme in the β subunit and stimulates the catalytic conversion of GTP to cGMP several hundred fold. Several endogenous factors have been identified that modulate sGC function in vitro and in vivo. In previous work, we determined that protein disulfide isomerase (PDI interacts with sGC in a redox-dependent manner in vitro and that PDI inhibited NO-stimulated activity in cells. To our knowledge, this was the first report of a physical interaction between sGC and a thiol-redox protein. To characterize this interaction between sGC and PDI, we first identified peptide linkages between sGC and PDI, using a lysine cross-linking reagent and recently developed mass spectrometry analysis. Together with Flag-immunoprecipitation using sGC domain deletions, wild-type (WT and mutated PDI, regions of sGC involved in this interaction were identified. The observed data were further explored with computational modeling to gain insight into the interaction mechanism between sGC and oxidized PDI. Our results indicate that PDI interacts preferentially with the catalytic domain of sGC, thus providing a mechanism for PDI inhibition of sGC. A model in which PDI interacts with either the α or the β catalytic domain is proposed.

  5. AcEST: BP913898 [AcEST

    Lifescience Database Archive (English)

    Full Text Available Or... 201 1e-51 sp|Q5R8S7|PPIA_PONPY Peptidyl-prolyl cis-trans isomerase A OS=Po... 201 1e-51 sp|P62941|PPIA...l-prolyl cis-trans isomerase OS=Po... 256 4e-67 tr|B3FNQ1|B3FNQ1_HEVBR Cyclophili..._POPJC Peptidyl-prolyl cis-trans isomerase OS=Po... 253 5e-66 tr|A8CYN7|A8CYN7_GERHY Peptidyl-prolyl cis-tra...ns isomerase OS=Ge... 252 8e-66 tr|A9P8L4|A9P8L4_POPTR Peptidyl-prolyl cis-trans isomerase OS=Po....r|A9PJ47|A9PJ47_POPJC Peptidyl-prolyl cis-trans isomerase OS=Po... 251 2e-65 tr|A9P9C8|A9P9C8_POPTR Peptidyl

  6. CRYSTAL-STRUCTURE OF RECOMBINANT HUMAN TRIOSEPHOSPHATE ISOMERASE AT 2.8 ANGSTROM RESOLUTION - TRIOSEPHOSPHATE ISOMERASE-RELATED HUMAN GENETIC-DISORDERS AND COMPARISON WITH THE TRYPANOSOMAL ENZYME

    NARCIS (Netherlands)

    MANDE, SC; MAINFROID, [No Value; KALK, KH; GORAJ, K; MARTIAL, JA; HOL, WGJ

    1994-01-01

    The crystal structure of recombinant human triosephosphate isomerase (hTIM) has been determined complexed with the transition-state analogue 2-phosphoglycolate at a resolution of 2.8 Angstrom. After refinement, the R-factor is 16.7% with good geometry. The asymmetric unit contains 1 complete dimer o

  7. Giardial triosephosphate isomerase as possible target of the cytotoxic effect of omeprazole in Giardia lamblia.

    Science.gov (United States)

    Reyes-Vivas, Horacio; de la Mora-de la Mora, Ignacio; Castillo-Villanueva, Adriana; Yépez-Mulia, Lilian; Hernández-Alcántara, Gloria; Figueroa-Salazar, Rosalia; García-Torres, Itzhel; Gómez-Manzo, Saúl; Méndez, Sara T; Vanoye-Carlo, América; Marcial-Quino, Jaime; Torres-Arroyo, Angélica; Oria-Hernández, Jesús; Gutiérrez-Castrellón, Pedro; Enríquez-Flores, Sergio; López-Velázquez, Gabriel

    2014-12-01

    Giardiasis is highly prevalent in the developing world, and treatment failures with the standard drugs are common. This work deals with the proposal of omeprazole as a novel antigiardial drug, focusing on a giardial glycolytic enzyme used to follow the cytotoxic effect at the molecular level. We used recombinant technology and enzyme inactivation to demonstrate the capacity of omeprazole to inactivate giardial triosephosphate isomerase, with no adverse effects on its human counterpart. To establish the specific target in the enzyme, we used single mutants of every cysteine residue in triosephosphate isomerase. The effect on cellular triosephosphate isomerase was evaluated by following the remnant enzyme activity on trophozoites treated with omeprazole. The interaction of omeprazole with giardial proteins was analyzed by fluorescence spectroscopy. The susceptibility to omeprazole of drug-susceptible and drug-resistant strains of Giardia lamblia was evaluated to demonstrate its potential as a novel antigiardial drug. Our results demonstrate that omeprazole inhibits giardial triosephosphate isomerase in a species-specific manner through interaction with cysteine at position 222. Omeprazole enters the cytoplasmic compartment of the trophozoites and inhibits cellular triosephosphate isomerase activity in a dose-dependent manner. Such inhibition takes place concomitantly with the cytotoxic effect caused by omeprazole on trophozoites. G. lamblia triosephosphate isomerase (GlTIM) is a cytoplasmic protein which can help analyses of how omeprazole works against the proteins of this parasite and in the effort to understand its mechanism of cytotoxicity. Our results demonstrate the mechanism of giardial triosephosphate isomerase inhibition by omeprazole and show that this drug is effective in vitro against drug-resistant and drug-susceptible strains of G. lamblia.

  8. Genetic and functional aspects of linoleate isomerase in Lactobacillus acidophilus.

    Science.gov (United States)

    Macouzet, Martin; Robert, Normand; Lee, Byong H

    2010-08-01

    While the remarkable health effects of conjugated linoleic acid (CLA) catalyzed from alpha-linoleic acid by the enzyme linoleate isomerase (LI, EC 5.2.1.5) are well recognized, how widely this biochemical activity is present and the mechanisms of its regulation in lactic acid bacteria are unknown. Although certain strains of Lactobacillus acidophilus can enrich CLA in fermented dairy products, it is unknown if other strains share this capacity. Due to its immense economic importance, this work aimed to investigate genetic aspects of CLA production in L. acidophilus for the first time. The genomic DNA from industrial and type strains of L. acidophilus were subjected to PCR and immunoblot analyses using the putative LI gene of L. reuteri ATCC 55739 as probe. The CLA production ability was estimated by gas chromatography of the biomass extracts. The presumptive LI gene from L. acidophilus ATCC 832 was isolated and sequenced. The resulting sequence shared 71% identity with that of L. reuteri and at least 99% with reported sequences from other L. acidophilus strains. All the strains accumulated detectable levels of CLA and tested positive by PCR and immunoblotting. However, no apparent correlation was observed between the yields and the hybridization patterns. The results suggest that LI activity might be common among L. acidophilus and related species and provide a new tool for screening potential CLA producers.

  9. Stereoselectivity of chalcone isomerase with chalcone derivatives: a computational study.

    Science.gov (United States)

    Yao, Yuan; Zhang, Hui; Li, Ze-Sheng

    2013-11-01

    Chalcone isomerase (CHI) catalyzes the intramolecular cyclization of chalcones into flavonoids. The activity of CHI is essential for the biosynthesis of flavonoids precursors of floral pigments and phenylpropanoid plant defense compounds. In the present study, we explored the detailed binding structures and binding free energies for two different active site conformations of CHI with s-cis/s-trans conformers of three chalcone compounds by performing molecular dynamics (MD) simulations and binding free energy calculations. The computational results indicate that s-cis/s-trans conformers of chalcone compounds are orientated in the similar binding position in the active site of CHI and stabilized by the different first hydrogen bond network and the same second hydrogen bond network. The first hydrogen bond network results in much lower binding affinity of s-trans conformer of chalcone compound with CHI than that of s-cis conformer. The conformational change of the active site residue T48 from indirectly interacting with the substrate via the second hydrogen bond network to directly forming the hydrogen bond with the substrates cannot affect the binding mode of both conformers of chalcone compounds, but remarkably improves the binding affinity. These results show that CHI has a strong stereoselectivity. The calculated binding free energies for three chalcone compounds with CHI are consistent with the experimental activity data. In addition, several valuable insights are suggested for future rational design and discovery of high-efficiency mutants of CHI.

  10. Catalytic mechanism of a retinoid isomerase essential for vertebrate vision.

    Science.gov (United States)

    Kiser, Philip D; Zhang, Jianye; Badiee, Mohsen; Li, Qingjiang; Shi, Wuxian; Sui, Xuewu; Golczak, Marcin; Tochtrop, Gregory P; Palczewski, Krzysztof

    2015-06-01

    Visual function in vertebrates is dependent on the membrane-bound retinoid isomerase RPE65, an essential component of the retinoid cycle pathway that regenerates 11-cis-retinal for rod and cone opsins. The mechanism by which RPE65 catalyzes stereoselective retinoid isomerization has remained elusive because of uncertainty about how retinoids bind to its active site. Here we present crystal structures of RPE65 in complex with retinoid-mimetic compounds, one of which is in clinical trials for the treatment of age-related macular degeneration. The structures reveal the active site retinoid-binding cavity located near the membrane-interacting surface of the enzyme as well as an Fe-bound palmitate ligand positioned in an adjacent pocket. With the geometry of the RPE65-substrate complex clarified, we delineate a mechanism of catalysis that reconciles the extensive biochemical and structural research on this enzyme. These data provide molecular foundations for understanding a key process in vision and pharmacological inhibition of RPE65 with small molecules.

  11. Crystal Structure of Triosephosphate Isomerase from Trypanosoma cruzi in Hexane

    Science.gov (United States)

    Gao, Xiu-Gong; Maldonado, Ernesto; Perez-Montfort, Ruy; Garza-Ramos, Georgina; Tuena de Gomez-Puyou, Marietta; Gomez-Puyou, Armando; Rodriguez-Romero, Adela

    1999-08-01

    To gain insight into the mechanisms of enzyme catalysis in organic solvents, the x-ray structure of some monomeric enzymes in organic solvents was determined. However, it remained to be explored whether the structure of oligomeric proteins is also amenable to such analysis. The field acquired new perspectives when it was proposed that the x-ray structure of enzymes in nonaqueous media could reveal binding sites for organic solvents that in principle could represent the starting point for drug design. Here, a crystal of the dimeric enzyme triosephosphate isomerase from the pathogenic parasite Trypanosoma cruzi was soaked and diffracted in hexane and its structure solved at 2- angstrom resolution. Its overall structure and the dimer interface were not altered by hexane. However, there were differences in the orientation of the side chains of several amino acids, including that of the catalytic Glu-168 in one of the monomers. No hexane molecules were detected in the active site or in the dimer interface. However, three hexane molecules were identified on the surface of the protein at sites, which in the native crystal did not have water molecules. The number of water molecules in the hexane structure was higher than in the native crystal. Two hexanes localized at <4 angstrom from residues that form the dimer interface; they were in close proximity to a site that has been considered a potential target for drug design.

  12. Cis-trans isomerizations of beta-carotene and lycopene: a theoretical study.

    Science.gov (United States)

    Guo, Wen-Hsin; Tu, Cheng-Yi; Hu, Ching-Han

    2008-09-25

    The all-trans to mono-cis isomerizations of polyenes and two C40H56 carotenes, beta-carotene and lycopene, at the ground singlet (S0) and triplet (T1) states are studied by means of quantum chemistry computations. At the S0 state of polyenes containing n acetylene units (Pn), we find that the energy barrier of the central C=C rotation decreases with n. In contrast, however, at the T 1 state, the rotational barrier increases with n. For the C40H56 carotenes, the rotational barriers of lycopene are lower than those of their beta-carotene counterparts. This difference renders the rotational rates of lycopene to be 1-2 orders of magnitude higher than those of beta-carotene at room temperature. For both these carotenes, the barrier is lowest for the rotation toward the 13-cis isomer. The relative abundances are in the following order: all-trans > 9-cis > 13-cis > 15-cis. Although the 5-cis isomer of lycopene has the lowest energy among the cis isomers, its formation from the all-trans form is restricted, owing to a very large rotational barrier. The possible physiological implications of this study are discussed.

  13. The cis-trans Isomerisation of Homologous 2-Hydroxycycloalkanecarboxylic Acids under Basic Conditions

    Institute of Scientific and Technical Information of China (English)

    GYARMATI, Cs. Zsuzsanna; P(A)LINK(O), István; BOKROS, Attila; MARTINEK, A. Tamás; BERN(A)TH, Gábor

    2006-01-01

    The cis→trans isomerisation of homologous 2-hydroxycycloalkanecarboxylic acids in strongly basic aqueous solution was studied starting from the cis isomers. It was found that the cyclopentane, cyclohexane and cycloheptane homologues afforded synthetically useful amounts of the trans acids and the procedure resulted in relatively small quantities of the corresponding olefinic acids. In contrast, the isomerisation of the cis-2-hydroxycyclooctanecarboxylic acid produced roughly equal amounts of the cis and trans isomers and the 1-cyclooctenecarboxylic acid at equilibrium. Molecular modelling with the PM3 semiempirical method of the reactants, products and the intermediates applying explicit water molecules as reaction medium gave a fair estimate for the rate sequence of the idealised (dehydration-free) isomerisation reactions in aqueous base solution.

  14. Modelling of the cis-trans partitioning in the photoisomerizations of cyanines and stilbene derivatives

    Science.gov (United States)

    Caselli, M.; Momicchioli, F.; Ponterini, G.

    1993-12-01

    In the course of photoisomerization, polymethine cyanines as well as stilbene and its derivates decay from the S 1 potential energy minimum, corresponding to the perpendicular geometry, to yield either cis or trans ground-state molecules. The fraction of cis isomers obtained, α, spans a larger range of values for symmetric cyanines than for stilbene derivatives. It is argued that such different behaviour for the two classes of compounds should be traceable to the electronically different nature of their S 1 perp species. Making use of radiationless transition theory results, it is shown the relative location of the S 1 minimum and S 0 maximum along the internal rotation coordinate is crucial to the evaluation of α: even small differences between these critical twisting angles, which are more reasonably expected for polymethine cyanines than for stilbene-like compounds, may cause strong deviations from equipartitioning (α=0.5).

  15. PBOND: Web Server for the Prediction of Proline and Non-Proline cis / trans Isomerization

    Institute of Scientific and Technical Information of China (English)

    Konstantinos P. Exarchos; Themis P. Exarchos; Costas Papaloukas; Anastassios N. Troganis; Dimitrios I. Fotiadis

    2009-01-01

    PBOND is a web server that predicts the conformation of the peptide bond be-tween any two amino acids. PBOND classifies the peptide bonds into one out of four classes, namely cis imide(cis-Pro), cis amide(cis-nonPro), trans imide (trans-Pro)and trans amide(trans-nonPro). Moreover, for every prediction a reliability index is computed. The underlying structure of the server consists of three stages:(1)feature extraction,(2)feature selection and(3)peptide bond clas-sification. PBOND can handle both single sequences as well as multiple sequences for batch processing. The predictions can either be directly downloaded from the web site or returned via e-mail. The PBOND web server is freely available at http://195.251.198.21/pbond.html.

  16. Determinación de Cis-/Trans-Estilbenos en cera de panal de abeja

    OpenAIRE

    González Cámara, Yolanda

    2013-01-01

    Las abejas juegan un papel fundamental no solo en lo relacionado con la producción de la colmena sino también en la polinización de plantas. Por todo ello, lo que las afecte negativamente, tendrá sin duda una gran repercusión en la Agricultura y el Medio Ambiente. La investigación sobre el tema se centra actualmente en la búsqueda de compuestos naturales o sintéticos alternativos al uso de antibióticos prohibidos actualmente para el tratamiento de infecciones. Un ejemplo claro es el uso...

  17. Cis/trans Coordination in olefin metathesis by static and molecular dynamic DFT calculations

    KAUST Repository

    Poater, Albert

    2014-05-25

    In regard to [(N-heterocyclic carbene)Ru]-based catalysts, it is still a matter of debate if the substrate binding is preferentially cis or trans to the N-heterocyclic carbene ligand. By means of static and molecular dynamic DFT calculations, a simple olefin, like ethylene, is shown to be prone to the trans binding. Bearing in mind the higher reactivity of trans isomers in olefin metathesis, this insight helps to construct small alkene substrates with increased reactivity. © 2014 Springer Science+Business Media New York.

  18. cis,trans,cis-1,2,3,4-Tetrakis[2-(ethylsulfanylphenyl]cyclobutane

    Directory of Open Access Journals (Sweden)

    Barbara Sohr

    2016-01-01

    Full Text Available The title cyclobutane derivative, C36H40S4, formed serendipitously through a photochemically initiated [2 + 2] cycloaddition. The asymmetric unit contains half a molecule with the 2-(ethylsulfanylphenyl substituents in a cis configuration, the other half of the molecule being generated by the application of a twofold rotation operation. The substituents in both halves of the molecules are in a trans arrangement relative to each other. The cyclobutane ring shows angular and torsional strains, with C—C—C bond angles of 89.80 (8 and 88.40 (8°, and an average absolute torsion angle of 14.28 (10°. The angle of pucker in the ring is 20.27 (12°. The Ccb—Ccb—Cb angles between the cyclobutane (cb ring atoms and the attached benzene (b ring atoms are widened and range from 115.19 (10 to 121.66 (10°. A weak intramolecular C—H...S hydrogen-bonding interaction between one of the cyclobutane ring H atoms and the S atom may help to establish the molecular conformation. No specific intermolecular interactions are found.

  19. AcEST: DK946305 [AcEST

    Lifescience Database Archive (English)

    Full Text Available yl cis-trans isomerase OS=Po... 250 3e-65 tr|B3FNQ1|B3FNQ1_HEVBR Cyclophilin OS=H...9 5e-65 tr|A9PIY0|A9PIY0_POPJC Peptidyl-prolyl cis-trans isomerase OS=Po... 248 1e-64 tr|A8CYN7|A8CYN7_GERHY...merase OS=Th... 248 2e-64 tr|A9P8L4|A9P8L4_POPTR Peptidyl-prolyl cis-trans isomerase OS=Po...... 247 3e-64 tr|A9PJ47|A9PJ47_POPJC Peptidyl-prolyl cis-trans isomerase OS=Po... 246 8e-64 tr|A9P9...C8|A9P9C8_POPTR Peptidyl-prolyl cis-trans isomerase OS=Po... 246 8e-64 tr|Q8L5T1|Q8L5T1_BETVE Peptidyl-proly

  20. Testing electrostatic complementarity in enzyme catalysis: hydrogen bonding in the ketosteroid isomerase oxyanion hole.

    Directory of Open Access Journals (Sweden)

    Daniel A Kraut

    2006-04-01

    Full Text Available A longstanding proposal in enzymology is that enzymes are electrostatically and geometrically complementary to the transition states of the reactions they catalyze and that this complementarity contributes to catalysis. Experimental evaluation of this contribution, however, has been difficult. We have systematically dissected the potential contribution to catalysis from electrostatic complementarity in ketosteroid isomerase. Phenolates, analogs of the transition state and reaction intermediate, bind and accept two hydrogen bonds in an active site oxyanion hole. The binding of substituted phenolates of constant molecular shape but increasing pK(a models the charge accumulation in the oxyanion hole during the enzymatic reaction. As charge localization increases, the NMR chemical shifts of protons involved in oxyanion hole hydrogen bonds increase by 0.50-0.76 ppm/pK(a unit, suggesting a bond shortening of 0.02 A/pK(a unit. Nevertheless, there is little change in binding affinity across a series of substituted phenolates (DeltaDeltaG = -0.2 kcal/mol/pK(a unit. The small effect of increased charge localization on affinity occurs despite the shortening of the hydrogen bonds and a large favorable change in binding enthalpy (DeltaDeltaH = -2.0 kcal/mol/pK(a unit. This shallow dependence of binding affinity suggests that electrostatic complementarity in the oxyanion hole makes at most a modest contribution to catalysis of 300-fold. We propose that geometrical complementarity between the oxyanion hole hydrogen-bond donors and the transition state oxyanion provides a significant catalytic contribution, and suggest that KSI, like other enzymes, achieves its catalytic prowess through a combination of modest contributions from several mechanisms rather than from a single dominant contribution.

  1. Peri/Epicellular Protein Disulfide Isomerase Sustains Vascular Lumen Caliber Through an Anticonstrictive Remodeling Effect.

    Science.gov (United States)

    Tanaka, Leonardo Y; Araújo, Haniel A; Hironaka, Gustavo K; Araujo, Thaís L S; Takimura, Celso K; Rodriguez, Andres I; Casagrande, Annelise S; Gutierrez, Paulo S; Lemos-Neto, Pedro Alves; Laurindo, Francisco R M

    2016-03-01

    Whole-vessel remodeling critically determines lumen caliber in vascular (patho)physiology, and it is reportedly redox-dependent. We hypothesized that the cell-surface pool of the endoplasmic reticulum redox chaperone protein disulfide isomerase-A1 (peri/epicellular=pecPDI), which is known to support thrombosis, also regulates disease-associated vascular architecture. In human coronary atheromas, PDI expression inversely correlated with constrictive remodeling and plaque stability. In a rabbit iliac artery overdistension model, there was unusually high PDI upregulation (≈25-fold versus basal, 14 days postinjury), involving both intracellular and pecPDI. PecPDI neutralization with distinct anti-PDI antibodies did not enhance endoplasmic reticulum stress or apoptosis. In vivo pecPDI neutralization with PDI antibody-containing perivascular gel from days 12 to 14 post injury promoted 25% decrease in the maximally dilated arteriographic vascular caliber. There was corresponding whole-vessel circumference loss using optical coherence tomography without change in neointima, which indicates constrictive remodeling. This was accompanied by decreased hydrogen peroxide generation. Constrictive remodeling was corroborated by marked changes in collagen organization, that is, switching from circumferential to radial fiber orientation and to a more rigid fiber type. The cytoskeleton architecture was also disrupted; there was a loss of stress fiber coherent organization and a switch from thin to medium thickness actin fibers, all leading to impaired viscoelastic ductility. Total and PDI-associated expressions of β1-integrin, and levels of reduced cell-surface β1-integrin, were diminished after PDI antibody treatment, implicating β1-integrin as a likely pecPDI target during vessel repair. Indeed, focal adhesion kinase phosphorylation, a downstream β1-integrin effector, was decreased by PDI antibody. Thus, the upregulated pecPDI pool tunes matrix/cytoskeleton reshaping to

  2. Escherichia coli rpiA gene encoding ribose phosphate isomerase A

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Maigaard, Marianne

    1993-01-01

    The rpiA gene encoding ribose phosphate isomerase A was cloned from phage 1A2(471) of the Kohara gene library. Subcloning, restriction, and complementation analyses revealed an 1,800-bp SspI-generated DNA fragment that contained the entire control and coding sequences. This DNA fragment was seque......The rpiA gene encoding ribose phosphate isomerase A was cloned from phage 1A2(471) of the Kohara gene library. Subcloning, restriction, and complementation analyses revealed an 1,800-bp SspI-generated DNA fragment that contained the entire control and coding sequences. This DNA fragment...... was confirmed by amino-terminal analysis of partially purified ribose phosphate isomerase A. Our data indicate that the enzyme is composed of two identical subunits. The 5' end of the rpiA-specified transcript was analyzed by primer extension, which revealed a well-conserved -10 region 34 bp upstream...... strains harboring the rpiA gene in a multicopy plasmid contained up to 42-fold as much ribose phosphate isomerase A activity as the haploid strain....

  3. Escherichia coli rpiA> gene encoding ribose phosphate isomerase A

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Maigaard, Marianne

    1993-01-01

    The rpiA gene encoding ribose phosphate isomerase A was cloned from phage 1A2(471) of the Kohara gene library. Subcloning, restriction, and complementation analyses revealed an 1,800-bp SspI-generated DNA fragment that contained the entire control and coding sequences. This DNA fragment was seque...

  4. Screening and selection of wild strains for L-arabinose isomerase production

    Directory of Open Access Journals (Sweden)

    R. M. Manzo

    2013-12-01

    Full Text Available The majority of L-arabinose isomerases have been isolated by recombinant techniques, but this methodology implies a reduced technological application. For this reason, 29 bacterial strains, some of them previously characterized as L-arabinose isomerase producers, were assayed as L-arabinose fermenting strains by employing conveniently designed culture media with 0.5% (w/v L-arabinose as main carbon source. From all evaluated bacterial strains, Enterococcus faecium DBFIQ ID: E36, Enterococcus faecium DBFIQ ID: ETW4 and Pediococcus acidilactici ATCC ID: 8042 were, in this order, the best L-arabinose fermenting strains. Afterwards, to assay L-arabinose metabolization and L-arabinose isomerase activity, cell-free extract and saline precipitated cell-free extract of the three bacterial cultures were obtained and the production of ketoses was determined by the cysteine carbazole sulfuric acid method. Results showed that the greater the L-arabinose metabolization ability, the higher the enzymatic activity achieved, so Enterococcus faecium DBFIQ ID: E36 was selected to continue with production, purification and characterization studies. This work thus describes a simple microbiological method for the selection of L-arabinose fermenting bacteria for the potential production of the enzyme L-arabinose isomerase.

  5. Rapid expansion of the protein disulfide isomerase gene family facilitates the folding of venom peptides

    DEFF Research Database (Denmark)

    Safavi-Hemami, Helena; Li, Qing; Jackson, Ronneshia L.

    2016-01-01

    Formation of correct disulfide bonds in the endoplasmic reticulum is a crucial step for folding proteins destined for secretion. Protein disulfide isomerases (PDIs) play a central role in this process. We report a previously unidentified, hypervariable family of PDIs that represents the most dive...

  6. Experimental validation of in silico model-predicted isocitrate dehydrogenase and phosphomannose isomerase from Dehalococcoides mccartyi.

    Science.gov (United States)

    Islam, M Ahsanul; Tchigvintsev, Anatoli; Yim, Veronica; Savchenko, Alexei; Yakunin, Alexander F; Mahadevan, Radhakrishnan; Edwards, Elizabeth A

    2016-01-01

    Gene sequences annotated as proteins of unknown or non-specific function and hypothetical proteins account for a large fraction of most genomes. In the strictly anaerobic and organohalide respiring Dehalococcoides mccartyi, this lack of annotation plagues almost half the genome. Using a combination of bioinformatics analyses and genome-wide metabolic modelling, new or more specific annotations were proposed for about 80 of these poorly annotated genes in previous investigations of D. mccartyi metabolism. Herein, we report the experimental validation of the proposed reannotations for two such genes (KB1_0495 and KB1_0553) from D. mccartyi strains in the KB-1 community. KB1_0495 or DmIDH was originally annotated as an NAD(+)-dependent isocitrate dehydrogenase, but biochemical assays revealed its activity primarily with NADP(+) as a cofactor. KB1_0553, also denoted as DmPMI, was originally annotated as a hypothetical protein/sugar isomerase domain protein. We previously proposed that it was a bifunctional phosphoglucose isomerase/phosphomannose isomerase, but only phosphomannose isomerase activity was identified and confirmed experimentally. Further bioinformatics analyses of these two protein sequences suggest their affiliation to potentially novel enzyme families within their respective larger enzyme super families.

  7. Identification of Triosephosphate Isomerase as a Novel Allergen in Octopus fangsiao

    Science.gov (United States)

    A 28 kDa-protein was purified from octopus (Octopus fangsiao) and identified to be triosephosphate isomerase (TIM). The purified TIM is a glycoprotein with 1.7% carbohydrates and the isoelectric point is 7.6. TIM aggregated after heating above 45 °C, and the secondary structure was altered in extre...

  8. Structural and Biochemical Characterization of the Human Cyclophilin Family of Peptidyl-Prolyl Isomerases

    Energy Technology Data Exchange (ETDEWEB)

    Davis, Tara L.; Walker, John R.; Campagna-Slater, Valérie; Finerty, Jr., Patrick J.; Paramanathan, Ragika; Bernstein, Galina; MacKenzie, Farrell; Tempel, Wolfram; Ouyang, Hui; Lee, Wen Hwa; Eisenmesser, Elan Z.; Dhe-Paganon, Sirano (Toronto); (Colorado)

    2011-12-14

    Peptidyl-prolyl isomerases catalyze the conversion between cis and trans isomers of proline. The cyclophilin family of peptidyl-prolyl isomerases is well known for being the target of the immunosuppressive drug cyclosporin, used to combat organ transplant rejection. There is great interest in both the substrate specificity of these enzymes and the design of isoform-selective ligands for them. However, the dearth of available data for individual family members inhibits attempts to design drug specificity; additionally, in order to define physiological functions for the cyclophilins, definitive isoform characterization is required. In the current study, enzymatic activity was assayed for 15 of the 17 human cyclophilin isomerase domains, and binding to the cyclosporin scaffold was tested. In order to rationalize the observed isoform diversity, the high-resolution crystallographic structures of seven cyclophilin domains were determined. These models, combined with seven previously solved cyclophilin isoforms, provide the basis for a family-wide structure:function analysis. Detailed structural analysis of the human cyclophilin isomerase explains why cyclophilin activity against short peptides is correlated with an ability to ligate cyclosporin and why certain isoforms are not competent for either activity. In addition, we find that regions of the isomerase domain outside the proline-binding surface impart isoform specificity for both in vivo substrates and drug design. We hypothesize that there is a well-defined molecular surface corresponding to the substrate-binding S2 position that is a site of diversity in the cyclophilin family. Computational simulations of substrate binding in this region support our observations. Our data indicate that unique isoform determinants exist that may be exploited for development of selective ligands and suggest that the currently available small-molecule and peptide-based ligands for this class of enzyme are insufficient for isoform

  9. In silico analysis suggests that PH0702 and PH0208 encode for methylthioribose-1-phosphate isomerase and ribose-1,5-bisphosphate isomerase, respectively, rather than aIF2Bβ and aIF2Bδ.

    Science.gov (United States)

    Gogoi, Prerana; Srivastava, Ambuj; Jayaprakash, Prajisha; Jeyakanthan, Jeyaraman; Kanaujia, Shankar Prasad

    2016-01-01

    The overall process of protein biosynthesis across all domains of life is similar; however, detailed insights reveal a range of differences in the proteins involved. For decades, the process of protein translation in archaea has been considered to be closer to eukaryotes than to bacteria. In archaea, however, several homologues of eukaryotic proteins involved in translation initiation have not yet been identified; one of them being the initiation factor eIF2B consisting of five subunits (α, β, γ, δ and ε). Three open reading frames (PH0440, PH0702 and PH0208) in Pyrococcus horikoshii have been proposed to encode for the α-, β- and δ-subunits of aIF2B, respectively. The crystal structure of PH0440 shows similarity toward the α-subunit of eIF2B. However, the capability of PH0702 and PH0208 to function as the β- and δ-subunits of eIF2B, respectively, remains uncertain. In this study, we have taken up the task of annotating PH0702 and PH0208 using bioinformatics methods. The phylogenetic analysis of protein sequences belonging to IF2B-like family along with PH0702 and PH0208 revealed that PH0702 belonged to methylthioribose-1-phosphate isomerase (MTNA) group of proteins, whereas, PH0208 was found to be clustered in the group of ribose-1,5-bisphosphate isomerase (R15PI) proteins. A careful analysis of protein sequences and structures available for eIF2B, MTNA and R15PI confirms that PH0702 and PH0208 contain residues essential for the enzymatic activity of MTNA and R15PI, respectively. Additionally, the protein PH0208 comprises of the residues required for the dimer formation which is essential for the biological activity of R15PI. This prompted us to examine all eIF2B-like proteins from archaea and to annotate their function. The results reveal that majority of these proteins are homologues of the α-subunit of eIF2B, even though they lack the residues essential for their functional activity. A better understanding of the mechanism of GTP exchange during

  10. Identification of GutQ from Escherichia coli as a D-arabinose 5-phosphate isomerase.

    Science.gov (United States)

    Meredith, Timothy C; Woodard, Ronald W

    2005-10-01

    The glucitol operon (gutAEBDMRQ) of Escherichia coli encodes a phosphoenolpyruvate:sugar phosphotransferase system that metabolizes the hexitol D-glucitol (sorbitol). The functions for all but the last gene, gutQ, have been previously assigned. The high sequence similarity between GutQ and KdsD, a D-arabinose 5-phosphate isomerase (API) from the 3-deoxy-D-manno-octulosonate (KDO)-lipopolysaccharide (LPS) biosynthetic pathway, suggested a putative activity, but its role within the context of the gut operon remained unclear. Accordingly, the enzyme was cloned, overexpressed, and characterized. Recombinant GutQ was shown to indeed be a second copy of API from the E. coli K-12 genome with biochemical properties similar to those of KdsD, catalyzing the reversible aldol-ketol isomerization between D-ribulose 5-phosphate (Ru5P) and D-arabinose 5-phosphate (A5P). Genomic disruptions of each API gene were constructed in E. coli K-12. TCM11[(deltakdsD)] was capable of sustaining essential LPS synthesis at wild-type levels, indicating that GutQ functions as an API inside the cell. The gut operon remained inducible in TCM7[(deltagutQ)], suggesting that GutQ is not directly involved in d-glucitol catabolism. The conditional mutant TCM15[(deltagutQdeltakdsD)] was dependent on exogenous A5P both for LPS synthesis/growth and for upregulation of the gut operon. The phenotype was suppressed by complementation in trans with a plasmid encoding a functional copy of GutQ or by increasing the amount of A5P in the medium. As there is no obvious obligatory role for GutQ in the metabolism of d-glucitol and there is no readily apparent link between D-glucitol metabolism and LPS biosynthesis, it is suggested that A5P is not only a building block for KDO biosynthesis but also may be a regulatory molecule involved in expression of the gut operon.

  11. Synthesis, Crystal Structures, Cis-Trans Conformation and Substituent Effects of Manganese(Ⅱ) and Cobalt(Ⅱ) Complexes Having 3,8-Dithiophen or 3,8-Di-3-methylthiophen-1,10-phenanthroline%3,8-二噻吩和3,8-二甲基噻吩-1,10-菲啰啉的锰(Ⅱ)和钴(Ⅱ)配合物的晶体结构、顺反异构及取代基效应研究

    Institute of Scientific and Technical Information of China (English)

    胡斌; 颜流水; 张爱琴; 黄伟

    2012-01-01

    本文报道了1个基于3,8-二噻吩-l,10-菲啰啉(dtphen)的双核锰(Ⅱ)配合物1 [trans-Mn2Cl4(dtphen)2]和2个基于3,8-二甲基噻吩-1,10-菲啰啉(dmtphen)的单核锰(Ⅱ)和单核钴(Ⅱ)配合物2和3(分子式分别为[cis-MnCl2(dmtphen)2]和[cis-CoCl2(dmtphen)2])的合成、波谱和晶体结构表征.其中,2个锰(Ⅱ)配合物的分子结构呈现不同的配位模式,由于噻吩环上甲基引入所产生的位阻效应,导致单核配合物2和3中,两配体中的噻吩环相对于l,10-菲啰啉环呈现相同的反式/反式分子构型,其二面角分布在14.1(1)°~51.5(1)°.而对于双核配合物1,由于没有甲基位阻的影响,其相应芳环之间二面角减少至2.0(1)°~20.2(1)°,且配体呈现顺式/反式分子构型.%A dinuclear 3,8-dithiophen substituted 1,10-phenanthroline (dtphen) manganese(Ⅱ) complex and a pair of 3,8-di-3-methyhhiophen substituted 1,10-phenanthroline (dmtphen) manganese (Ⅱ) and cobalt (Ⅱ) complexes,formulated as [trans-Mn2Cl4(dtphen)2] (1),[cis-MnCl2(dmtphen)2] (2) and [cis-CoCl2(dmtphen)2] (3),were synthesized and characterized by elemental analysis,FTIR,UV-Vis spectra and X-ray single-crystal structural analysis.The two Mn(Ⅱ) complexes exhibit diverse coordination models in the whole molecular structures.In mononuclear complexe 2 and 3,both dmtphen ligands have similar trans/trans conformation but different dihedral angles ranging from 14.1(1)°~51.5(1)° because of the spatial crowding effects of methyl group.In contrast,the dihedral angles of dtphen ligands in dinuclear complex 1 are only within 2.0(1)°~20.2(1)° and the two side thiophene rings in the dtphen ligand adopt the cis/trans conformation relative to the central phen unit.CCDC:870021,1; 870022,2; 870023,3.

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

    Science.gov (United States)

    Lobley, Carina M C; Aller, Pierre; Douangamath, Alice; Reddivari, Yamini; Bumann, Mario; Bird, Louise E; Nettleship, Joanne E; Brandao-Neto, Jose; Owens, Raymond J; O'Toole, Paul W; Walsh, Martin A

    2012-12-01

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

  13. Crystal structure of Streptomyces diastaticus No. 7 strain M1033 xylose isomerase

    Institute of Scientific and Technical Information of China (English)

    朱学勇; 龚为民; 牛立文; 滕脉坤; 徐庆平; 伍传金; 崔涛; 王玉珍; 王淳

    1996-01-01

    The crystal structures of Streptomyces diastaticus No. 7 strain M1033 xylose isomerase (SDXyI) have been analysed and refined at 0.19nm. The crystal space group is I222, with unit cell dimensions of a=9.884 ran, b=9.393nm and c=8.798nm. Based on the coordinates of the Streptomyces rubiginosus xylose isomerase (SRXyI), the initial model of SDXyl was built up by the dose packing analysing and R-factor searching and refined by PROLSQ to a final R-factor of 0.177 with the rms deviations of bond lengths and bond angles of 0.001 9nm and 2.1°, respectively. No significant global conformation change existed between SRXyI and SDXyI except the local conformation in the active site.

  14. Diversifying Selection Underlies the Origin of Allozyme Polymorphism at the Phosphoglucose Isomerase Locus in Tigriopus californicus

    OpenAIRE

    Schoville, Sean D.; Flowers, Jonathan M.; Ronald S Burton

    2012-01-01

    The marine copepod Tigriopus californicus lives in intertidal rock pools along the Pacific coast, where it exhibits strong, temporally stable population genetic structure. Previous allozyme surveys have found high frequency private alleles among neighboring subpopulations, indicating that there is limited genetic exchange between populations. Here we evaluate the factors responsible for the diversification and maintenance of alleles at the phosphoglucose isomerase (Pgi) locus by evaluating pa...

  15. BIOPHYSICS. Comment on "Extreme electric fields power catalysis in the active site of ketosteroid isomerase".

    Science.gov (United States)

    Chen, Deliang; Savidge, Tor

    2015-08-28

    Fried et al. (Reports, 19 December 2014, p. 1510) demonstrate electric field-dependent acceleration of biological catalysis using ketosteroid isomerase as a prototypic example. These findings were not extended to aqueous solution because water by itself has field fluctuations that are too large and fast to provide a catalytic effect. Given physiological context, when water electrostatic interactions are considered, electric fields play a less important role in the catalysis.

  16. Identification, expression, and characterization of the highly conserved D-xylose isomerase in animals

    Institute of Scientific and Technical Information of China (English)

    Ming Ding; Yigang Teng; Qiuyu Yin; Wei Chen; Fukun Zhao

    2009-01-01

    D-xylose is a necessary sugar for animals. The xylanase from a mollusk, Ampullaria crossean, was previously reported by our laboratory. This xylanase can degrade the xylan into D-xylose. But there is still a gap in our knowledge on its metabolic pathway. The question is how does the xylose enter the pentose pathway? With the help of genomic databases and bioinformatic tools, we found that some animals, such as bacteria, have a highly conserved D-xylose isomerase (EC 5.3.1.5). The xylose isomerase from a sea squirt, Ciona intestinali, was heterogeneously expressed in Escherichia coli and purified to confirm its function. The recombinant enzyme had good thermal stability in the presence of Mg2+. At the optimum temperature and optimum pH environment, its specific activity on D-xylose was 0.331μmol/mg/min. This enzyme exists broadly in many animals, but it disappeared in the genome of Amphibia-like Xenopus laevis. Its sequence was highly conserved. The xylose isomerases from animals are very interesting proteins for the study of evolution.

  17. Silencing of xylose isomerase and cellulose synthase by siRNA inhibits encystation in Acanthamoeba castellanii.

    Science.gov (United States)

    Aqeel, Yousuf; Siddiqui, Ruqaiyyah; Khan, Naveed Ahmed

    2013-03-01

    A key challenge in the successful treatment of Acanthamoeba infections is its ability to transform into a dormant cyst form that is resistant to physiological conditions and pharmacological therapies, resulting in recurrent infections. The carbohydrate linkage analysis of cyst walls of Acanthamoeba castellanii showed variously linked sugar residues, including xylofuranose/xylopyranose, glucopyranose, mannopyranose, and galactopyranose. Here, it is shown that exogenous xylose significantly reduced A. castellanii differentiation in encystation assays (P < 0.05 using paired t test, one-tailed distribution). Using small interfering RNA (siRNA) probes against xylose isomerase and cellulose synthase, as well as specific inhibitors, the findings revealed that xylose isomerase and cellulose synthase activities are crucial in the differentiation of A. castellanii. Inhibition of both enzymes using siRNA against xylose isomerase and cellulose synthase but not scrambled siRNA attenuated A. castellanii metamorphosis, as demonstrated by the arrest of encystation of A. castellanii. Neither inhibitor nor siRNA probes had any effect on the viability and extracellular proteolytic activities of A. castellanii.

  18. Purification and characterization of an extremely stable glucose isomerase from Geobacillus thermodenitrificans TH2.

    Science.gov (United States)

    Konak, L; Kolcuoğlu, Y; Ozbek, E; Colak, A; Ergenoglu, B

    2014-01-01

    The D-glucose/D-xylose isomerase was purified from a thermophilic bacterium, Geobacillus thermodenitrificans TH2, by precipitating with heat shock and using Q-Sepharose ion exchange column chromatography, and then characterized. The purified enzyme had a single band having molecular weight of 49 kDa on SDS-PAGE. In the presence of D-glucose as a substrate, the optimum temperature and pH of the enzyme were found to be 80 degrees C and 7.5, respectively. The purified xylose isomerase of G. thermodenitrificans TH2 was extremely stable at pH 7.5 after 96 h incubation at 4 degrees C and 50 degrees C. When the thermal stability profile was analyzed, it was determined that the purified enzyme was extremely stable during incubation periods of 4 months and 4 days at 4 degrees C and 50 degrees C, respectively. The K(m) and V(max) values of the purified xylose isomerase from G. thermodenitrificans TH2 were calculated as 32 mM and 4.68 micromol/min per mg of protein, respectively. Additionally, it was detected that some metal ions affected the enzyme activity at different ratios. The enzyme was active and stable at high temperatures and nearly neutral pHs which are desirable for the usage in the food and ethanol industry.

  19. The Role of S-Nitrosylation and S-Glutathionylation of Protein Disulphide Isomerase in Protein Misfolding and Neurodegeneration

    Directory of Open Access Journals (Sweden)

    M. Halloran

    2013-01-01

    Full Text Available Neurodegenerative diseases involve the progressive loss of neurons, and a pathological hallmark is the presence of abnormal inclusions containing misfolded proteins. Although the precise molecular mechanisms triggering neurodegeneration remain unclear, endoplasmic reticulum (ER stress, elevated oxidative and nitrosative stress, and protein misfolding are important features in pathogenesis. Protein disulphide isomerase (PDI is the prototype of a family of molecular chaperones and foldases upregulated during ER stress that are increasingly implicated in neurodegenerative diseases. PDI catalyzes the rearrangement and formation of disulphide bonds, thus facilitating protein folding, and in neurodegeneration may act to ameliorate the burden of protein misfolding. However, an aberrant posttranslational modification of PDI, S-nitrosylation, inhibits its protective function in these conditions. S-nitrosylation is a redox-mediated modification that regulates protein function by covalent addition of nitric oxide- (NO- containing groups to cysteine residues. Here, we discuss the evidence for abnormal S-nitrosylation of PDI (SNO-PDI in neurodegeneration and how this may be linked to another aberrant modification of PDI, S-glutathionylation. Understanding the role of aberrant S-nitrosylation/S-glutathionylation of PDI in the pathogenesis of neurodegenerative diseases may provide insights into novel therapeutic interventions in the future.

  20. The Potato Sucrose Transporter StSUT1 Interacts with a DRM-Associated Protein Disulfide Isomerase

    Institute of Scientific and Technical Information of China (English)

    Undine Krügel; Hong-Xia He; Konstanze Gier; Jana Reins; Izabela Chincinska; Bernhard Grimm; Waltraud X. Schulze; Christina Kühn

    2012-01-01

    Organization of proteins into complexes is crucial for many cellular functions.Recently,the SUT1 protein was shown to form homodimeric complexes,to be associated with lipid raft-like microdomains in yeast as well as in plants and to undergo endocytosis in response to brefeldin A.We therefore aimed to identify SUT1-interacting proteins that might be involved in dimerization,endocytosis,or targeting of SUT1 to raft-like microdomains.Therefore,we identified potato membrane proteins,which are associated with the detergent-resistant membrane (DRM) fraction.Among the proteins identified,we clearly confirmed StSUT1 as part of DRM in potato source leaves.We used the yeast two-hybrid split ubiquitin system (SUS) to systematically screen for interaction between the sucrose transporter StSUT1 and other membraneassociated or soluble proteins in vivo.The SUS screen was followed by immunoprecipitation using affinity-purified StSUT1-specific peptide antibodies and mass spectrometric analysis of co-precipitated proteins.A large overlap was observed between the StSUT1-interacting proteins identified in the co-immunoprecipitation and the detergent-resistant membrane fraction.One of the SUT1-interacting proteins,a protein disulfide isomerase (PDI),interacts also with other sucrose transporter proteins.A potential role of the PDI as escort protein is discussed.

  1. Acrosin-binding protein (ACRBP) and triosephosphate isomerase (TPI) are good markers to predict boar sperm freezing capacity.

    Science.gov (United States)

    Vilagran, Ingrid; Castillo, Judit; Bonet, Sergi; Sancho, Sílvia; Yeste, Marc; Estanyol, Josep M; Oliva, Rafael

    2013-09-15

    Sperm cryopreservation is the most efficient method for storing boar sperm samples for a long time. However, one of the inconveniences of this method is the large variation between and within boars in the cryopreservation success of their sperm. The aim of the present work was thus to find reliable and useful predictive biomarkers of the good and poor capacity to withstand the freeze-thawing process in boar ejaculates. To find these biomarkers, the amount of proteins present in the total proteome in sperm cells were compared between good freezability ejaculates (GFE) and poor freezability ejaculates (PFE) using the two-dimensional difference gel electrophoresis technique. Samples were classified as GFE and PFE using progressive motility and viability of the sperm at 30 and 240 minutes after thawing, and the proteomes from each group, before starting cryopreservation protocols, were compared. Because two proteins, acrosin binding protein (ACRBP) and triosephosphate isomerase (TPI), presented the highest significant differences between GFE and PFE groups in two-dimensional difference gel electrophoresis assessment, Western blot analyses for ACRBP and TPI were also performed for validation. ACRBP normalized content was significantly lower in PFE than in GFE (P sperm viability and motility was confirmed using Pearson's linear correlation. In conclusion, ACRBP and TPI can be used as markers of boar sperm freezability before starting the cryopreservation procedure, thereby avoiding unnecessary costs involved in this practice.

  2. Identification of Amino Acids that Account for Long-Range Interactions in Two Triosephosphate Isomerases from Pathogenic Trypanosomes

    Energy Technology Data Exchange (ETDEWEB)

    García-Torres, Itzhel; Cabrera, Nallely; Torres-Larios, Alfredo; Rodríguez-Bolaños, Mónica; Díaz-Mazariegos, Selma; Gómez-Puyou, Armando; Perez-Montfort, Ruy (UNAM-Mexico)

    2012-04-02

    For a better comprehension of the structure-function relationship in proteins it is necessary to identify the amino acids that are relevant for measurable protein functions. Because of the numerous contacts that amino acids establish within proteins and the cooperative nature of their interactions, it is difficult to achieve this goal. Thus, the study of protein-ligand interactions is usually focused on local environmental structural differences. Here, using a pair of triosephosphate isomerase enzymes with extremely high homology from two different organisms, we demonstrate that the control of a seventy-fold difference in reactivity of the interface cysteine is located in several amino acids from two structurally unrelated regions that do not contact the cysteine sensitive to the sulfhydryl reagent methylmethane sulfonate, nor the residues in its immediate vicinity. The change in reactivity is due to an increase in the apparent pKa of the interface cysteine produced by the mutated residues. Our work, which involved grafting systematically portions of one protein into the other protein, revealed unsuspected and multisite long-range interactions that modulate the properties of the interface cysteines and has general implications for future studies on protein structure-function relationships.

  3. Bioproduction of D-Tagatose from D-Galactose Using Phosphoglucose Isomerase from Pseudomonas aeruginosa PAO1.

    Science.gov (United States)

    Patel, Manisha J; Patel, Arti T; Akhani, Rekha; Dedania, Samir; Patel, Darshan H

    2016-07-01

    Pseudomonas aeruginosa PAO1 phosphoglucose isomerase was purified as an active soluble form by a single-step purification using Ni-NTA chromatography that showed homogeneity on SDS-PAGE with molecular mass ∼62 kDa. The optimum temperature and pH for the maximum isomerization activity with D-galactose were 60 °C and 7.0, respectively. Generally, sugar phosphate isomerases show metal-independent activity but PA-PGI exhibited metal-dependent isomerization activity with aldosugars and optimally catalyzed the D-galactose isomerization in the presence of 1.0 mM MnCl2. The apparent Km and Vmax for D-galactose under standardized conditions were calculated to be 1029 mM (±31.30 with S.E.) and 5.95 U/mg (±0.9 with S.E.), respectively. Equilibrium reached after 180 min with production of 567.51 μM D-tagatose from 1000 mM of D-galactose. Though, the bioconversion ratio is low but it can be increased by immobilization and enzyme engineering. Although various L-arabinose isomerases have been characterized for bioproduction of D-tagatose, P. aeruginosa glucose phosphate isomerase is distinguished from the other L-arabinose isomerases by its optimal temperature (60 °C) for D-tagatose production being mesophilic bacteria, making it an alternate choice for bulk production.

  4. Characterization of a F280N variant of L-arabinose isomerase from Geobacillus thermodenitrificans identified as a D-galactose isomerase.

    Science.gov (United States)

    Kim, Baek-Joong; Hong, Seung-Hye; Shin, Kyung-Chul; Jo, Ye-Seul; Oh, Deok-Kun

    2014-11-01

    The double-site variant (C450S-N475K) L-arabinose isomerase (L-AI) from Geobacillus thermodenitrificans catalyzes the isomerization of D-galactose to D-tagatose, a functional sweetener. Using a substrate-docking homology model, the residues near to D-galactose O6 were identified as Met186, Phe280, and Ile371. Several variants obtained by site-directed mutagenesis of these three residues were analyzed, and a triple-site (F280N) variant enzyme exhibited the highest activity for D-galactose isomerization. The k cat/K m of the triple-site variant enzyme for D-galactose was 2.1-fold higher than for L-arabinose, whereas the k cat/K m of the double-site variant enzyme for L-arabinose was 43.9-fold higher than for D-galactose. These results suggest that the triple-site variant enzyme is a D-galactose isomerase. The conversion rate of D-galactose to D-tagatose by the triple-site variant enzyme was approximately 3-fold higher than that of the double-site variant enzyme for 30 min. However, the conversion yields of L-arabinose to L-ribulose by the triple-site and double-site variant enzymes were 10.6 and 16.0 % after 20 min, respectively. The triple-site variant enzyme exhibited increased specific activity, turnover number, catalytic efficiency, and conversion rate for D-galactose isomerization compared to the double-site variant enzyme. Therefore, the amino acid at position 280 determines the substrate specificity for D-galactose and L-arabinose, and the triple-site variant enzyme has the potential to produce D-tagatose on an industrial scale.

  5. The chitin biosynthesis pathway in Entamoeba and the role of glucosamine-6-P isomerase by RNA interference.

    Science.gov (United States)

    Samanta, Sintu Kumar; Ghosh, Sudip K

    2012-11-01

    Entamoeba histolytica, the causative agent of amoebiasis, infects through its cyst form. A thick chitin wall protects the cyst from the harsh environment outside of the body. It is known that chitin is synthesized only during encystation, but the chitin synthesis pathway (CSP) of Entamoeba is not well characterized. In this report, we have identified the genes involved in chitin biosynthesis from the Entamoeba genome database and verified their expression profile at the transcriptional level in encysting Entamoeba invadens. Semi-quantitative RT-PCR (sqRT-PCR) analysis showed that all the chitin pathway genes are entirely absent or transcribed at low levels in trophozoites. The mRNA expression of most of the CSP genes reached their maximum level between 9 and 12h after the in vitro initiation of encystation. Double-stranded RNA-mediated silencing of glucosamine-6-P isomerase (Gln6Pi) reduced chitin synthesis to 62-64%, which indicates that Gln6Pi might be a key enzyme for regulating chitin synthesis in Entamoeba. The study of different enzymes involved in glycogen metabolism revealed that stored glycogen is converted to glucose during encystation. It is clear from the sqRT-PCR analysis that the rate of glycolysis decreases as encystation proceeds. Encystation up-regulates the expression of glycogen phosphorylase, which is responsible for glycogen degradation. The significant decrease in chitin synthesis in encysting cells treated with a specific inhibitor of glycogen phosphorylase indicates that the glucose obtained from the degradation of stored glycogen in trophozoites might be one of the major sources of glucose for chitin synthesis.

  6. Triosephosphate isomerase: energetics of the reaction catalyzed by the yeast enzyme expressed in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Nickbarg, E.B.; Knowles, J.R.

    1988-08-09

    Triosephosphate isomerase from bakers' yeast, expressed in Escherichia coli strain DF502(p12), has been purified to homogeneity. The kinetics of the reaction in each direction have been determined at pH 7.5 and 30 degrees C. Deuterium substitution at the C-2 position of substrate (R)-glyceraldehyde phosphate and at the 1-pro-R position of substrate dihydroxyacetone phosphate results in kinetic isotope effects on kcat of 1.6 and 3.4, respectively. The extent of transfer of tritium from (1(R)-TH)dihydroxyacetone phosphate to product (R)-glyceraldehyde phosphate during the catalyzed reaction is only 3% after 66% conversion to product, indicating that the enzymic base that mediates proton transfer is in rapid exchange with solvent protons. When the isomerase-catalyzed reaction is run in tritiated water in each direction, radioactivity is incorporated both into the remaining substrate and into the product. In the exchange-conversion experiment with dihydroxyacetone phosphate as substrate, the specific radioactivity of remaining dihydroxyacetone phosphate rises as a function of the extent of reaction with a slope of about 0.3, while the specific radioactivity of the products is 54% that of the solvent. In the reverse direction with (R)-glyceraldehyde phosphate as substrate, the specific radioactivity of the product formed is only 11% that of the solvent, while the radioactivity incorporated into the remaining substrate (R)-glyceraldehyde phosphate also rises as a function of the extent of reaction with a slope of 0.3. These results have been analyzed according to the protocol described earlier to yield the free energy profile of the reaction catalyzed by the yeast isomerase.

  7. Structural basis for featuring of steroid isomerase activity in alpha class glutathione transferases.

    Science.gov (United States)

    Tars, Kaspars; Olin, Birgit; Mannervik, Bengt

    2010-03-19

    Glutathione transferases (GSTs) are abundant enzymes catalyzing the conjugation of hydrophobic toxic substrates with glutathione. In addition to detoxication, human GST A3-3 displays prominent steroid double-bond isomerase activity; e.g. transforming Delta(5)-androstene-3-17-dione into Delta(4)-androstene-3-17-dione (AD). This chemical transformation is a crucial step in the biosynthesis of steroids, such as testosterone and progesterone. In contrast to GST A3-3, the homologous GST A2-2 does not show significant steroid isomerase activity. We have solved the 3D structures of human GSTs A2-2 and A3-3 in complex with AD. In the GST A3-3 crystal structure, AD was bound in an orientation suitable for the glutathione (GSH)-mediated catalysis to occur. In GST A2-2, however, AD was bound in a completely different orientation with its reactive double bond distant from the GSH-binding site. The structures illustrate how a few amino acid substitutions in the active site spectacularly alter the binding mode of the steroid substrate in relation to the conserved catalytic groups and an essentially fixed polypeptide chain conformation. Furthermore, AD did not bind to the GST A2-2-GSH complex. Altogether, these results provide a first-time structural insight into the steroid isomerase activity of any GST and explain the 5000-fold difference in catalytic efficiency between GSTs A2-2 and A3-3. More generally, the structures illustrate how dramatic diversification of functional properties can arise via minimal structural alterations. We suggest a novel structure-based mechanism of the steroid isomerization reaction.

  8. Glucose(xylose isomerase production by Streptomyces sp. CH7 grown on agricultural residues

    Directory of Open Access Journals (Sweden)

    Kankiya Chanitnun

    2012-09-01

    Full Text Available Streptomyces sp. CH7 was found to efficiently produce glucose(xylose isomerase when grown on either xylan or agricultural residues. This strain produced a glucose(xylose isomerase activity of roughly 1.8 U/mg of protein when it was grown in medium containing 1% xylose as a carbon source. Maximal enzymatic activities of about 5 and 3 U/mg were obtained when 1% xylan and 2.5% corn husks were used, respectively. The enzyme was purified from a mycelial extract to 16-fold purity with only two consecutive column chromatography steps using Macro-prep DEAE and Sephacryl-300, respectively. The approximate molecular weight of the purified enzyme is 170 kDa, and it has four identical subunits of 43.6 kDa as estimated by SDS-PAGE. Its Km values for glucose and xylose were found to be 258.96 and 82.77 mM, respectively, and its Vmax values are 32.42 and 63.64 μM/min/mg, respectively. The purified enzyme is optimally active at 85ºC and pH 7.0. It is stable at pH 5.5-8.5 and at temperatures up to 60ºC after 30 min. These findings indicate that glucose(xylose isomerase from Streptomyces sp. CH7 has the potential for industrial applications, especially for high-fructose syrup production and bioethanol fermentation from hemicellulosic hydrolysates by Saccharomyces cerevisiae.

  9. Effect of gamma irradiation on whole-cell glucose isomerase. Pt. 1

    Energy Technology Data Exchange (ETDEWEB)

    Bachman, S.; Gebicka, L.

    1984-03-01

    Gamma-rays induced inactivation of Actinoplanes missouriensis and Streptomyces olivaceus glucose isomerase has been studied. This enzyme exhibits high resistance against ionizing radiation. The D/sub 37/ value was found to be equal to 131 kGy for Actinoplanes missouriensis cells and 88 kGy for Streptomyces olivaceus cells when irradiated in the dry state in the presence of air. Mg/sup 2 +/ ions do not affect the radiosensitivity of the enzyme in cells, while the addition of Co/sup 2 +/ ions to the cell suspension increases its stability against ionizing radiation.

  10. Crystal structure and substrate specificity of D-galactose-6-phosphate isomerase complexed with substrates.

    Directory of Open Access Journals (Sweden)

    Woo-Suk Jung

    Full Text Available D-Galactose-6-phosphate isomerase from Lactobacillus rhamnosus (LacAB; EC 5.3.1.26, which is encoded by the tagatose-6-phosphate pathway gene cluster (lacABCD, catalyzes the isomerization of D-galactose-6-phosphate to D-tagatose-6-phosphate during lactose catabolism and is used to produce rare sugars as low-calorie natural sweeteners. The crystal structures of LacAB and its complex with D-tagatose-6-phosphate revealed that LacAB is a homotetramer of LacA and LacB subunits, with a structure similar to that of ribose-5-phosphate isomerase (Rpi. Structurally, LacAB belongs to the RpiB/LacAB superfamily, having a Rossmann-like αβα sandwich fold as has been identified in pentose phosphate isomerase and hexose phosphate isomerase. In contrast to other family members, the LacB subunit also has a unique α7 helix in its C-terminus. One active site is distinctly located at the interface between LacA and LacB, whereas two active sites are present in RpiB. In the structure of the product complex, the phosphate group of D-tagatose-6-phosphate is bound to three arginine residues, including Arg-39, producing a different substrate orientation than that in RpiB, where the substrate binds at Asp-43. Due to the proximity of the Arg-134 residue and backbone Cα of the α6 helix in LacA to the last Asp-172 residue of LacB with a hydrogen bond, a six-carbon sugar-phosphate can bind in the larger pocket of LacAB, compared with RpiB. His-96 in the active site is important for ring opening and substrate orientation, and Cys-65 is essential for the isomerization activity of the enzyme. Two rare sugar substrates, D-psicose and D-ribulose, show optimal binding in the LacAB-substrate complex. These findings were supported by the results of LacA activity assays.

  11. Immobilization of glucose isomerase onto radiation synthesized P(AA-co-AMPS) hydrogel and its application

    OpenAIRE

    2014-01-01

    Isomerization of glucose to fructose was carried out using Glucose isomerase (GI) that immobilized by entrapment into Poly(acrylic acid) P(AA) and Poly(acrylic acid-co-2-Acrylamido 2-methyl Propane sulfonic acid) P(AA-co-AMPS) polymer networks, the enzyme carriers were prepared by radiation induced copolymerization in the presence of (Methylene-bisacrylamide) (MBAA) as a crosslinking agent. The maximum gel fraction of pure P(AA) and P(AA-co-AMPS) hydrogel was found to be 95.2% and 89.6% for P...

  12. Redox-coupled structural changes of the catalytic a' domain of protein disulfide isomerase.

    Science.gov (United States)

    Inagaki, Koya; Satoh, Tadashi; Yagi-Utsumi, Maho; Le Gulluche, Anne-Charlotte; Anzai, Takahiro; Uekusa, Yoshinori; Kamiya, Yukiko; Kato, Koichi

    2015-09-14

    Protein disulfide isomerase functions as a folding catalyst in the endoplasmic reticulum. Its b' and a' domains provide substrate-binding sites and undergo a redox-dependent domain rearrangement coupled to an open-closed structural change. Here we determined the first solution structure of the a' domain in its oxidized form and thereby demonstrate that oxidation of the a' domain induces significant conformational changes not only in the vicinity of the active site but also in the distal b'-interfacial segment. Based on these findings, we propose that this conformational transition triggers the domain segregation coupled with the exposure of the hydrophobic surface.

  13. Endoglin、肽基脯氨酰顺反异构酶和微管不稳定蛋白在非小细胞肺癌组织中的表达及其临床意义%Expression and clinical significance of endoglin, peptidy1 proly1 cistrans isomerase and stathmin in non-small cell lung carcinoma

    Institute of Scientific and Technical Information of China (English)

    潘丹; 杨小敏; 朱海乐

    2015-01-01

    Objective To investigate the expression of microvessel density (MVD) marked with endoglin,peptidy1 proly1 cis-trans isomerase (Pin1) and stathmin in non-small cell lung carcinoma (NSCLC) and to explore its relevance to the clinicopathologic features.Methods The expression of MVD marked with endoglin,Pin1 and stathmin was detected by immunohistochemistry in 42 cases of NSCLC tissues and 20 cases of non-cancerous adjacent lung tissues.Results The expression of MVD marked with endoglin was 41.26 ± 2.32 in NSCLC tissues,and 23.04 ± 1.44 in non-cancerous adjacent lung tissues (P <0.01).The expression of MVD marked with endoglin were related NSCLC to TNM stage and lymph node metastasis (P < 0.05).The expression rate of Pin1 in NSCLC tissues was higher than that in non-cancerous adjacent lung tissues (83.33% vs.25.00%,P < 0.05).The expressions of Pin1 was related NSCLC to TNM stage and lymph node metastasis (P < 0.05).The expression rate of stathmin in NSCLC tissues was higher than that in non-cancerous adjacent lung tissues (64.29% vs.30.00%,P < 0.05).The expressions of stathmin was related NSCLC to histologic grade,TNM stage and lymph node metastasis (P <0.05).Conclusion The overexpression of endoglin,Pin1 and stathmin may play important roles in the occurrence and development of NSCLC,and they can be used as reference index of the malignant degree and poor prognosis of NSCLC.%目的 探讨Endoglin标记的微血管密度计数(MVD)、肽基脯氨酰顺反异构酶(Pin1)和微管不稳定蛋白(Stathmin)在非小细胞肺癌(NSCLC)组织中的表达及与NSCLC临床病理特征的关系.方法 利用免疫组织化学方法检测42例NSCLC组织和20例癌旁组织中Endoglin标记的MVD、Pin1和Stathmin的表达.结果 Endoglin标记的MVD在NSCLC癌组织中表达强度为41.26±2.32,明显高于在癌旁组织中表达强度(23.04±1.44),两者差异有统计学意义(P<0.01),Endoglin标记的MVD表达强度与NSCLC的TNM分期、

  14. The Crystal Structure of Yeast Protein Disulfide Isomerase Suggests Cooperativity Between Its Active Sites

    Energy Technology Data Exchange (ETDEWEB)

    Tian,G.; Xiang, S.; Noiva, R.; Lennarz, W.; Schindelin, H.

    2006-01-01

    Protein disulfide isomerase plays a key role in catalyzing the folding of secretory proteins. It features two catalytically inactive thioredoxin domains inserted between two catalytically active thioredoxin domains and an acidic C-terminal tail. The crystal structure of yeast PDI reveals that the four thioredoxin domains are arranged in the shape of a twisted 'U' with the active sites facing each other across the long sides of the 'U.' The inside surface of the 'U' is enriched in hydrophobic residues, thereby facilitating interactions with misfolded proteins. The domain arrangement, active site location, and surface features strikingly resemble the Escherichia coli DsbC and DsbG protein disulfide isomerases. Biochemical studies demonstrate that all domains of PDI, including the C-terminal tail, are required for full catalytic activity. The structure defines a framework for rationalizing the differences between the two active sites and their respective roles in catalyzing the formation and rearrangement of disulfide bonds.

  15. Characterization of a monoclonal antibody that specifically inhibits triosephosphate isomerase activity of Taenia solium.

    Science.gov (United States)

    Víctor, Sanabria-Ayala; Yolanda, Medina-Flores; Araceli, Zavala-Carballo; Lucía, Jiménez; Abraham, Landa

    2013-08-01

    In the present study, we obtained and characterized partially a monoclonal antibody (4H11D10B11 mAb) against triosephosphate isomerase from Taenia solium (TTPI). This antibody recognized the enzyme by both ELISA and western blot and was able to inhibit its enzymatic activity in 74%. Moreover, the antigen-binding fragments (Fabs), products of digestion of the monoclonal antibody with papain, retained almost the same inhibitory effect. We determined the binding site by ELISA; synthetic peptides containing sequences from different non-conserved regions of the TTPI were confronted to the 4H11D10B11 mAb. The epitope recognized by the monoclonal antibody was located on peptide TTPI-56 (ATPAQAQEVHKVVRDWIRKHVDAGIADKARI), and an analysis of mimotopes, obtained with the 4H11D10B11 mAb, suggests that the epitope spans the sequence WIRKHVDAGIAD, residues 193-204 of the enzyme. This epitope is located within helix 6, next to loop 6, an essential active loop during catalysis. The antibody did not recognize triosephosphate isomerase from man and pig, definitive and intermediary hosts of T. solium, respectively. Furthermore, it did not bind to the catalytic site, since kinetic analysis demonstrated that inhibition had a non-competitive profile.

  16. Theileria parasites secrete a prolyl isomerase to maintain host leukocyte transformation.

    Science.gov (United States)

    Marsolier, J; Perichon, M; DeBarry, J D; Villoutreix, B O; Chluba, J; Lopez, T; Garrido, C; Zhou, X Z; Lu, K P; Fritsch, L; Ait-Si-Ali, S; Mhadhbi, M; Medjkane, S; Weitzman, J B

    2015-04-16

    Infectious agents develop intricate mechanisms to interact with host cell pathways and hijack their genetic and epigenetic machinery to change host cell phenotypic states. Among the Apicomplexa phylum of obligate intracellular parasites, which cause veterinary and human diseases, Theileria is the only genus that transforms its mammalian host cells. Theileria infection of bovine leukocytes induces proliferative and invasive phenotypes associated with activated signalling pathways, notably JNK and AP-1 (ref. 2). The transformed phenotypes are reversed by treatment with the theilericidal drug buparvaquone. We used comparative genomics to identify a homologue of the peptidyl-prolyl isomerase PIN1 in T. annulata (TaPIN1) that is secreted into the host cell and modulates oncogenic signalling pathways. Here we show that TaPIN1 is a bona fide prolyl isomerase and that it interacts with the host ubiquitin ligase FBW7, leading to its degradation and subsequent stabilization of c-JUN, which promotes transformation. We performed in vitro and in silico analysis and in vivo zebrafish xenograft experiments to demonstrate that TaPIN1 is directly inhibited by the anti-parasite drug buparvaquone (and other known PIN1 inhibitors) and is mutated in a drug-resistant strain. Prolyl isomerization is thus a conserved mechanism that is important in cancer and is used by Theileria parasites to manipulate host oncogenic signalling.

  17. Analysis of bacterial xylose isomerase gene diversity using gene-targeted metagenomics.

    Science.gov (United States)

    Nurdiani, Dini; Ito, Michihiro; Maruyama, Toru; Terahara, Takeshi; Mori, Tetsushi; Ugawa, Shin; Takeyama, Haruko

    2015-08-01

    Bacterial xylose isomerases (XI) are promising resources for efficient biofuel production from xylose in lignocellulosic biomass. Here, we investigated xylose isomerase gene (xylA) diversity in three soil metagenomes differing in plant vegetation and geographical location, using an amplicon pyrosequencing approach and two newly-designed primer sets. A total of 158,555 reads from three metagenomic DNA replicates for each soil sample were classified into 1127 phylotypes, detected in triplicate and defined by 90% amino acid identity. The phylotype coverage was estimated to be within the range of 84.0-92.7%. The xylA gene phylotypes obtained were phylogenetically distributed across the two known xylA groups. They shared 49-100% identities with their closest-related XI sequences in GenBank. Phylotypes demonstrating analysis, suggesting soil-specific xylA genotypes and taxonomic compositions. The differences among xylA members and their compositions in the soil were strongly correlated with 16S rRNA variation between soil samples, also assessed by amplicon pyrosequencing. This is the first report of xylA diversity in environmental samples assessed by amplicon pyrosequencing. Our data provide information regarding xylA diversity in nature, and can be a basis for the screening of novel xylA genotypes for practical applications.

  18. Sucrose isomerase and its mutants from Erwinia rhapontici can synthesise α-arbutin.

    Science.gov (United States)

    Zhou, Xing; Zheng, Yuantao; Wei, Xingming; Yang, Kedi; Yang, Xiangkai; Wang, Yuting; Xu, Liming; Du, Liqin; Huang, Ribo

    2011-10-01

    Sucrose isomerase (SI) from Erwinia rhapontici is an intramolecular isomerase that is normally used to synthesise isomaltulose from sucrose by a mechanism of intramolecular transglycosylation. In this study, it was found that SI could synthesise α-arbutin using hydroquinone and sucrose as substrates, via an intermolecular transglycosylation reaction. Five phenylalanine residues (F185, F186, F205, F297, and F321) in the catalytic pocket of SI were chosen for sitedirected mutagenesis. Mutants F185I, F321I, and F321W, whose hydrolytic activities were enhanced after the mutation, could synthesise α-arbutin through intermolecular transglycosylation with a more than two-fold increase in the molar transfer ratio compared with wild type SI. The F297A mutant showed a strong ability to synthesise a novel α-arbutin derivative and a four-fold increase in its specific activity for intermolecular transglycosylation over the wild type. Our findings may lead to a new way to synthesise novel glucoside products such as α-arbutin derivatives by simply manipulating the Phe residues in the catalytic pocket. From the structure superposition, our strategy of manipulating these Phe residues may be applicable to other similar transglycosylating enzymes.

  19. Post-duplication charge evolution of phosphoglucose isomerases in teleost fishes through weak selection on many amino acid sites

    Directory of Open Access Journals (Sweden)

    Sato Yukuto

    2007-10-01

    Full Text Available Abstract Background The partitioning of ancestral functions among duplicated genes by neutral evolution, or subfunctionalization, has been considered the primary process for the evolution of novel proteins (neofunctionalization. Nonetheless, how a subfunctionalized protein can evolve into a more adaptive protein is poorly understood, mainly due to the limitations of current analytical methods, which can detect only strong selection for amino acid substitutions involved in adaptive molecular evolution. In this study, we employed a comparative evolutionary approach to this question, focusing on differences in the structural properties of a protein, specifically the electric charge, encoded by fish-specific duplicated phosphoglucose isomerase (Pgi genes. Results Full-length cDNA cloning, RT-PCR based gene expression analyses, and comparative sequence analyses showed that after subfunctionalization with respect to the expression organ of duplicate Pgi genes, the net electric charge of the PGI-1 protein expressed mainly in internal tissues became more negative, and that of PGI-2 expressed mainly in muscular tissues became more positive. The difference in net protein charge was attributable not to specific amino acid sites but to the sum of various amino acid sites located on the surface of the PGI molecule. Conclusion This finding suggests that the surface charge evolution of PGI proteins was not driven by strong selection on individual amino acid sites leading to permanent fixation of a particular residue, but rather was driven by weak selection on a large number of amino acid sites and consequently by steady directional and/or purifying selection on the overall structural properties of the protein, which is derived from many modifiable sites. The mode of molecular evolution presented here may be relevant to various cases of adaptive modification in proteins, such as hydrophobic properties, molecular size, and electric charge.

  20. The Caenorhabditis elegans parvulin gene subfamily and their expression under cold or heat stress along with the fkb subfamily.

    Science.gov (United States)

    Fasseas, Michael K; Dimou, Maria; Katinakis, Panagiotis

    2012-07-06

    Parvulins and FKBPs are members of the peptidyl-prolyl cis/trans isomerases (PPIase) enzyme family whose role is to catalyze the interconversion between the cis trans forms of a peptide bond preceding internal proline residues in a polypeptide substrate. Members of the parvulin subfamily have been found to be involved in a variety of diseases, including Alzheimer's disease and cancer and are also considered possible antiparasitic targets. Genes Y110A2AL.13 (pin-1) and Y48C3A.16 (pin-4) were found in the worm's genome, possibly encoding parvulins. One is homologous to human and fly PIN1 whereas the other is homologous to human and fly PIN4. Both were expressed in Escherichia coli, purified and found to have in vitro PPIase activity. Expression levels of both genes, as well as the fkb genes (that encode FK506-binding proteins) were measured during development and under cold or heat stress conditions. The results revealed a potential role for these genes under temperature-related stress. RNAi silencing was performed for wild type and mutant strain worms under normal and cold or heat stress conditions. A reduced lifespan was observed when pin-4 dsRNA was fed to the fkb-5 deficient worms. Our work presents a first attempt to characterize the Caenorhabditis elegans parvulins and may present an interesting starting point for further experimentation concerning their role, along with the FKBP subfamily, in nematode physiology and their possible use as antiparasitic targets.

  1. Kinetic measurements of phosphoglucose isomerase and phosphomannose isomerase by direct analysis of phosphorylated aldose-ketose isomers using tandem mass spectrometry

    Science.gov (United States)

    Gao, Hong; Chen, Ye; Leary, Julie A.

    2005-02-01

    A mass spectrometry based method for the direct determination of kinetic constants for phosphoglucose isomerase (PGI) and phosphomannose isomerase (PMI) is described. PGI catalyzes the interconversion between glucose-6-phosphate (Glc6P) and fructose-6-phosphate (Fru6P) and PMI performs the same function between mannose-6-phosphate (Man6P) and Fru6P. These two enzymes are essential in the pathways of glycolytic or oxidative metabolism of carbohydrates and have been considered as potential therapeutic targets. Traditionally, they are assayed either by spectrophotometric detection of Glc6P with one or more coupling enzymes or by a colorimetric detection of Fru6P. However, no suitable assay for Man6P has been developed yet to study the reaction of PMI in the direction from Fru6P to Man6P. In the work presented herein, a general assay for the isomeric substrate-product pair between Glc6P and Fru6P or between Man6P and Fru6P was developed, with the aim of directly studying the kinetics of PGI and PMI in both directions. The 6-phosphorylated aldose and ketose isomers were distinguished based on their corresponding tandem mass spectra (MS2) obtained on a quadrupole ion trap mass spectrometer, and a multicomponent quantification method was utilized to determine the composition of binary mixtures. Using this method, the conversion between Fru6P and Glc6P and that between Fru6P and Man6P are directly monitored. The equilibrium constants for the reversible reactions catalyzed by PGI and PMI are measured to be 0.3 and 1.1, respectively, and the kinetic parameters for both substrates of PGI and PMI are also determined. The values of KM and Vmax for Fru6P as substrate of PMI are reported to be 0.15 mM and 7.78 [mu]mol/(min mg), respectively. All other kinetic parameters measured correlate well with those obtained using traditional methods, demonstrating the accuracy and reliability of this assay.

  2. EST Table: CK528352 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available CK528352 rswfa0_006832.y1 10/09/29 100 %/165 aa dbj|BAD90848.1| cyclophilin-like pr... 10/08/28 66 %/168 aa Y49A3A.5#CE22213#WBGene00000877#locus:cyn-1#Peptidyl-prolyl cis-trans isomerase#status:Confirmed#UniProt:P52

  3. Gclust Server: 70270 [Gclust Server

    Lifescience Database Archive (English)

    Full Text Available 70270 Ter_Tery_2705 Cluster Sequences - 517 peptidyl-prolyl cis-trans isomerase, cy...clophilin type 1 1.00e+00 0.0 0.0 4.0 0.0 0.0 0.0 Show 70270 Cluster ID 70270 Sequence ID Ter_Tery_2705 Link

  4. Dicty_cDB: SLA264 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available -propyl cis-trans isomerase (CYP1) gene, complete cds. 52 3e-22 4 AJ537762 |AJ537762.1 Timarcha... balearica EST, clone Timarcha3B3. 88 5e-22 3 AJ537662 |AJ537662.1 Timarcha balearica EST, clone Timarcha

  5. Kinase-dead ATM protein is highly oncogenic and can be preferentially targeted by Topo-isomerase I inhibitors.

    Science.gov (United States)

    Yamamoto, Kenta; Wang, Jiguang; Sprinzen, Lisa; Xu, Jun; Haddock, Christopher J; Li, Chen; Lee, Brian J; Loredan, Denis G; Jiang, Wenxia; Vindigni, Alessandro; Wang, Dong; Rabadan, Raul; Zha, Shan

    2016-06-15

    Missense mutations in ATM kinase, a master regulator of DNA damage responses, are found in many cancers, but their impact on ATM function and implications for cancer therapy are largely unknown. Here we report that 72% of cancer-associated ATM mutations are missense mutations that are enriched around the kinase domain. Expression of kinase-dead ATM (Atm(KD/-)) is more oncogenic than loss of ATM (Atm(-/-)) in mouse models, leading to earlier and more frequent lymphomas with Pten deletions. Kinase-dead ATM protein (Atm-KD), but not loss of ATM (Atm-null), prevents replication-dependent removal of Topo-isomerase I-DNA adducts at the step of strand cleavage, leading to severe genomic instability and hypersensitivity to Topo-isomerase I inhibitors. Correspondingly, Topo-isomerase I inhibitors effectively and preferentially eliminate Atm(KD/-), but not Atm-proficientor Atm(-/-) leukemia in animal models. These findings identify ATM kinase-domain missense mutations as a potent oncogenic event and a biomarker for Topo-isomerase I inhibitor based therapy.

  6. Cloning and Overexpression of the Triosephosphate Isomerase Genes from Psychrophilic and Thermophilic Bacteria. Structural Comparison of the Predicted Protein Sequences

    NARCIS (Netherlands)

    Rentier-Delrue, Françoise; Mande, Shekhar C.; Moyens, Sylvianne; Terpstra, Peter; Mainfroid, Véronique; Goraj, Karine; Lion, Michelle; Hol, Wim G.J.; Martial, Joseph A.

    1993-01-01

    We focused on the temperature adaptation of triosephosphate isomerase (TIM; E.C. 5.3.1.1.) by comparing the structure of TIMs isolated from bacterial organisms living in either cold or hot environments. The TIM gene from psychrophilic bacteria Moraxella sp. TA137 was cloned and its nucleotide sequen

  7. Physical proximity and functional association of glycoprotein 1balpha and protein-disulfide isomerase on the platelet plasma membrane

    NARCIS (Netherlands)

    Burgess, J K; Hotchkiss, K A; Suter, C; Dudman, N P; Szöllösi, J; Chesterman, C N; Chong, B H; Hogg, P J

    2000-01-01

    Platelet function is influenced by the platelet thiol-disulfide balance. Platelet activation resulted in 440% increase in surface protein thiol groups. Two proteins that presented free thiol(s) on the activated platelet surface were protein-disulfide isomerase (PDI) and glycoprotein 1balpha (GP1balp

  8. A unique arabinose 5-phosphate isomerase found within a genomic island associated with the uropathogenicity of Escherichia coli CFT073.

    Science.gov (United States)

    Mosberg, Joshua A; Yep, Alejandra; Meredith, Timothy C; Smith, Sara; Wang, Pan-Fen; Holler, Tod P; Mobley, Harry L T; Woodard, Ronald W

    2011-06-01

    Previous studies showed that deletion of genes c3405 to c3410 from PAI-metV, a genomic island from Escherichia coli CFT073, results in a strain that fails to compete with wild-type CFT073 after a transurethral cochallenge in mice and is deficient in the ability to independently colonize the mouse kidney. Our analysis of c3405 to c3410 suggests that these genes constitute an operon with a role in the internalization and utilization of an unknown carbohydrate. This operon is not found in E. coli K-12 but is present in a small number of pathogenic E. coli and Shigella boydii strains. One of the genes, c3406, encodes a protein with significant homology to the sugar isomerase domain of arabinose 5-phosphate isomerases but lacking the tandem cystathionine beta-synthase domains found in the other arabinose 5-phosphate isomerases of E. coli. We prepared recombinant c3406 protein, found it to possess arabinose 5-phosphate isomerase activity, and characterized this activity in detail. We also constructed a c3406 deletion mutant of E. coli CFT073 and demonstrated that this deletion mutant was still able to compete with wild-type CFT073 in a transurethral cochallenge in mice and could colonize the mouse kidney. These results demonstrate that the presence of c3406 is not essential for a pathogenic phenotype.

  9. Probing the structure of human protein disulfide isomerase by chemical cross-linking combined with mass spectrometry

    DEFF Research Database (Denmark)

    Peng, Li; Rasmussen, Morten Ib; Chailyan, Anna;

    2014-01-01

    Protein disulfide-isomerase (PDI) is a four-domain flexible protein that catalyzes the formation of disulfide bonds in the endoplasmic reticulum. Here we have analyzed native PDI purified from human placenta by chemical cross-linking followed by mass spectrometry (CXMS). In addition to PDI the sa...

  10. Synthesis, Crystal Structure, and Biological Activity of cis/trans Amide Rotomers of (Z-N′-(2-Oxoindolin-3-ylideneformohydrazide

    Directory of Open Access Journals (Sweden)

    Hatem A. Abdel-Aziz

    2014-01-01

    Full Text Available (Z-N′-(2-Oxoindolin-3-ylideneformohydrazide (2 was synthesized by the reaction of (Z-3-hydrazonoindolin-2-one (1 with formic acid under reflux. The structure of 2 was characterized by IR, Mass, 1H NMR, and X-ray crystal structure determination. Interestingly, compound 2 appeared in DMSO-d6 as cis and trans amide rotomers in 25% and 75%, respectively. The X-ray analysis showed the Z geometrical isomer of 2 around –C=N– for cis and trans amide rotomers. The crystal of 2 belongs to monoclinic, space group P21/c, with a=4.5206 (1 Å, b=22.4747 (7 Å, c=17.3637 (5 Å, β=103.752 (1°, Z=8, V=1713.57 (8 Å3, Dc=1.467 Mg m−3, μ=0.11 mm−1, F(000=784, R=0.047, and wR=0.123 for 3798 observed reflections with I>2σ(I. Compound 2 exhibited a moderate activity in its antimicrobial evaluation against E. coli and P. aeruginosa and a good activity against S. aureus close to that of the standard drug ciprofloxacin. The in vitro anticancer activity of 2 was evaluated against two human tumor cell lines, namely, HepG2 hepatocellular carcinoma and MCF-7 breast cancer. HepG2 cancer cell line was more susceptible to compound 2 than MCF-7.

  11. Photophysics of the Red Chromophore of HcRed: Evidence for Cis-Trans Isomerization and Protonation-State Changes

    Energy Technology Data Exchange (ETDEWEB)

    Cotlet, M.; Mudalige, K.; Habuchi, S.; Goodwin, P.M.; Pai, R.K.; De Schryver, F.

    2010-03-15

    HcRed is a dimeric intrinsically fluorescent protein with origins in the sea anemone Heteractis crispa. This protein exhibits deep red absorption and emission properties. Using a combination of ensemble and single molecule methods and by varying environmental parameters such as temperature and pH, we found spectroscopic evidence for the presence of two ground state conformers, trans and cis chromophores that are in thermal equilibrium and that follow different excited-state pathways upon exposure to light. The photocycle of HcRed appears to be a combination of both kindling proteins and bright emitting GFP/GFP-like proteins: the trans chromophore undergoes light driven isomerization followed by radiative relaxation with a fluorescence lifetime of 0.5 ns. The cis chromophore exhibits a photocycle similar to bright GFPs and GFP-like proteins such as enhanced GFP, enhanced YFP or DsRed, with radiative relaxation with a fluorescence lifetime of 1.5 ns, singlet-triplet deactivation on a microsecond time scale and solvent controlled protonation/deprotonation in tens of microseconds. Using single molecule spectroscopy, we identify trans and cis conformers at the level of individual moieties and show that it is possible that the two conformers can coexist in a single protein due to the dimeric nature of HcRed.

  12. Effects of thionation and fluorination on cis-trans isomerization in tertiary amides: An investigation of N -alkylglycine (Peptoid) rotamers

    DEFF Research Database (Denmark)

    Engel-Andreasen, Jens; Wich, Kathrine; Laursen, Jonas S.;

    2015-01-01

    Peptoids constitute a class of peptidomimetics with potential as protease resistant, biologically active ligands. To harness the full potential of such compounds, however, detailed predictive insight into their propensity to adopt well-defined secondary structures is highly desirable. In this wor...

  13. A theoretical view on the thermodynamic cis-trans equilibrium of dihalo ruthenium olefin metathesis (pre-)catalysts

    KAUST Repository

    Pump, Eva

    2015-02-24

    Abstract: This work was conducted to provide an overview on the position of the thermodynamic cis–trans equilibrium of 85 conventional and X-chelated alkylidene-ruthenium complexes (X=O, S, Se, N, P, Cl, I, Br). The reported energies (ΔE) were obtained through single-point calculations with M06 functional and TZVP basis set from BP86/SVP-optimized cis- and trans-dichloro geometries and using the polarizable continuum model to simulate the influence of the solvent. Dichloromethane and toluene were selected as examples for solvents with high and low dielectric constants. The obtained relative stabilities of the cis- and trans-dihalo derivatives of the respective alkylidene complexes will serve for a better explanation of their catalytic activity as has been disclosed herein with selected examples.Graphical abstract: [Figure not available: see fulltext.

  14. A theoretical study of porphyrin isomers and their core-modified analogues: cis-trans isomerism, tautomerism and relative stabilities

    Indian Academy of Sciences (India)

    M Punnagai; Saju Joseph; G Narahari Sastry

    2004-08-01

    Semiempirical (AM1 and PM3) and density functional theory (DFT) calculations were performed on about 50 porphyrin isomers with 25 each of 1,2 (syn) and 1,3 (anti) tautomeric forms. The corresponding oxa- and thia-core-modified analogues were also computed. The variations of relative energies and stabilities of the core-modified analogues were compared with parent porphyrin 1 and the corresponding oxa- and thia-analogues. The trends in relative energies are not significantly changed while going from parent system to oxa- and thia-core-modified porphyrins in case of both syn and anti tautomers. Isomers of types [2.2.0.0], [3.0.1.0], [3.1.0.0], and [4.0.0.0] are destabilized due to the absence of methine bridge, which results in angle strain for tetrapyrroles. Isomers having [2.1.1.0], [2.1.0.1], [2.0.2.0] and [2.2.0.0] connectivity, the isomers, are more stable compared to the corresponding isomers in both syn and anti forms of parent and core-modified analogues.

  15. Reduced Dimension DVR Study of cis-trans Isomerization in the S_1 State of C_2H_2

    CERN Document Server

    Baraban, Joshua H; Steeves, Adam H; Stanton, John F; Field, Robert W

    2011-01-01

    Isomerization between the cis and trans conformers of the S1 state of acetylene is studied using a reduced dimension DVR calculation. Existing DVR techniques are combined with a high accuracy potential energy surface and a kinetic energy operator derived from FG theory to yield an effective but simple Hamiltonian for treating large amplitude motions. The spectroscopic signatures of the S1 isomerization are discussed, with emphasis on the vibrational aspects. The presence of a low barrier to isomerization causes distortion of the trans vibrational level structure and the appearance of nominally electronically forbidden \\~A1A2 \\leftarrow X 1{\\Sigma}+g transitions to vibrational levels of the cis conformer. Both of these effects are modeled in agreement with experimental results, and the underlying mechanisms of tunneling and state mixing are elucidated by use of the calculated vibrational wavefunctions.

  16. Bioethanol production from steam-pretreated corn stover through an isomerase mediated process.

    Science.gov (United States)

    De Bari, Isabella; Cuna, Daniela; Di Matteo, Vincenzo; Liuzzi, Federico

    2014-03-25

    Agricultural by-products such as corn stover are considered strategic raw materials for the production of second-generation bioethanol from renewable and non-food sources. This paper describes the conversion of steam-pretreated corn stover to ethanol utilising a multi-step process including enzymatic hydrolysis, isomerisation, and fermentation of mixed hydrolysates with native Saccharomyces cerevisiae. An immobilised isomerase enzyme was used for the xylose isomerisation along with high concentrations of S. cerevisiae. The objective was to assess the extent of simultaneity of the various conversion steps, through a detailed analysis of process time courses, and to test this process scheme for the conversion of lignocellulosic hydrolysates containing several inhibitors of the isomerase enzyme (e.g. metal ions, xylitol and glycerol). The process was tested on two types of hydrolysate after acid-catalysed steam pretreatment: (a) the water soluble fraction (WSF) in which xylose was the largest carbon source and (b) the entire slurry, containing both cellulose and hemicellulose carbohydrates, in which glucose predominated. The results indicated that the ethanol concentration rose when the inoculum concentration was increased in the range 10-75 g/L. However, when xylose was the largest carbon source, the metabolic yields were higher than 0.51g(ethanol)/g(consumed) sugars probably due to the use of yeast internal cellular resources. This phenomenon was not observed in the fermentation of mixed hydrolysates obtained from the entire pretreated product and in which glucose was the largest carbon source. The ethanol yield from biomass suspensions with dry matter (DM) concentrations of 11-12% (w/v) was 70% based on total sugars (glucose, xylose, galactose). The results suggest that xylulose uptake was more effective in mixed hydrolysates containing glucose levels similar to, or higher than, xylose. Analysis of the factors that limit isomerase activity in lignocellulosic

  17. Chemotactic Activity of Cyclophilin A in the Skin Mucus of Yellow Catfish (Pelteobagrus fulvidraco and Its Active Site for Chemotaxis

    Directory of Open Access Journals (Sweden)

    Farman Ullah Dawar

    2016-08-01

    Full Text Available Fish skin mucus is a dynamic barrier for invading pathogens with a variety of anti-microbial enzymes, including cyclophilin A (CypA, a multi-functional protein with peptidyl-prolyl cis/trans isomerase (PPIase activity. Beside various other immunological functions, CypA induces leucocytes migration in vitro in teleost. In the current study, we have discovered several novel immune-relevant proteins in yellow catfish skin mucus by mass spectrometry (MS. The CypA present among them was further detected by Western blot. Moreover, the CypA present in the skin mucus displayed strong chemotactic activity for yellow catfish leucocytes. Interestingly, asparagine (like arginine in mammals at position 69 was the critical site in yellow catfish CypA involved in leucocyte attraction. These novel efforts do not only highlight the enzymatic texture of skin mucus, but signify CypA to be targeted for anti-inflammatory therapeutics.

  18. Genetic control of chalcone isomerase activity in flowers of Dianthus caryophyllus.

    Science.gov (United States)

    Forkmann, G; Dangelmayr, B

    1980-06-01

    In flowers of Dianthus caryophyllus (carnation), the gene I is concerned with a discrete step in flavonoid biosynthesis, Genotypes with recessive (ii) alleles produce yellow flowers, which contain the chalcone isosalipurposide (naringenin-chalcone-2'-glucoside) as the major petal pigment, but in genotypes with wild-type alleles flavonols and anthocyanins can be formed and the flowers are white or red. Enzymatic measurements on petal extracts of four strains with different flower coloration revealed a clear correlation between accumulation of chalcone in recessive genotypes and deficiency of chalcone isomerase (E.C. 5.5.1.6) activity. From the chemogenetic and enzymological evidence it can be concluded that naringenin-chalcone is the first product of the synthesis of the flavonoid skeleton and that only the conversion of naringenin-chalcone to naringenin furnishes the substrate for the further reactions to flavonol and anthocyanin.

  19. In silico cloning and B/T cell epitope prediction of triosephosphate isomerase from Echinococcus granulosus.

    Science.gov (United States)

    Wang, Fen; Ye, Bin

    2016-10-01

    Cystic echinococcosis is a worldwide zoonosis caused by Echinococcus granulosus. Because the methods of diagnosis and treatment for cystic echinococcosis were limited, it is still necessary to screen target proteins for the development of new anti-hydatidosis vaccine. In this study, the triosephosphate isomerase gene of E. granulosus was in silico cloned. The B cell and T cell epitopes were predicted by bioinformatics methods. The cDNA sequence of EgTIM was composition of 1094 base pairs, with an open reading frame of 753 base pairs. The deduced amino acid sequences were composed of 250 amino acids. Five cross-reactive epitopes, locating on 21aa-35aa, 43aa-57aa, 94aa-107aa, 115-129aa, and 164aa-183aa, could be expected to serve as candidate epitopes in the development of vaccine against E. granulosus. These results could provide bases for gene cloning, recombinant expression, and the designation of anti-hydatidosis vaccine.

  20. Inheritance and subcellular localization of triose-phosphate isomerase in dwarf mountain pine (Pinus mugo).

    Science.gov (United States)

    Odrzykoski, I J

    2001-01-01

    Several trees with expected heterozygous phenotype for triose-phosphate isomerase (TPI) were discovered in a population of dwarf mountain pine (Pinus mugo Turra) from southern Poland. As the inheritance of this enzyme in pines has not been reported, segregation of allelic variants was tested in eight trees with putative heterozygous phenotypes for two loci, TpiA and TPIB: Linkage between these and some other isozyme loci were studied and evidence for linkage has been found between TpiA and PgdA (r = 0.10) and between TpiB and DiaD (r = 0.36), but in single trees only. The subcellular localization of TPI isozymes was determined by comparing isoenzymes from the total extract with those found in fraction enriched in plastids, prepared by differential gradient centrifugation of cellular organelles. The more slowly migrating TPI-B isozyme is located in plastids.

  1. Mechanisms of Neuroprotection by Protein Disulphide Isomerase in Amyotrophic Lateral Sclerosis

    Directory of Open Access Journals (Sweden)

    Adam K. Walker

    2011-01-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is a devastating neurodegenerative disease characterised by the progressive loss of motor neurons, leading to paralysis and death within several years of onset. Although protein misfolding is a key feature of ALS, the upstream triggers of disease remain elusive. Recently, endoplasmic reticulum (ER stress was identified as an early and central feature in ALS disease models as well as in human patient tissues, indicating that ER stress could be an important process in disease pathogenesis. One important chaperone induced by ER stress is protein disulphide isomerase (PDI, which is both upregulated and posttranslationally inhibited by S-nitrosylation in ALS. In this paper, we present evidence from studies of genetics, model organisms, and patient tissues which indicate an active role for PDI and ER stress in ALS disease processes.

  2. Domain a' of protein disulfide isomerase plays key role in inhibiting alpha-synuclein fibril formation.

    Science.gov (United States)

    Cheng, Han; Wang, Lei; Wang, Chih-chen

    2010-07-01

    alpha-Synuclein (alpha Syn) is the main component of Lewy bodies formed in midbrain dopaminergic neurons which is a pathological characteristic of Parkinson's disease. It has been recently showed to induce endoplasmic reticulum (ER) stress and impair ER functions. However, the mechanism of how ER responds to alpha Syn toxicity is poorly understood. In the present study, we found that protein disulfide isomerase (PDI), a stress protein abundant in ER, effectively inhibits alpha Syn fibril formation in vitro. In PDI molecule with a structure of abb'xa'c, domain a' was found to be essential and sufficient for PDI to inhibit alpha Syn fibril formation. PDI was further found to be more avid for binding with intermediate species formed during alpha Syn fibril formation, and the binding was more intensive in the later lag phase. Our results provide new insight into the role of PDI in protecting ER from the deleterious effects of misfolded protein accumulation in many neurodegenerative diseases.

  3. Extreme electric fields power catalysis in the active site of ketosteroid isomerase.

    Science.gov (United States)

    Fried, Stephen D; Bagchi, Sayan; Boxer, Steven G

    2014-12-19

    Enzymes use protein architecture to impose specific electrostatic fields onto their bound substrates, but the magnitude and catalytic effect of these electric fields have proven difficult to quantify with standard experimental approaches. Using vibrational Stark effect spectroscopy, we found that the active site of the enzyme ketosteroid isomerase (KSI) exerts an extremely large electric field onto the C=O chemical bond that undergoes a charge rearrangement in KSI's rate-determining step. Moreover, we found that the magnitude of the electric field exerted by the active site strongly correlates with the enzyme's catalytic rate enhancement, enabling us to quantify the fraction of the catalytic effect that is electrostatic in origin. The measurements described here may help explain the role of electrostatics in many other enzymes and biomolecular systems.

  4. Increase Renaturation Yield of Reteplase Using the Recombinant Human Protein Disulfide Isomerase

    Institute of Scientific and Technical Information of China (English)

    Zhao Youchun(赵友春); Wang Ge; Kong Yang; Wang Yanbing; Zhang Changkai; Chen Chao; Liang Bufeng

    2004-01-01

    Reteplase, the recombinant type of novel tissue plasminogen activator (t-PA) variant, is a promising thrombolytics in clinics. Expressed in the form of an inclusion body, reteplase consists of about 40 % of the total intracellular proteins of Escherichia coli. The recombinant human protein disulfide isomerase (rhPDI) is used to increase the chance for the correct matching of the 18 hydrosulfide groups of the reteplase molecule in the renaturation process and it increase is the reteplase renaturation yield from 1%~2% to 15%~20% with a the purity aboue 99% and the specific activity of 5(105 IU/mg is reached. This novel method can reduce significantly the cost of production.

  5. Substrate specificity of platypus venom L-to-D-peptide isomerase.

    Science.gov (United States)

    Bansal, Paramjit S; Torres, Allan M; Crossett, Ben; Wong, Karen K Y; Koh, Jennifer M S; Geraghty, Dominic P; Vandenberg, Jamie I; Kuchel, Philip W

    2008-04-04

    The L-to-D-peptide isomerase from the venom of the platypus (Ornithorhyncus anatinus) is the first such enzyme to be reported for a mammal. In delineating its catalytic mechanism and broader roles in the animal, its substrate specificity was explored. We used N-terminal segments of defensin-like peptides DLP-2 and DLP-4 and natriuretic peptide OvCNP from the venom as substrates. The DLP analogues IMFsrs and ImFsrs (srs is a solubilizing chain; lowercase letters denote D-amino acid) were effective substrates for the isomerase; it appears to recognize the N-terminal tripeptide sequence Ile-Xaa-Phe-. A suite of 26 mutants of these hexapeptides was synthesized by replacing the second residue (Met) with another amino acid, viz. Ala, alpha-aminobutyric acid, Ile, Leu, Lys, norleucine, Phe, Tyr, and Val. It was shown that mutant peptides incorporating norleucine and Phe are substrates and exhibit L- or D-amino acid isomerization, but mutant peptides that contain residues with shorter, beta-branched or long side chains with polar terminal groups, viz. Ala, alpha-aminobutyric acid, Ile, Val, Leu, Lys, and Tyr, respectively, are not substrates. It was demonstrated that at least three N-terminal amino acid residues are absolutely essential for L-to-D-isomerization; furthermore, the third amino acid must be a Phe residue. None of the hexapeptides based on LLH, the first three residues of OvCNP, were substrates. A consistent 2-base mechanism is proposed for the isomerization; abstraction of a proton by 1 base is concomitant with delivery of a proton by the conjugate acid of a second base.

  6. Improved xylose fermentation of Kluyveromyces marxianus at elevated temperature through construction of a xylose isomerase pathway.

    Science.gov (United States)

    Wang, Rongliang; Li, Lulu; Zhang, Biao; Gao, Xiaolian; Wang, Dongmei; Hong, Jiong

    2013-08-01

    To improve the xylose fermentation ability of Kluyveromyces marxianus, a xylose assimilation pathway through xylose isomerase was constructed. The genes encoding xylose reductase (KmXyl1) and xylitol dehydrogenase (KmXyl2) were disrupted in K. marxianus YHJ010 and the resultant strain was named YRL002. A codon-optimized xylose isomerase gene from Orpinomyces was transformed into K. marxianus YRL002 and expressed under GAPDH promoter. The transformant was adapted in the SD medium containing 1 % casamino acid with 2 % xylose as sole carbon source. After 32 times of trans-inoculation, a strain named YRL005, which can grow at a specific growth rate of 0.137/h with xylose as carbon source, was obtained. K. marxianus YRL005 could ferment 30.15 g/l of xylose and produce 11.52 g/l ethanol with a yield of 0.38 g/g, production rate of 0.069 g/l/h at 42 °C, and also could ferment 16.60 g/l xylose to produce 5.21 g/l ethanol with a yield of 0.31 g/g, and production rate of 0.054 g/l h at 45 °C. Co-fermentation with 2 % glucose could not improve the amount and yield of ethanol fermented from xylose obviously, but it could improve the production rate. Furthermore, K. marxianus YRL005 can ferment with the corn cob hydrolysate, which contained 20.04 g/l xylose to produce 8.25 g/l ethanol. It is a good platform to construct thermo-tolerant xylose fermentation yeast.

  7. Immobilization of glucose isomerase onto radiation synthesized P(AA-co-AMPS hydrogel and its application

    Directory of Open Access Journals (Sweden)

    H. Kamal

    2014-04-01

    Full Text Available Isomerization of glucose to fructose was carried out using Glucose isomerase (GI that immobilized by entrapment into Poly(acrylic acid P(AA and Poly(acrylic acid-co-2-Acrylamido 2-methyl Propane sulfonic acid P(AA-co-AMPS polymer networks, the enzyme carriers were prepared by radiation induced copolymerization in the presence of (Methylene-bisacrylamide (MBAA as a crosslinking agent. The maximum gel fraction of pure P(AA and P(AA-co-AMPS hydrogel was found to be 95.2% and 89.6% for P(AA and P(AA-co-AMPS, respectively at a total dose of 20 kGy. Effects of immobilization conditions such as radiation dose, MBAA concentration, comonomer composition and amount of GI were investigated. The influence of reaction conditions on the activity of immobilized GI were studied, the optimum pH value of the reaction solution is 7.5 and reaction temperature is 65 °C. The immobilized GI into P(AA-co-AMPS and P(AA polymer networks retained 81% and 69%, respectively of its initial activity after recycled for 15 times while it retained 87% and 71%, respectively of its initial activity after stored at 4 °C for 48 days. The Km values of free and immobilized GI onto P(AA-co-AMPS and onto P(AA matrices were found to be 34, 29.2 and 14.5 mg/mL, respectively while the Vmax Values calculated to be 3.87, 1.6 and 0.79 mg/mL min, respectively. GI entrapped into P(AA-co-AMPS hydrogel show promising behavior that may be useful as the newly glucose isomerase reactor in biomedical applications.

  8. Analysis of the relationship between Chalcone Isomerase gene expression level and rutin production in Ficus deltoidea var. deltoidea and F. deltoidea var. angustifolia

    Science.gov (United States)

    Najid, Najihah Mohd; Zain, Che Radziah Che Mohd; Zainal, Zamri

    2016-11-01

    Ficus deltoidea (moraceae) is a herbal plant with medicinal values. Previous studies reported that the F. deltoidea contains a high level of bioactive compounds such as flavonoids. A cDNA encodes for chalcone isomerase was identified from F. deltoidea, designated as FdCHI, which involved in the isomerization of naringenin chalcone to naringenin. Naringenin is a key branch point for the synthesis of rutin, which is believed involved in defense mechanism in the plant. Therefore, we hypothesized that there might be a direct relationship between FdCHI expression level and rutin production in leaves of F. deltoidea var. deltoidea (FDD) and F. deltoidea var. angustifolia (FDA). Our result showed that expression level of FdCHI in leaves FDD was greater than FDA. Analysis of High Performance Liquid Chromatography (HPLC) revealed that rutin was only detected in FDA leaves. Based on the results between FdCHI expression and rutin production, this study concluded that there is no relationship between FdCHI expression and rutin production in leaves of FDA and FDD.

  9. Protein disulfide isomerase-like protein 1-1 controls endosperm development through regulation of the amount and composition of seed proteins in rice.

    Directory of Open Access Journals (Sweden)

    Yeon Jeong Kim

    Full Text Available Protein disulfide isomerase (PDI is a chaperone protein involved in oxidative protein folding by acting as a catalyst and assisting folding in the endoplasmic reticulum (ER. A genome database search showed that rice contains 19 PDI-like genes. However, their functions are not clearly identified. This paper shows possible functions of rice PDI-like protein 1-1 (PDIL1-1 during seed development. Seeds of the T-DNA insertion PDIL1-1 mutant, PDIL1-1Δ, identified by genomic DNA PCR and western blot analysis, display a chalky phenotype and a thick aleurone layer. Protein content per seed was significantly lower and free sugar content higher in PDIL1-1Δ mutant seeds than in the wild type. Proteomic analysis of PDIL1-1Δ mutant seeds showed that PDIL1-1 is post-translationally regulated, and its loss causes accumulation of many types of seed proteins including glucose/starch metabolism- and ROS (reactive oxygen species scavenging-related proteins. In addition, PDIL1-1 strongly interacts with the cysteine protease OsCP1. Our data indicate that the opaque phenotype of PDIL1-1Δ mutant seeds results from production of irregular starch granules and protein body through loss of regulatory activity for various proteins involved in the synthesis of seed components.

  10. Effects of /sup 45/Ca on murine skeletal muscle. 1. Alterations of glycogen, phosphorylase and phosphohexose isomerase levels

    Energy Technology Data Exchange (ETDEWEB)

    Asotra, K.; Katoch, S.S.; Krishan, K.; Malhotra, R.K. (Himachal Pradesh Univ., Simla (India). Dept. of Bio-sciences)

    1983-01-01

    Adult Swiss albino mice weighing 16+-1 g were injected with 3.7x10/sup 4/ Bq and 7.4x10/sup 4/ Bq/g body weight of /sup 45/Ca. Mice of both dose groups were autopsied on days 1, 3, 5, 7, 14 and 28 after /sup 45/Ca administration. Diaphragm and gastrocnemius in the /sup 45/Ca-treated and normal mice were analyzed for quantitation of glycogen as well as bioassay of phosphorylase and phosphohexose isomerase activities. Internal irradiation with the two doses of /sup 45/Ca resulted in glycogen accumulation in both the muscles. /sup 45/Ca-treated diaphragm showed greater radioresponse but a slower recovery than gastrocnemius with respect to glycogen accumulation. A decline in the rates of glycogenolysis and glycolysis indicated by decreased phosphorylase and phosphohexose isomerase activities appeared to be responsible for glycogen accumulation in skeletal muscle on account of /sup 45/Ca treatment.

  11. Effect of manganese ions on ethanol fermentation by xylose isomerase expressing Saccharomyces cerevisiae under acetic acid stress.

    Science.gov (United States)

    Ko, Ja Kyong; Um, Youngsoon; Lee, Sun-Mi

    2016-12-01

    The efficient fermentation of lignocellulosic hydrolysates in the presence of inhibitors is highly desirable for bioethanol production. Among the inhibitors, acetic acid released during the pretreatment of lignocellulose negatively affects the fermentation performance of biofuel producing organisms. In this study, we evaluated the inhibitory effects of acetic acid on glucose and xylose fermentation by a high performance engineered strain of xylose utilizing Saccharomyces cerevisiae, SXA-R2P-E, harboring a xylose isomerase based pathway. The presence of acetic acid severely decreased the xylose fermentation performance of this strain. However, the acetic acid stress was alleviated by metal ion supplementation resulting in a 52% increased ethanol production rate under 2g/L of acetic acid stress. This study shows the inhibitory effect of acetic acid on an engineered isomerase-based xylose utilizing strain and suggests a simple but effective method to improve the co-fermentation performance under acetic acid stress for efficient bioethanol production.

  12. The equilibrium unfolding of triosephosphate isomerase from t. cruzi in guanidinium hydrochloride is a four state process. Intrinsic fluorescence studies

    OpenAIRE

    Edgar Vázquez Contreras; Brenda Guadalupe Sánchez Rebollar; María Elena Chánez Cárdenas

    2004-01-01

    Equilibrium and kinetic folding pathways of several homologous proteins have been studied. Early studies concluded that the folding routes of homologous proteins follow fundamentally similar pathways, and that the folding of a certain conformation is conserved throughout evolution. However, there are examples of homologous proteins that unfold by different routes. Regarding triosephosphate isomerase (TIM), unfolding studies with enzymes from different sources, have shown: 1) two-state behavio...

  13. Optimization of Fermentation Medium for the Production of Glucose Isomerase Using Streptomyces sp. SB-P1

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    Sheetal Bhasin

    2012-01-01

    Full Text Available The combination of medium ingredients has a profound influence on the metabolic pathways running in the microorganism which regulates the production of numerous metabolites. Glucose isomerase (GI, an enzyme with huge potential in the market, can isomerise glucose into fructose. GI is used widely for the production of High-Fructose Corn Syrup (HFCS. HFCS is used as a sweetener in food and pharmaceutical industries. Streptomyces are well-known producers of numerous enzymes including glucose isomerase. An array of 75 isolates was screened for the production of glucose isomerase. The isolate Streptomyces sp. SB-P1 was found to produce maximum amount of extracellular GI. Sucrose and raffinose among pure carbon sources and corn cob and wheat husk among crude agro residues were found to yield high enzyme titers. Potassium nitrate among pure nitrogen sources and soy residues among crude sources gave maximum production. Quantitative effect of carbon, nitrogen, and inducer on GI was also determined. Plackett-Burman design was used to study the effect of different medium ingredients. Sucrose and xylose as carbon sources and peptone and soy residues as nitrogen sources proved to be beneficial for GI production.

  14. The prolyl isomerase Pin1 acts synergistically with CDK2 to regulate the basal activity of estrogen receptor α in breast cancer.

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    Chiara Lucchetti

    Full Text Available In hormone receptor-positive breast cancers, most tumors in the early stages of development depend on the activity of the estrogen receptor and its ligand, estradiol. Anti-estrogens, such as tamoxifen, have been used as the first line of therapy for over three decades due to the fact that they elicit cell cycle arrest. Unfortunately, after an initial period, most cells become resistant to hormonal therapy. Peptidylprolyl isomerase 1 (Pin1, a protein overexpressed in many tumor types including breast, has been demonstrated to modulate ERalpha activity and is involved in resistance to hormonal therapy. Here we show a new mechanism through which CDK2 drives an ERalpha-Pin1 interaction under hormone- and growth factor-free conditions. The PI3K/AKT pathway is necessary to activate CDK2, which phosphorylates ERalphaSer294, and mediates the binding between Pin1 and ERalpha. Site-directed mutagenesis demonstrated that ERalphaSer294 is essential for Pin1-ERalpha interaction and modulates ERalpha phosphorylation on Ser118 and Ser167, dimerization and activity. These results open up new drug treatment opportunities for breast cancer patients who are resistant to anti-estrogen therapy.

  15. Heterologous expression and biochemical characterization of glucose isomerase from Thermobifida fusca.

    Science.gov (United States)

    Deng, Hui; Chen, Sheng; Wu, Dan; Chen, Jian; Wu, Jing

    2014-06-01

    Glucose isomerase (GIase) catalyzes the isomerization of D-glucose to D-fructose. The GIase from Thermobifida fusca WSH03-11 was expressed in Escherichia coli BL21(DE3), and the purified enzyme took the form of a tetramer in solution and displayed a pI value of 5.05. The temperature optimum of GIase was 80 °C and its half life was about 2 h at 80 °C or 15 h at 70 °C. The pH optimum of GIase was 10 and the enzyme retained 95 % activity over the pH range of 5-10 after incubating at 4 °C for 24 h. Kinetic studies showed that the K m and K cat values of the enzyme are 197 mM and 1,688 min(-1), respectively. The maximum conversion yield of glucose (45 %, w/v) to fructose of the enzyme was 53 % at pH 7.5 and 70 °C. The present study provides the basis for the industrial application of recombinant T. fusca GIase in the production of high fructose syrup.

  16. Inhibition of glucosephosphate isomerase allozymes of the mosquitofish, Gambusia holbrooki, by mercury

    Energy Technology Data Exchange (ETDEWEB)

    Kramer, V.J.; Newman, M.C. (Univ. of Georgia, Aiken, SC (United States). Savannah River Ecology Lab.)

    1994-01-01

    Frequencies of allozyme genotypes are being used as population-level indicators of environmental heavy-metal contamination. A genotype of glucose phosphate isomerase, Gpi-2[sup 38/38], of mosquitofish (Gambusia holbrooki) has been identified as mercury-sensitive in an acute toxicity assay. Partially purified preparations of GPI-2 38/38 and GPI-2 100/100 were assayed to determine differences in maximum gluconeogenic reaction velocity at seven mercury (added as HgCl[sub 2]) concentrations, 15 to 960 nM Hg. Log-Probit analysis of the inhibition curves indicated that the log (IC50) (log[sub 10] of the Hg concentration causing a 50% reduction in reaction velocity) for GPI-2 100/100 initial uninhibited reaction velocity was greater than that of GPI-2 38/38. Although the mechanism of inhibition was not experimentally determined, under the assumption of noncompetitive interaction between Hg and GPI-2, the inhibitor dissociation constants (95% asymptotic C.I.) for GPI-2 100/100 and GPI-2 38/38 were estimated from the log (IC50) as 204 nM Hg (155---269 nM Hg) and 479 nM Hg (363-617 nM Hg), respectively. These results suggested that Hg susceptibility related to the Gpi-2[sup 38/38] genotype in acute toxicity assays was likely not due to enhanced Hg inhibition of GPI-2 38/38.

  17. Mannose phosphate isomerase regulates fibroblast growth factor receptor family signaling and glioma radiosensitivity.

    Directory of Open Access Journals (Sweden)

    Aurélie Cazet

    Full Text Available Asparagine-linked glycosylation is an endoplasmic reticulum co- and post-translational modification that enables the transit and function of receptor tyrosine kinase (RTK glycoproteins. To gain insight into the regulatory role of glycosylation enzymes on RTK function, we investigated shRNA and siRNA knockdown of mannose phosphate isomerase (MPI, an enzyme required for mature glycan precursor biosynthesis. Loss of MPI activity reduced phosphorylation of FGFR family receptors in U-251 and SKMG-3 malignant glioma cell lines and also resulted in significant decreases in FRS2, Akt, and MAPK signaling. However, MPI knockdown did not affect ligand-induced activation or signaling of EGFR or MET RTKs, suggesting that FGFRs are more susceptible to MPI inhibition. The reductions in FGFR signaling were not caused by loss of FGF ligands or receptors, but instead were caused by interference with receptor dimerization. Investigations into the cellular consequences of MPI knockdown showed that cellular programs driven by FGFR signaling, and integral to the clinical progression of malignant glioma, were impaired. In addition to a blockade of cellular migration, MPI knockdown also significantly reduced glioma cell clonogenic survival following ionizing radiation. Therefore our results suggest that targeted inhibition of enzymes required for cell surface receptor glycosylation can be manipulated to produce discrete and limited consequences for critical client glycoproteins expressed by tumor cells. Furthermore, this work identifies MPI as a potential enzymatic target for disrupting cell surface receptor-dependent survival signaling and as a novel approach for therapeutic radiosensitization.

  18. Protein disulphide isomerase as a target for nanoparticle-mediated sensitisation of cancer cells to radiation

    Science.gov (United States)

    Taggart, L. E.; McMahon, S. J.; Butterworth, K. T.; Currell, F. J.; Schettino, G.; Prise, K. M.

    2016-05-01

    Radiation resistance and toxicity in normal tissues are limiting factors in the efficacy of radiotherapy. Gold nanoparticles (GNPs) have been shown to be effective at enhancing radiation-induced cell death, and were initially proposed to physically enhance the radiation dose deposited. However, biological responses of GNP radiosensitization based on physical assumptions alone are not predictive of radiosensitisation and therefore there is a fundamental research need to determine biological mechanisms of response to GNPs alone and in combination with ionising radiation. This study aimed to identify novel mechanisms of cancer cell radiosensitisation through the use of GNPs, focusing on their ability to induce cellular oxidative stress and disrupt mitochondrial function. Using N-acetyl-cysteine, we found mitochondrial oxidation to be a key event prior to radiation for the radiosensitisation of cancer cells and suggests the overall cellular effects of GNP radiosensitisation are a result of their interaction with protein disulphide isomerase (PDI). This investigation identifies PDI and mitochondrial oxidation as novel targets for radiosensitisation.

  19. Protein disulfide-isomerase interacts with a substrate protein at all stages along its folding pathway.

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    Alistair G Irvine

    Full Text Available In contrast to molecular chaperones that couple protein folding to ATP hydrolysis, protein disulfide-isomerase (PDI catalyzes protein folding coupled to formation of disulfide bonds (oxidative folding. However, we do not know how PDI distinguishes folded, partly-folded and unfolded protein substrates. As a model intermediate in an oxidative folding pathway, we prepared a two-disulfide mutant of basic pancreatic trypsin inhibitor (BPTI and showed by NMR that it is partly-folded and highly dynamic. NMR studies show that it binds to PDI at the same site that binds peptide ligands, with rapid binding and dissociation kinetics; surface plasmon resonance shows its interaction with PDI has a Kd of ca. 10(-5 M. For comparison, we characterized the interactions of PDI with native BPTI and fully-unfolded BPTI. Interestingly, PDI does bind native BPTI, but binding is quantitatively weaker than with partly-folded and unfolded BPTI. Hence PDI recognizes and binds substrates via permanently or transiently unfolded regions. This is the first study of PDI's interaction with a partly-folded protein, and the first to analyze this folding catalyst's changing interactions with substrates along an oxidative folding pathway. We have identified key features that make PDI an effective catalyst of oxidative protein folding - differential affinity, rapid ligand exchange and conformational flexibility.

  20. Effects of a Buried Cysteine-To-Serine Mutation on Yeast Triosephosphate Isomerase Structure and Stability

    Directory of Open Access Journals (Sweden)

    Adela Rodríguez-Romero

    2012-08-01

    Full Text Available All the members of the triosephosphate isomerase (TIM family possess a cystein residue (Cys126 located near the catalytically essential Glu165. The evolutionarily conserved Cys126, however, does not seem to play a significant role in the catalytic activity. On the other hand, substitution of this residue by other amino acid residues destabilizes the dimeric enzyme, especially when Cys is replaced by Ser. In trying to assess the origin of this destabilization we have determined the crystal structure of Saccharomyces cerevisiae TIM (ScTIM at 1.86 Å resolution in the presence of PGA, which is only bound to one subunit. Comparisons of the wild type and mutant structures reveal that a change in the orientation of the Ser hydroxyl group, with respect to the Cys sulfhydryl group, leads to penetration of water molecules and apparent destabilization of residues 132–138. The latter results were confirmed by means of Molecular Dynamics, which showed that this region, in the mutated enzyme, collapses at about 70 ns.

  1. Experiments testing the abatement of radiation damage in D-xylose isomerase crystals with cryogenic helium.

    Science.gov (United States)

    Hanson, B Leif; Harp, Joel M; Kirschbaum, Kristin; Schall, Constance A; DeWitt, Ken; Howard, Andrew; Pinkerton, A Alan; Bunick, Gerard J

    2002-11-01

    Helium is a more efficient cryogen than nitrogen, and for macromolecular data collection at high-flux beamlines will deliver lower temperatures. An open-flow helium cryostat developed at the University of Toledo (the Pinkerton Device) has been used for macromolecular data collection. This device differs from standard commercial He cryostats by having a much narrower aperture providing a high velocity stream of He around the crystal that maximizes convective and conductive heat exchange between the crystal and the cryogen. This paper details a series of experiments conducted at the IMCA-CAT 17ID beamline using one crystal for each experimental condition to examine whether helium at 16 K provided better radiation-damage abatement compared with nitrogen at 100 K. These studies used matched high-quality crystals (0.94 A diffraction resolution) of D-xylose isomerase derived from the commercial material Gensweet SGI. Comparisons show that helium indeed abates the indicators of radiation damage, in this case resulting in longer crystal diffractive lifetimes. The overall trend suggests that crystals maintain order and that high-resolution data are less affected by increased radiation load when crystals are cooled with He rather than N(2). This is probably the result of a lower effective temperature at the crystal with concomitant reduction in free-radical diffusion. Other features, such as an apparent phase transition in macromolecular crystals at lower temperatures, require investigation to broaden the utility of He use.

  2. Understanding protein lids: structural analysis of active hinge mutants in triosephosphate isomerase.

    Science.gov (United States)

    Kursula, I; Salin, M; Sun, J; Norledge, B V; Haapalainen, A M; Sampson, N S; Wierenga, R K

    2004-04-01

    The conformational switch from open to closed of the flexible loop 6 of triosephosphate isomerase (TIM) is essential for the catalytic properties of TIM. Using a directed evolution approach, active variants of chicken TIM with a mutated C-terminal hinge tripeptide of loop 6 have been generated (Sun,J. and Sampson,N.S., Biochemistry, 1999, 38, 11474-11481). In chicken TIM, the wild-type C-terminal hinge tripeptide is KTA. Detailed enzymological characterization of six variants showed that some of these (LWA, NPN, YSL, KTK) have decreased catalytic efficiency, whereas others (KVA, NSS) are essentially identical with wild-type. The structural characterization of these six variants is reported. No significant structural differences compared with the wild-type are found for KVA, NSS and LWA, but substantial structural adaptations are seen for NPN, YSL and KTK. These structural differences can be understood from the buried position of the alanine side chain in the C-hinge position 3 in the open conformation of wild-type loop 6. Replacement of this alanine with a bulky side chain causes the closed conformation to be favored, which correlates with the decreased catalytic efficiency of these variants. The structural context of loop 6 and loop 7 and their sequence conservation in 133 wild-type sequences is also discussed.

  3. Understanding protein lids: kinetic analysis of active hinge mutants in triosephosphate isomerase.

    Science.gov (United States)

    Sun, J; Sampson, N S

    1999-08-31

    In previous work we tested what three amino acid sequences could serve as a protein hinge in triosephosphate isomerase [Sun, J., and Sampson, N. S. (1998) Protein Sci. 7, 1495-1505]. We generated a genetic library encoding all 8000 possible 3 amino acid combinations at the C-terminal hinge and selected for those combinations of amino acids that formed active mutants. These mutants were classified into six phylogenetic families. Two families resembled wild-type hinges, and four families represented new types of hinges. In this work, the kinetic characteristics and thermal stabilities of mutants representing each of these families were determined in order to understand what properties make an efficient protein hinge, and why all of the families are not observed in nature. From a steady-state kinetic analysis of our mutants, it is clear that the partitioning between protonation of intermediate to form product and intermediate release from the enzyme surface to form methylglyoxal (a decomposition product) is not affected. The two most impaired mutants undergo a change in rate-limiting step from enediol formation to dihydroxyacetone phosphate binding. Thus, it appears that k(cat)/K(m)'s are reduced relative to wild type as a result of slower Michaelis complex formation and dissociation, rather than increased loop opening speed.

  4. Effects of a Buried Cysteine-To-Serine Mutation on Yeast Triosephosphate Isomerase Structure and Stability

    Science.gov (United States)

    Hernández-Santoyo, Alejandra; Domínguez-Ramírez, Lenin; Reyes-López, César A.; González-Mondragón, Edith; Hernández-Arana, Andrés; Rodríguez-Romero, Adela

    2012-01-01

    All the members of the triosephosphate isomerase (TIM) family possess a cystein residue (Cys126) located near the catalytically essential Glu165. The evolutionarily conserved Cys126, however, does not seem to play a significant role in the catalytic activity. On the other hand, substitution of this residue by other amino acid residues destabilizes the dimeric enzyme, especially when Cys is replaced by Ser. In trying to assess the origin of this destabilization we have determined the crystal structure of Saccharomyces cerevisiae TIM (ScTIM) at 1.86 Å resolution in the presence of PGA, which is only bound to one subunit. Comparisons of the wild type and mutant structures reveal that a change in the orientation of the Ser hydroxyl group, with respect to the Cys sulfhydryl group, leads to penetration of water molecules and apparent destabilization of residues 132–138. The latter results were confirmed by means of Molecular Dynamics, which showed that this region, in the mutated enzyme, collapses at about 70 ns. PMID:22949845

  5. Structural and biochemical characterization of a recombinant triosephosphate isomerase from Rhipicephalus (Boophilus) microplus.

    Science.gov (United States)

    Moraes, Jorge; Arreola, Rodrigo; Cabrera, Nallely; Saramago, Luiz; Freitas, Daniela; Masuda, Aoi; da Silva Vaz, Itabajara; Tuena de Gomez-Puyou, Marietta; Perez-Montfort, Ruy; Gomez-Puyou, Armando; Logullo, Carlos

    2011-06-01

    Triosephosphate isomerase (TIM) is an enzyme with a role in glycolysis and gluconeogenesis by catalyzing the interconversion between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. This enzyme has been used as a target in endoparasite drug development. In this work we cloned, expressed, purified and studied kinetic and structural characteristics of TIM from tick embryos, Rhipicephalus (Boophilus) microplus (BmTIM). The Km and Vmax of the recombinant BmTIM with glyceraldehyde 3-phosphate as substrate, were 0.47 mM and 6031 μmol min⁻¹ mg protein⁻¹, respectively. The resolution of the diffracted crystal was estimated to be 2.4 Å and the overall data showed that BmTIM is similar to other reported dimeric TIMs. However, we found that, in comparison to other TIMs, BmTIM has the highest content of cysteine residues (nine cysteine residues per monomer). Only two cysteines could make disulfide bonds in monomers of BmTIM. Furthermore, BmTIM was highly sensitive to the action of the thiol reagents dithionitrobenzoic acid and methyl methane thiosulfonate, suggesting that there are five cysteines exposed in each dimer and that these residues could be employed in the development of species-specific inhibitors.

  6. Structural and biochemical characterization of a recombinant triosephosphate isomerase from Rhipicephalus (Boophilus) microplus

    Energy Technology Data Exchange (ETDEWEB)

    Moraes, Jorge; Arreola, Rodrigo; Cabrera, Nallely; Saramago, Luiz; Freitas, Daniela; Masuda, Aoi; da Silva Vaz Jr., Itabajara; Tuena de Gomez-Puyou, Marietta; Perez-Montfort, Ruy; Gomez-Puyou, Armando; Logullo, Carlos (UNICAMP); (UFRGS-Brazil); (UNAM-Mexico)

    2012-02-06

    Triosephosphate isomerase (TIM) is an enzyme with a role in glycolysis and gluconeogenesis by catalyzing the interconversion between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. This enzyme has been used as a target in endoparasite drug development. In this work we cloned, expressed, purified and studied kinetic and structural characteristics of TIM from tick embryos, Rhipicephalus (Boophilus) microplus (BmTIM). The Km and Vmax of the recombinant BmTIM with glyceraldehyde 3-phosphate as substrate, were 0.47 mM and 6031 {micro}mol min{sup -1} mg protein{sup -1}, respectively. The resolution of the diffracted crystal was estimated to be 2.4 {angstrom} and the overall data showed that BmTIM is similar to other reported dimeric TIMs. However, we found that, in comparison to other TIMs, BmTIM has the highest content of cysteine residues (nine cysteine residues per monomer). Only two cysteines could make disulfide bonds in monomers of BmTIM. Furthermore, BmTIM was highly sensitive to the action of the thiol reagents dithionitrobenzoic acid and methyl methane thiosulfonate, suggesting that there are five cysteines exposed in each dimer and that these residues could be employed in the development of species-specific inhibitors.

  7. Cloning and Characterization of a Lycium chinense Carotenoid Isomerase Gene Enhancing Carotenoid Accumulation in Transgenic Tobacco

    Institute of Scientific and Technical Information of China (English)

    李招娣; 季静; 王罡

    2015-01-01

    Carotenoid isomerase(CRTISO)is a key enzyme that catalyzes the conversion of cis-lycopene to all-trans lycopene. In this study, we isolated and characterized the CRTISO gene from Lycium chinense (LcCRTISO) for the first time. The open reading frame of LcCRTISO was 1 815 bp encoding a protein of 604 amino acids with a molecular mass of 66.24 kDa. Amino acid sequence analysis revealed that the LcCRTISO had a high level of simi-larity to other CRTISO. Phylogenetic analysis displayed that LcCRTISO kept a closer relationship with the CRTISO of plants than with those of other species. Semi-quantitative PCR analysis indicated that LcCRTISO gene was expressed in all tissues tested with the highest expression in maturing fruits. The overexpression of LcCRTISO gene in transgenic tobacco resulted in an increase of total carotenoids in the leaves withβ-carotene and lutein being the predominants. The results obtained here clearly suggested that the LcCRTISO gene was a promising candidate for carotenoid production.

  8. DNA sequence analysis of the triose phosphate isomerase gene from isolates of Giardia lamblia

    Institute of Scientific and Technical Information of China (English)

    卢思奇; 文建凡; 李继红; 王凤云

    2002-01-01

    Objective To confirm the genetic relation between Giardia lamblia (G. lamblia) isolates from different geographic regions of China and other countries. Methods Genomic DNA were extracted from the trophozoites or cysts of Giardia lamblia. The triose phosphate isomerase (tim) gene was amplified using polymerase chain reaction (PCR) technique. PCR products were digested with endonuclease and sequenced. The data of sequencing were analyzed with the DNAstar software and compared with that of the isolates acquired from GenBank. Results Of nine isolates of Giardia lamblia from China (C1, C2, CH2 and CH3), Cambodia (CAM), Australia (A1 and A2) and America (BP and CDC), respectively, 3 (A1, A2 and CAM) fit into Group 1 (WB), 2 (CH2 and CH3)) into Group 2, and 4 (C1, C2, BP and CDC) into Group 3 (GS). The results confirmed the genetic relatedness of G. lamblia isolates from all over the world. Conclusion Genotyping isolates of G. Lamblia provides important information for establishing the phylogenetic relationship or for the epidemiological evaluation of the spreading of this organism.

  9. The unfolded protein response and the role of protein disulphide isomerase in neurodegeneration.

    Directory of Open Access Journals (Sweden)

    Emma ePerri

    2016-01-01

    Full Text Available The maintenance and regulation of proteostasis is a critical function for post-mitotic neurons and dysregulation of proteostasis is increasingly implicated in neurodegenerative diseases. Despite having different clinical manifestations, these disorders share similar pathology; an accumulation of misfolded proteins in neurons and subsequent disruption to cellular proteostasis. The endoplasmic reticulum (ER is an important component of proteostasis, and when the accumulation of misfolded proteins occurs within the ER, this disturbs ER homeostasis, giving rise to ER stress. This triggers the unfolded protein response (UPR, distinct signalling pathways that whilst initially protective, are pro-apoptotic if ER stress is prolonged. ER stress is increasingly implicated in neurodegenerative diseases, and emerging evidence highlights the complexity of the UPR in these disorders, with both protective and detrimental components being described. Protein Disulphide Isomerase (PDI is an ER chaperone induced during ER stress that is responsible for the formation of disulphide bonds in proteins. Whilst initially considered to be protective, recent studies have revealed unconventional roles for PDI in neurodegenerative diseases, distinct from its normal function in the UPR and the ER, although these mechanisms remain poorly defined. However specific aspects of PDI function may offer the potential to be exploited therapeutically in the future. This review will focus on the evidence linking ER stress and the UPR to neurodegenerative diseases, with particular emphasis on the emerging functions ascribed to PDI in these conditions.

  10. Cloning and characterization of a sucrose isomerase from Erwinia rhapontici NX-5 for isomaltulose hyperproduction.

    Science.gov (United States)

    Li, Sha; Cai, Heng; Qing, Yujia; Ren, Ben; Xu, Hong; Zhu, Hongyang; Yao, Jun

    2011-01-01

    The sucrose isomerase (SIase) gene from an efficient strain of Erwinia rhapontici NX-5 for isomaltulose hyperproduction was cloned and overexpressed in Escherichia coli. Protein sequence alignment revealed that SIase was a member of the glycoside hydrolase 13 family. The molecular mass of the purified recombinant protein was estimated at 66 kDa by SDS-PAGE. The SIase had an optimal pH and temperature of 5.0 and 30 °C, respectively, with a K (m) of 257 mmol/l and V (max) of 48.09 μmol/l/s for sucrose. To the best of our knowledge, the recombinant SIase has the most acidic optimum pH for isomaltulose synthesis. When the recombinant E. coli (pET22b- palI) cells were used for isomaltulose synthesis, almost complete conversion of sucrose (550 g/l solution) to isomaltulose was achieved in 1.5 h with high isomaltulose yields (87%). The immobilized E. coli cells remained stable for more than 30 days in a "batch"-type enzyme reactor. This indicated that the recombinant SIase could continuously and efficiently produce isomaltulose.

  11. Purification and characterization of a highly selective sucrose isomerase from Erwinia rhapontici NX-5.

    Science.gov (United States)

    Ren, Ben; Li, Sha; Xu, Hong; Feng, Xiao-Hai; Cai, Heng; Ye, Qi

    2011-06-01

    A highly selective sucrose isomerase (SIase) was purified to homogeneity from the cell-free extract of Erwinia rhapontici NX-5 with a recovery of 27.7% and a fold purification of 213.6. The purified SIase showed a high specific activity of 427.1 U mg(-1) with molecular weight of 65.6 kDa. The K (m) for sucrose was 222 mM while V (max) was 546 U mg(-1). The optimum pH and temperature for SIase activity were 6.0 and 30 °C, respectively. The purified SIase was stable in the temperature range of 10-40 °C and retained 65% of the enzyme activity after 2 weeks' storage at 30 °C. The SIase activity was enhanced by Mg(2+) and Mn(2+), inhibited by Ca(2+), Cu(2+), Zn(2+), and Co(2+), completely inhibited by Hg(2+) and Ag(2+). The purified SIase was strongly inhibited by SDS, while partially inhibited by dimethylformamide, tetrahydrofuran, and PMSF. Additionally, glucose and fructose acted as competitive inhibitors for purified SIase.

  12. The xylose isomerase gene from Thermoanaerobacterium thermosulfurogenes allows effective selection of transgenic plant cells using D-xylose as the selection agent.

    Science.gov (United States)

    Haldrup, A; Petersen, S G; Okkels, F T

    1998-05-01

    The xylose isomerase gene (xylA) from Thermoanaerobacterium thermosulfurogenes (formerly Clostridium thermosulfurogenes) has been expressed in three plant species (potato, tobacco, and tomato) and transgenic plants have been selected on xylose-containing medium. The xylose isomerase gene was transferred to the target plant by Agrobacterium-mediated transformation. The xylose isomerase gene was expressed using the enhanced cauliflower mosaic virus (CaMV) 35S promoter and the omega' translation enhancer sequence from tobacco mosaic virus. Unoptimized selection studies showed that, in potato and tomato, the xylose isomerase selection was more efficient than the established kanamycin resistance selection, whereas in tobacco the opposite was observed. Efficiency may be increased by optimization. The xylose isomerase system enables the transgenic cells to utilize xylose as a carbohydrate source. It is an example of a positive selection system because transgenic cells proliferate while non-transgenic cells are starved but still survive. This contrasts to antibiotic or herbicide resistance where transgenic cells survive on a selective medium but non-transgenic cells are killed. The results give access to a new selection method which is devoid of the disadvantages of antibiotic or herbicide selection.

  13. Survival of glucose phosphate isomerase null somatic cells and germ cells in adult mouse chimaeras.

    Science.gov (United States)

    Keighren, Margaret A; Flockhart, Jean H; West, John D

    2016-05-15

    The mouse Gpi1 gene encodes the glycolytic enzyme glucose phosphate isomerase. Homozygous Gpi1(-/-) null mouse embryos die but a previous study showed that some homozygous Gpi1(-/-) null cells survived when combined with wild-type cells in fetal chimaeras. One adult female Gpi1(-/-)↔Gpi1(c/c) chimaera with functional Gpi1(-/-) null oocytes was also identified in a preliminary study. The aims were to characterise the survival of Gpi1(-/-) null cells in adult Gpi1(-/-)↔Gpi1(c/c) chimaeras and determine if Gpi1(-/-) null germ cells are functional. Analysis of adult Gpi1(-/-)↔Gpi1(c/c) chimaeras with pigment and a reiterated transgenic lineage marker showed that low numbers of homozygous Gpi1(-/-) null cells could survive in many tissues of adult chimaeras, including oocytes. Breeding experiments confirmed that Gpi1(-/-) null oocytes in one female Gpi1(-/-)↔Gpi1(c/c) chimaera were functional and provided preliminary evidence that one male putative Gpi1(-/-)↔Gpi1(c/c) chimaera produced functional spermatozoa from homozygous Gpi1(-/-) null germ cells. Although the male chimaera was almost certainly Gpi1(-/-)↔Gpi1(c/c), this part of the study is considered preliminary because only blood was typed for GPI. Gpi1(-/-) null germ cells should survive in a chimaeric testis if they are supported by wild-type Sertoli cells. It is also feasible that spermatozoa could bypass a block at GPI, but not blocks at some later steps in glycolysis, by using fructose, rather than glucose, as the substrate for glycolysis. Although chimaera analysis proved inefficient for studying the fate of Gpi1(-/-) null germ cells, it successfully identified functional Gpi1(-/-) null oocytes and revealed that some Gpi1(-/-) null cells could survive in many adult tissues.

  14. Carotenoid isomerase is key determinant of petal color of Calendula officinalis.

    Science.gov (United States)

    Kishimoto, Sanae; Ohmiya, Akemi

    2012-01-02

    Orange petals of calendula (Calendula officinalis) accumulate red carotenoids with the cis-configuration at the C-5 or C-5' position (5-cis-carotenoids). We speculated that the orange-flowered calendula is a carotenoid isomerase (crtiso) loss-of-function mutant that impairs the cis-to-trans conversion of 5-cis-carotenoids. We compared the sequences and enzyme activities of CRTISO from orange- and yellow-flowered calendulas. Four types of CRTISO were expressed in calendula petals. The deduced amino acid sequence of one of these genes (CoCRTISO1) was different between orange- and yellow-flowered calendulas, whereas the sequences of the other three CRTISOs were identical between these plants. Analysis of the enzymatic activities of the CoCRTISO homologs showed that CoCRTISO1-Y, which was expressed in yellow petals, converted carotenoids from the cis-to-trans-configuration, whereas both CoCRTISO1-ORa and 1-ORb, which were expressed in orange petals, showed no activity with any of the cis-carotenoids we tested. Moreover, the CoCRTISO1 genotypes of the F2 progeny obtained by crossing orange and yellow lines linked closely to petal color. These data indicate that CoCRTISO1 is a key regulator of the accumulation of 5-cis-carotenoids in calendula petals. Site-directed mutagenesis showed that the deletion of Cys-His-His at positions 462-464 in CoCRTISO1-ORa and a Gly-to-Glu amino acid substitution at position 450 in CoCRTISO1-ORb abolished enzyme activity completely, indicating that these amino acid residues are important for the enzymatic activity of CRTISO.

  15. Functional Analysis of the Isopentenyl Diphosphate Isomerase of Salvia miltiorrhiza via Color Complementation and RNA Interference

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    Xianan Zhang

    2015-11-01

    Full Text Available Isopentenyl diphosphate isomerase (IPI catalyzes the isomerization between the common terpene precursor substances isopentenyl diphosphate (IPP and dimethylallyl diphosphate (DMAPP during the terpenoid biosynthesis process. In this study, tissue expression analysis revealed that the expression level of the Salvia miltiorrhiza IPI1 gene (SmIPI1 was higher in the leaves than in the roots and stems. Furthermore, color complementation and RNA interference methods were used to verify the function of the SmIPI1 gene from two aspects. A recombinant SmIPI1 plasmid was successfully constructed and transferred into engineered E. coli for validating the function of SmIPI1 through the color difference in comparison to the control group; the observed color difference indicated that SmIPI1 served in promoting the accumulation of lycopene. Transformant hairy root lines with RNA interference of SmIPI1 were successfully constructed mediated by Agrobacterium rhizogenes ACCC 10060. RNA interference hairy roots had a severe phenotype characterized by withering, deformity or even death. The mRNA expression level of SmIPI1 in the RSi3 root line was only 8.4% of that of the wild type. Furthermore the tanshinone content was too low to be detected in the RNA interference lines. These results suggest that SmIPI1 plays a critical role in terpenoid metabolic pathways. Addition of an exogenous SmIPI1 gene promoted metabolic flow toward the biosynthesis of carotenoids in E. coli, and SmIPI1 interference in S. miltiorrhiza hairy roots may cause interruption of the 2-C-methyl-D-erythritol-4-phosphate metabolic pathway.

  16. Substrate-Induced Dimerization of Engineered Monomeric Variants of Triosephosphate Isomerase from Trichomonas vaginalis.

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    Samuel Lara-Gonzalez

    Full Text Available The dimeric nature of triosephosphate isomerases (TIMs is maintained by an extensive surface area interface of more than 1600 Å2. TIMs from Trichomonas vaginalis (TvTIM are held in their dimeric state by two mechanisms: a ball and socket interaction of residue 45 of one subunit that fits into the hydrophobic pocket of the complementary subunit and by swapping of loop 3 between subunits. TvTIMs differ from other TIMs in their unfolding energetics. In TvTIMs the energy necessary to unfold a monomer is greater than the energy necessary to dissociate the dimer. Herein we found that the character of residue I45 controls the dimer-monomer equilibrium in TvTIMs. Unfolding experiments employing monomeric and dimeric mutants led us to conclude that dimeric TvTIMs unfold following a four state model denaturation process whereas monomeric TvTIMs follow a three state model. In contrast to other monomeric TIMs, monomeric variants of TvTIM1 are stable and unexpectedly one of them (I45A is only 29-fold less active than wild-type TvTIM1. The high enzymatic activity of monomeric TvTIMs contrast with the marginal catalytic activity of diverse monomeric TIMs variants. The stability of the monomeric variants of TvTIM1 and the use of cross-linking and analytical ultracentrifugation experiments permit us to understand the differences between the catalytic activities of TvTIMs and other marginally active monomeric TIMs. As TvTIMs do not unfold upon dimer dissociation, herein we found that the high enzymatic activity of monomeric TvTIM variants is explained by the formation of catalytic dimeric competent species assisted by substrate binding.

  17. Perturbation of the dimer interface of triosephosphate isomerase and its effect on Trypanosoma cruzi.

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    Vanesa Olivares-Illana

    Full Text Available BACKGROUND: Chagas disease affects around 18 million people in the American continent. Unfortunately, there is no satisfactory treatment for the disease. The drugs currently used are not specific and exert serious toxic effects. Thus, there is an urgent need for drugs that are effective. Looking for molecules to eliminate the parasite, we have targeted a central enzyme of the glycolytic pathway: triosephosphate isomerase (TIM. The homodimeric enzyme is catalytically active only as a dimer. Because there are significant differences in the interface of the enzymes from the parasite and humans, we searched for small molecules that specifically disrupt contact between the two subunits of the enzyme from Trypanosoma cruzi but not those of TIM from Homo sapiens (HTIM, and tested if they kill the parasite. METHODOLOGY/PRINCIPAL FINDINGS: Dithiodianiline (DTDA at nanomolar concentrations completely inactivates recombinant TIM of T. cruzi (TcTIM. It also inactivated HTIM, but at concentrations around 400 times higher. DTDA was also tested on four TcTIM mutants with each of its four cysteines replaced with either valine or alanine. The sensitivity of the mutants to DTDA was markedly similar to that of the wild type. The crystal structure of the TcTIM soaked in DTDA at 2.15 A resolution, and the data on the mutants showed that inactivation resulted from alterations of the dimer interface. DTDA also prevented the growth of Escherichia coli cells transformed with TcTIM, had no effect on normal E. coli, and also killed T. cruzi epimastigotes in culture. CONCLUSIONS/SIGNIFICANCE: By targeting on the dimer interface of oligomeric enzymes from parasites, it is possible to discover small molecules that selectively thwart the life of the parasite. Also, the conformational changes that DTDA induces in the dimer interface of the trypanosomal enzyme are unique and identify a region of the interface that could be targeted for drug discovery.

  18. Functional Analysis of the Isopentenyl Diphosphate Isomerase of Salvia miltiorrhiza via Color Complementation and RNA Interference.

    Science.gov (United States)

    Zhang, Xianan; Guan, Hongyu; Dai, Zhubo; Guo, Juan; Shen, Ye; Cui, Guanghong; Gao, Wei; Huang, Luqi

    2015-11-10

    Isopentenyl diphosphate isomerase (IPI) catalyzes the isomerization between the common terpene precursor substances isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) during the terpenoid biosynthesis process. In this study, tissue expression analysis revealed that the expression level of the Salvia miltiorrhiza IPI1 gene (SmIPI1) was higher in the leaves than in the roots and stems. Furthermore, color complementation and RNA interference methods were used to verify the function of the SmIPI1 gene from two aspects. A recombinant SmIPI1 plasmid was successfully constructed and transferred into engineered E. coli for validating the function of SmIPI1 through the color difference in comparison to the control group; the observed color difference indicated that SmIPI1 served in promoting the accumulation of lycopene. Transformant hairy root lines with RNA interference of SmIPI1 were successfully constructed mediated by Agrobacterium rhizogenes ACCC 10060. RNA interference hairy roots had a severe phenotype characterized by withering, deformity or even death. The mRNA expression level of SmIPI1 in the RSi3 root line was only 8.4% of that of the wild type. Furthermore the tanshinone content was too low to be detected in the RNA interference lines. These results suggest that SmIPI1 plays a critical role in terpenoid metabolic pathways. Addition of an exogenous SmIPI1 gene promoted metabolic flow toward the biosynthesis of carotenoids in E. coli, and SmIPI1 interference in S. miltiorrhiza hairy roots may cause interruption of the 2-C-methyl-D-erythritol-4-phosphate metabolic pathway.

  19. The secreted triose phosphate isomerase of Brugia malayi is required to sustain microfilaria production in vivo.

    Directory of Open Access Journals (Sweden)

    James P Hewitson

    2014-02-01

    Full Text Available Human lymphatic filariasis is a major tropical disease transmitted through mosquito vectors which take up microfilarial larvae from the blood of infected subjects. Microfilariae are produced by long-lived adult parasites, which also release a suite of excretory-secretory products that have recently been subject to in-depth proteomic analysis. Surprisingly, the most abundant secreted protein of adult Brugia malayi is triose phosphate isomerase (TPI, a glycolytic enzyme usually associated with the cytosol. We now show that while TPI is a prominent target of the antibody response to infection, there is little antibody-mediated inhibition of catalytic activity by polyclonal sera. We generated a panel of twenty-three anti-TPI monoclonal antibodies and found only two were able to block TPI enzymatic activity. Immunisation of jirds with B. malayi TPI, or mice with the homologous protein from the rodent filaria Litomosoides sigmodontis, failed to induce neutralising antibodies or protective immunity. In contrast, passive transfer of neutralising monoclonal antibody to mice prior to implantation with adult B. malayi resulted in 60-70% reductions in microfilarial levels in vivo and both oocyte and microfilarial production by individual adult females. The loss of fecundity was accompanied by reduced IFNγ expression by CD4⁺ T cells and a higher proportion of macrophages at the site of infection. Thus, enzymatically active TPI plays an important role in the transmission cycle of B. malayi filarial parasites and is identified as a potential target for immunological and pharmacological intervention against filarial infections.

  20. Protein disulfide isomerase inhibition blocks thrombin generation in humans by interfering with platelet factor V activation

    Science.gov (United States)

    Stopa, Jack D.; Neuberg, Donna; Puligandla, Maneka; Furie, Bruce; Zwicker, Jeffrey I.

    2017-01-01

    BACKGROUND: Protein disulfide isomerase (PDI) is required for thrombus formation. We previously demonstrated that glycosylated quercetin flavonoids such as isoquercetin inhibit PDI activity and thrombus formation in animal models, but whether extracellular PDI represents a viable anticoagulant target in humans and how its inhibition affects blood coagulation remain unknown. METHODS: We evaluated effects of oral administration of isoquercetin on platelet-dependent thrombin generation in healthy subjects and patients with persistently elevated anti-phospholipid antibodies. RESULTS: Following oral administration of 1,000 mg isoquercetin to healthy adults, the measured peak plasma quercetin concentration (9.2 μM) exceeded its IC50 for inhibition of PDI by isoquercetin in vitro (2.5 ± 0.4 μM). Platelet-dependent thrombin generation decreased by 51% in the healthy volunteers compared with baseline (P = 0.0004) and by 64% in the anti-phospholipid antibody cohort (P = 0.015) following isoquercetin ingestion. To understand how PDI affects thrombin generation, we evaluated substrates of PDI identified using an unbiased mechanistic-based substrate trapping approach. These studies identified platelet factor V as a PDI substrate. Isoquercetin blocked both platelet factor Va and thrombin generation with an IC50 of ~5 μM. Inhibition of PDI by isoquercetin ingestion resulted in a 53% decrease in the generation of platelet factor Va (P = 0.001). Isoquercetin-mediated inhibition was reversed with addition of exogenous factor Va. CONCLUSION: These studies show that oral administration of isoquercetin inhibits PDI activity in plasma and diminishes platelet-dependent thrombin generation predominantly by blocking the generation of platelet factor Va. These pharmacodynamic and mechanistic observations represent an important step in the development of a novel class of antithrombotic agents targeting PDI. TRIAL REGISTRATION: Clinicaltrials.gov (NCT01722669) FUNDING: National Heart

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

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    Leibly David J

    2011-10-01

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

  2. Structural effects of protein aging: terminal marking by deamidation in human triosephosphate isomerase.

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    Ignacio de la Mora-de la Mora

    Full Text Available Deamidation, the loss of the ammonium group of asparagine and glutamine to form aspartic and glutamic acid, is one of the most commonly occurring post-translational modifications in proteins. Since deamidation rates are encoded in the protein structure, it has been proposed that they can serve as molecular clocks for the timing of biological processes such as protein turnover, development and aging. Despite the importance of this process, there is a lack of detailed structural information explaining the effects of deamidation on the structure of proteins. Here, we studied the effects of deamidation on human triosephosphate isomerase (HsTIM, an enzyme for which deamidation of N15 and N71 has been long recognized as the signal for terminal marking of the protein. Deamidation was mimicked by site directed mutagenesis; thus, three mutants of HsTIM (N15D, N71D and N15D/N71D were characterized. The results show that the N71D mutant resembles, structurally and functionally, the wild type enzyme. In contrast, the N15D mutant displays all the detrimental effects related to deamidation. The N15D/N71D mutant shows only minor additional effects when compared with the N15D mutation, supporting that deamidation of N71 induces negligible effects. The crystal structures show that, in contrast to the N71D mutant, where minimal alterations are observed, the N15D mutation forms new interactions that perturb the structure of loop 1 and loop 3, both critical components of the catalytic site and the interface of HsTIM. Based on a phylogenetic analysis of TIM sequences, we propose the conservation of this mechanism for mammalian TIMs.

  3. Isolation of xylose isomerases by sequence- and function-based screening from a soil metagenomic library

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    Parachin Nádia

    2011-05-01

    Full Text Available Abstract Background Xylose isomerase (XI catalyses the isomerisation of xylose to xylulose in bacteria and some fungi. Currently, only a limited number of XI genes have been functionally expressed in Saccharomyces cerevisiae, the microorganism of choice for lignocellulosic ethanol production. The objective of the present study was to search for novel XI genes in the vastly diverse microbial habitat present in soil. As the exploitation of microbial diversity is impaired by the ability to cultivate soil microorganisms under standard laboratory conditions, a metagenomic approach, consisting of total DNA extraction from a given environment followed by cloning of DNA into suitable vectors, was undertaken. Results A soil metagenomic library was constructed and two screening methods based on protein sequence similarity and enzyme activity were investigated to isolate novel XI encoding genes. These two screening approaches identified the xym1 and xym2 genes, respectively. Sequence and phylogenetic analyses revealed that the genes shared 67% similarity and belonged to different bacterial groups. When xym1 and xym2 were overexpressed in a xylA-deficient Escherichia coli strain, similar growth rates to those in which the Piromyces XI gene was expressed were obtained. However, expression in S. cerevisiae resulted in only one-fourth the growth rate of that obtained for the strain expressing the Piromyces XI gene. Conclusions For the first time, the screening of a soil metagenomic library in E. coli resulted in the successful isolation of two active XIs. However, the discrepancy between XI enzyme performance in E. coli and S. cerevisiae suggests that future screening for XI activity from soil should be pursued directly using yeast as a host.

  4. Leishmania donovani triose phosphate isomerase: a potential vaccine target against visceral leishmaniasis.

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    Pramod K Kushawaha

    Full Text Available Visceral leishmaniasis (VL is one of the most important parasitic diseases with approximately 350 million people at risk. Due to the non availability of an ideal drug, development of a safe, effective, and affordable vaccine could be a solution for control and prevention of this disease. In this study, a potential Th1 stimulatory protein- Triose phosphate isomerase (TPI, a glycolytic enzyme, identified through proteomics from a fraction of Leishmania donovani soluble antigen ranging from 89.9-97.1 kDa, was assessed for its potential as a suitable vaccine candidate. The protein- L. donovani TPI (LdTPI was cloned, expressed and purified which exhibited the homology of 99% with L. infantum TPI. The rLdTPI was further evaluated for its immunogenicity by lymphoproliferative response (LTT, nitric oxide (NO production and estimation of cytokines in cured Leishmania patients/hamster. It elicited strong LTT response in cured patients as well as NO production in cured hamsters and stimulated remarkable Th1-type cellular responses including IFN-ã and IL-12 with extremely lower level of IL-10 in Leishmania-infected cured/exposed patients PBMCs in vitro. Vaccination with LdTPI-DNA construct protected naive golden hamsters from virulent L. donovani challenge unambiguously (∼90%. The vaccinated hamsters demonstrated a surge in IFN-ã, TNF-á and IL-12 levels but extreme down-regulation of IL-10 and IL-4 along with profound delayed type hypersensitivity and increased levels of Leishmania-specific IgG2 antibody. Thus, the results are suggestive of the protein having the potential of a strong candidate vaccine.

  5. Disulfide isomerase-like protein AtPDIL1–2 is a good candidate for trichlorophenol phytodetoxification

    Science.gov (United States)

    Peng, Ri-He; Qiu, Jin; Tian, Yong-Sheng; Gao, Jian-jie; Han, Hong-juan; Fu, Xiao-Yan; Zhu, Bo; Xu, Jing; Wang, Bo; Li, Zhen-jun; Wang, Li-juan; Yao, Quan-Hong

    2017-01-01

    Trichlorophenol (TCP) is a widely used and persistent environmentally toxic compound that poses a carcinogenic risk to humans. Phytoremediation is a proficient cleanup technology for organic pollutants. In this study, we found that the disulfide isomerase-like protein AtPDIL1–2 in plants is a good candidate for enhancing 2,4,6-TCP phytoremediation. The expression of AtPDIL1-2 in Arabidopsis was induced by 2,4,6-TCP. The heterologously expressed AtPDIL1-2 in Escherichia coli exhibited both oxidase and isomerase activities as protein disulfide isomerase and improved bacteria tolerance to 2,4,6-TCP. Further research revealed that transgenic tobacco overexpressing AtPDIL1-2 was more tolerant to high concentrations of 2,4,6-TCP and removed the toxic compound at far greater rates than the control plants. To elucidate the mechanism of action of AtPDIL1-2, we investigated the chemical interaction of AtPDIL1-2 with 2,4,6-TCP for the first time. HPLC analysis implied that AtPDIL1-2 exerts a TCP-binding activity. A suitable configuration of AtPDIL1-2-TCP binding was obtained by molecular docking studies using the AutoDock program. It predicted that the TCP binding site is located in the b-b′ domain of AtPDIL1-2 and that His254 of the protein is critical for the binding interaction. These findings imply that AtPDIL1-2 can be used for TCP detoxification by the way of overexpression in plants. PMID:28059139

  6. Display of Clostridium cellulovorans xylose isomerase on the cell surface of Saccharomyces cerevisiae and its direct application to xylose fermentation.

    Science.gov (United States)

    Ota, Miki; Sakuragi, Hiroshi; Morisaka, Hironobu; Kuroda, Kouichi; Miyake, Hideo; Tamaru, Yutaka; Ueda, Mitsuyoshi

    2013-01-01

    Xylose isomerase (XI) is a key enzyme in the conversion of D-xylose, which is a major component of lignocellulosic biomass, to D-xylulose. Genomic analysis of the bacterium Clostridium cellulovorans revealed the presence of XI-related genes. In this study, XI derived from C. cellulovorans was produced and displayed using the yeast cell-surface display system, and the xylose assimilation and fermentation properties of this XI-displaying yeast were examined. XI-displaying yeast grew well in medium containing xylose as the sole carbon source and directly produced ethanol from xylose under anaerobic conditions.

  7. The disulfide isomerase DsbC cooperates with the oxidase DsbA in a DsbD-independent manner

    OpenAIRE

    Vertommen, Didier; Depuydt, Matthieu; Pan, Jonathan; Leverrier, Pauline; Knoops, Laurent; Szikora, Jean-Pierre; Messens, Joris; Bardwell, James C. A.; Collet, Jean-Francois

    2007-01-01

    In Escherichia coli, DsbA introduces disulfide bonds into secreted proteins. DsbA is recycled by DsbB, which generates disulfides from quinone reduction. DsbA is not known to have any proofreading activity and can form incorrect disulfides in proteins with multiple cysteines. These incorrect disulfides are thought to be corrected by a protein disulfide isomerase, DsbC, which is kept in the reduced and active configuration by DsbD. The DsbC/DsbD isomerization pathway is considered to be isolat...

  8. The active centre of rabbit muscle triose phosphate isomerase. The site that is labelled by glycidol phosphate.

    Science.gov (United States)

    Miller, J C; Waley, S G

    1971-06-01

    1. Glycidol (2,3-epoxypropanol) phosphate is a specific irreversible inhibitor of rabbit muscle triose phosphate isomerase (EC 5.3.1.1); the site of attachment has now been studied. 2. The labelled enzyme was digested with pepsin and a modified peptide isolated. The sequence of the peptide is: Ala-Tyr-Glu-Pro-Val-Trp. 3. It is the glutamic acid residue in this peptide that is labelled: the peptide is thus a gamma-glutamyl ester derived from glycerol phosphoric acid. The same site is labelled by a mixture of glycidol and inorganic phosphate. 4. Kinetic and stereochemical features of these reactions are discussed.

  9. Protein-disulfide Isomerase Displaces the Cholera Toxin A1 Subunit from the Holotoxin without Unfolding the A1 Subunit*

    OpenAIRE

    Taylor, Michael; Banerjee, Tuhina; Ray, Supriyo; Tatulian, Suren A.; Teter, Ken

    2011-01-01

    Protein-disulfide isomerase (PDI) has been proposed to exhibit an “unfoldase” activity against the catalytic A1 subunit of cholera toxin (CT). Unfolding of the CTA1 subunit is thought to displace it from the CT holotoxin and to prepare it for translocation to the cytosol. To date, the unfoldase activity of PDI has not been demonstrated for any substrate other than CTA1. An alternative explanation for the putative unfoldase activity of PDI has been suggested by recent structural studies demons...

  10. Direct production of D-arabinose from D-xylose by a coupling reaction using D-xylose isomerase, D-tagatose 3-epimerase and D-arabinose isomerase.

    Science.gov (United States)

    Sultana, Ishrat; Mizanur, Rahman Md; Takeshita, Kei; Takada, Goro; Izumori, Ken

    2003-01-01

    Klebsiella pneumoniae 40bXX, a mutant strain that constitutively produces D-arabinose isomerase (D-AI), was isolated through a series of repeated subcultures from the parent strain on a mineral salt medium supplemented with L-Xylose as the sole carbon source. D-AI could be efficiently immobilized on chitopearl beads. The optimum temperature for the activity of the immobilized enzyme was 40 degrees C and the enzyme was stable up to 50 degrees C. The D-Al was active at pH 10.0 and was stable in the range of pH 6.0-11.0. The enzyme required manganese ions for maximum activity. Three immobilized enzymes, D-xylose isomerase (D-XI), D-tagatose 3-epimerase (D-TE and D-AI were used for the preparation of D-arabinose from D-xylose in a coupling reaction. After completion of the reaction, degradation of D-xylulose was carried out by Saccharomyces cerevisiae. The reaction mixture containing D-Xylose, D-ribulose and the product was then separated by ion exchange column chromatography. After crystallization, the product was checked by HPLC, IR spectroscopy, NMR spectroscopy and optical rotation measurements. Finally, 2.0 g of D-arabinose could be obtained from 5 g of the substrate.

  11. L-Ribose production from L-arabinose by immobilized recombinant Escherichia coli co-expressing the L-arabinose isomerase and mannose-6-phosphate isomerase genes from Geobacillus thermodenitrificans.

    Science.gov (United States)

    Kim, Kyoung-Rok; Seo, Eun-Sun; Oh, Deok-Kun

    2014-01-01

    L-Ribose is an important precursor for antiviral agents, and thus its high-level production is urgently demanded. For this aim, immobilized recombinant Escherichia coli cells expressing the L-arabinose isomerase and variant mannose-6-phosphate isomerase genes from Geobacillus thermodenitrificans were developed. The immobilized cells produced 99 g/l L-ribose from 300 g/l L-arabinose in 3 h at pH 7.5 and 60 °C in the presence of 1 mM Co(2+), with a conversion yield of 33 % (w/w) and a productivity of 33 g/l/h. The immobilized cells in the packed-bed bioreactor at a dilution rate of 0.2 h(-1) produced an average of 100 g/l L-ribose with a conversion yield of 33 % and a productivity of 5.0 g/l/h for the first 12 days, and the operational half-life in the bioreactor was 28 days. Our study is first verification for L-ribose production by long-term operation and feasible for cost-effective commercialization. The immobilized cells in the present study also showed the highest conversion yield among processes from L-arabinose as the substrate.

  12. Identification of glucoselysine-6-phosphate deglycase, an enzyme involved in the metabolism of the fructation product glucoselysine.

    OpenAIRE

    Wiame, Elsa; Lamosa, Pedro; Santos, Helena; Van Schaftingen, Emile

    2005-01-01

    The metabolism of the glycation product fructose-epsilon-lysine in Escherichia coli involves its ATP-dependent phosphorylation by a specific kinase (FrlD), followed by the conversion of fructoselysine 6-phosphate into glucose 6-phosphate and lysine by fructoselysine-6-phosphate deglycase (FrlB), which is distantly related to the isomerase domain of glucosamine-6-phosphate synthase. As shown in the present work, several bacterial operons comprise: (1) a homologue of fructoselysine-6-phosphate ...

  13. Parental Involvement

    OpenAIRE

    Ezra S Simon

    2008-01-01

    This study was conducted in Ghana to investigate, (1) factors that predict parental involvement, (2) the relationship between parental home and school involvement and the educational achievement of adolescents, (3) the relationship between parental authoritativeness and the educational achievement of adolescent students, (4) parental involvement serving as a mediator between their authoritativeness and the educational achievement of the students, and (5) whether parental involvement decreases...

  14. Soluble expression of human leukemia inhibitory factor with protein disulfide isomerase in Escherichia coli and its simple purification.

    Science.gov (United States)

    Song, Jung-A; Jung, A Song; Koo, Bon-Kyung; Chong, Seon-Ha; Kim, Kyunhoo; Choi, Dong Kyu; Thi Vu, Thu Trang; Nguyen, Minh Tan; Jeong, Boram; Ryu, Han-Bong; Kim, Injune; Jang, Yeon Jin; Robinson, Robert Charles; Choe, Han

    2013-01-01

    Human leukemia inhibitory factor (hLIF) is a multifunctional cytokine that is essential for maintaining the pluripotency of embryonic stem cells. hLIF may be also be useful in aiding fertility through its effects on increasing the implantation rate of fertilized eggs. Thus these applications in biomedical research and clinical medicine create a high demand for bioactive hLIF. However, production of active hLIF is problematic since eukaryotic cells demonstrate limited expression and prokaryotic cells produce insoluble protein. Here, we have adopted a hybrid protein disulfide isomerase design to increase the solubility of hLIF in Escherichia coli. Low temperature expression of hLIF fused to the b'a' domain of protein disulfide isomerase (PDIb'a') increased the soluble expression in comparison to controls. A simple purification protocol for bioactive hLIF was established that includes removal of the PDIb'a' domain by cleavage by TEV protease. The resulting hLIF, which contains one extra glycine residue at the N-terminus, was highly pure and demonstrated endotoxin levels below 0.05 EU/μg. The presence of an intramolecular disulfide bond was identified using mass spectroscopy. This purified hLIF effectively maintained the pluripotency of a murine embryonic stem cell line. Thus we have developed an effective method to produce a pure bioactive version of hLIF in E. coli for use in biomedical research.

  15. Crystal Structure of Escherichia coli L-Arabinose Isomerase (ECAI), The Putative Target of Biological Tagatose Production

    Energy Technology Data Exchange (ETDEWEB)

    Manjasetty,B.; Chance, M.

    2006-01-01

    Escherichia coli L-arabinose isomerase (ECAI; EC 5.3.1.4) catalyzes the isomerization of L-arabinose to L-ribulose in vivo. This enzyme is also of commercial interest as it catalyzes the conversion of D-galactose to D-tagatose in vitro. The crystal structure of ECAI was solved and refined at 2.6 Angstroms resolution. The subunit structure of ECAI is organized into three domains: an N-terminal, a central and a C-terminal domain. It forms a crystallographic trimeric architecture in the asymmetric unit. Packing within the crystal suggests the idea that ECAI can form a hexameric assembly. Previous electron microscopic and biochemical studies supports that ECAI is hexameric in solution. A comparison with other known structures reveals that ECAI adopts a protein fold most similar to E. coli fucose isomerase (ECFI) despite very low sequence identity 9.7%. The structural similarity between ECAI and ECFI with regard to number of domains, overall fold, biological assembly, and active site architecture strongly suggests that the enzymes have functional similarities. Further, the crystal structure of ECAI forms a basis for identifying molecular determinants responsible for isomerization of arabinose to ribulose in vivo and galactose to tagatose in vitro.

  16. Structural Basis for Redox Regulation of Cytoplasmic and Chloroplastic Triosephosphate Isomerases from Arabidopsis thaliana

    Science.gov (United States)

    López-Castillo, Laura M.; Jiménez-Sandoval, Pedro; Baruch-Torres, Noe; Trasviña-Arenas, Carlos H.; Díaz-Quezada, Corina; Lara-González, Samuel; Winkler, Robert; Brieba, Luis G.

    2016-01-01

    In plants triosephosphate isomerase (TPI) interconverts glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) during glycolysis, gluconeogenesis, and the Calvin-Benson cycle. The nuclear genome of land plants encodes two tpi genes, one gene product is located in the cytoplasm and the other is imported into the chloroplast. Herein we report the crystal structures of the TPIs from the vascular plant Arabidopsis thaliana (AtTPIs) and address their enzymatic modulation by redox agents. Cytoplasmic TPI (cTPI) and chloroplast TPI (pdTPI) share more than 60% amino acid identity and assemble as (β-α)8 dimers with high structural homology. cTPI and pdTPI harbor two and one accessible thiol groups per monomer respectively. cTPI and pdTPI present a cysteine at an equivalent structural position (C13 and C15 respectively) and cTPI also contains a specific solvent accessible cysteine at residue 218 (cTPI-C218). Site directed mutagenesis of residues pdTPI-C15, cTPI-C13, and cTPI-C218 to serine substantially decreases enzymatic activity, indicating that the structural integrity of these cysteines is necessary for catalysis. AtTPIs exhibit differential responses to oxidative agents, cTPI is susceptible to oxidative agents such as diamide and H2O2, whereas pdTPI is resistant to inhibition. Incubation of AtTPIs with the sulfhydryl conjugating reagents methylmethane thiosulfonate (MMTS) and glutathione inhibits enzymatic activity. However, the concentration necessary to inhibit pdTPI is at least two orders of magnitude higher than the concentration needed to inhibit cTPI. Western-blot analysis indicates that residues cTPI-C13, cTPI-C218, and pdTPI-C15 conjugate with glutathione. In summary, our data indicate that AtTPIs could be redox regulated by the derivatization of specific AtTPI cysteines (cTPI-C13 and pdTPI-C15 and cTPI-C218). Since AtTPIs have evolved by gene duplication, the higher resistance of pdTPI to redox agents may be an adaptive consequence to the

  17. Structural Basis for Redox Regulation of Cytoplasmic and Chloroplastic Triosephosphate Isomerases from Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Laura Margarita López-Castillo

    2016-12-01

    Full Text Available In plants triosephosphate isomerase (TPI interconverts glyceraldehyde 3-phosphate (G3P and dihydroxyacetone phosphate (DHAP during glycolysis, gluconeogenesis, and the Calvin-Benson cycle. The nuclear genome of land plants encodes two tpi genes, one gene product is located in the cytoplasm and the other is imported into the chloroplast. Herein we report the crystal structures of the TPIs from the vascular plant Arabidopsis thaliana (AtTPIs and address their enzymatic modulation by redox agents. Cytoplasmic TPI (cTPI and chloroplast TPI (pdTPI share more than 60% amino acid identity and assemble as (β-α8 dimers with high structural homology. cTPI and pdTPI harbor two and one accessible thiol groups per monomer respectively. cTPI and pdTPI present a cysteine at an equivalent structural position (C13 and C15 respectively and cTPI also contains a specific solvent accessible cysteine at residue 218 (cTPI-C218. Site directed mutagenesis of residues pdTPI-C15, cTPI-C13 and cTPI-C218 to serine substantially decreases enzymatic activity, indicating that the structural integrity of these cysteines is necessary for catalysis. AtTPIs exhibit differential responses to oxidative agents, cTPI is susceptible to oxidative agents such as diamide and H2O2, whereas pdTPI is resistant to inhibition. Incubation of AtTPIs with the sulfhydryl conjugating reagents methylmethane thiosulfonate (MMTS and glutathione inhibits enzymatic activity. However, the concentration necessary to inhibit pdTPI is at least two orders of magnitude higher than the concentration needed to inhibit cTPI. Western-blot analysis indicates that residues cTPI-C13, cTPI-C218, and pdTPI-C15 conjugate with glutathione. In summary, our data indicate that AtTPIs could be redox regulated by the derivatization of specific AtTPI cysteines (cTPI-C13 and pdTPI-C15 and cTPI-C218. Since AtTPIs have evolved by gene duplication, the higher resistance of pdTPI to redox agents may be an adaptive consequence to

  18. Proteomic analysis of stress-related proteins in transgenic broccoli harboring a gene for cytokinin production during postharvest senescence.

    Science.gov (United States)

    Liu, Mao-Sen; Li, Hui-Chun; Chang, You-Min; Wu, Min-Tze; Chen, Long-Fang Oliver

    2011-09-01

    Our previous study revealed a cytokinin-related retardation of post-harvest floret yellowing in transgenic broccoli (Brassica oleracea var. italica) that harbored the bacterial isopentenyltransferase (ipt) gene. We aimed to investigate the underlining mechanism of this delayed post-harvest senescence. We used 2D electrophoresis and liquid chromatography-electrospray ionization-mass spectrometry/mass spectrometry for a proteomics analysis of heads of ipt-transgenic and non-transgenic inbred lines of broccoli at harvest and after four days post-harvest storage. At harvest, we found an accumulation of stress-responsive proteins involved in maintenance of protein folding (putative protein disulfide isomerase, peptidyl-prolyl cis-trans isomerase and chaperonins), scavenging of reactive oxygen species (Mn superoxide dismutase), and stress protection [myrosinase-binding protein, jasmonate inducible protein, dynamin-like protein, NADH dehydrogenase (ubiquinone) Fe-S protein 1 and stress-inducible tetratricopeptide repeat-containing protein]. After four days' post-harvest storage of non-transgenic broccoli florets, the levels of proteins involved in protein folding and carbon fixation were decreased, which indicates cellular degradation and a change in metabolism toward senescence. In addition, staining for antioxidant enzyme activity of non-transgenic plants after post-harvest storage revealed a marked decrease in activity of Fe-superoxide dismutase and ascorbate peroxidase. Thus, the accumulation of stress-responsive proteins and antioxidant enzyme activity in ipt-transgenic broccoli are most likely associated with retardation of post-harvest senescence.

  19. STRUCTURE OF THE COMPLEX BETWEEN TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE AND N-HYDROXY-4-PHOSPHONO-BUTANAMIDE - BINDING AT THE ACTIVE-SITE DESPITE AN OPEN FLEXIBLE LOOP CONFORMATION

    NARCIS (Netherlands)

    VERLINDE, CLMJ; WITMANS, CJ; PIJNING, T; KALK, KH; HOL, WGJ; CALLENS, M; OPPERDOES, FR

    1992-01-01

    The structure of triosephosphate isomerase from Trypanosoma brucei complexed with the competitive inhibitor N-hydroxy-4-phosphono-butanamide was determined by X-ray crystallography to a resolution of 2.84 angstrom. Full occupancy binding of the inhibitor is observed only at one of the active sites o

  20. Growth and fermentation of D-xylose by Saccharomyces cerevisiae expressing a novel D-xylose isomerase originating from the bacterium Prevotella ruminicola TC2-24

    Science.gov (United States)

    Saccharomyces cerevisiae strains expressing xylose isomerase (XI) produce some of the highest reported ethanol yields from xylose. Unfortunately, most bacterial XIs that have been expressed in S. cerevisiae are not functional, require additional strain modification, and have low affinity for xylose...

  1. The multidrug resistance IncA/C transferable plasmid encodes a novel domain-swapped dimeric protein-disulfide isomerase.

    Science.gov (United States)

    Premkumar, Lakshmanane; Kurth, Fabian; Neyer, Simon; Schembri, Mark A; Martin, Jennifer L

    2014-01-31

    The multidrug resistance-encoding IncA/C conjugative plasmids disseminate antibiotic resistance genes among clinically relevant enteric bacteria. A plasmid-encoded disulfide isomerase is associated with conjugation. Sequence analysis of several IncA/C plasmids and IncA/C-related integrative and conjugative elements (ICE) from commensal and pathogenic bacteria identified a conserved DsbC/DsbG homolog (DsbP). The crystal structure of DsbP reveals an N-terminal domain, a linker region, and a C-terminal catalytic domain. A DsbP homodimer is formed through domain swapping of two DsbP N-terminal domains. The catalytic domain incorporates a thioredoxin-fold with characteristic CXXC and cis-Pro motifs. Overall, the structure and redox properties of DsbP diverge from the Escherichia coli DsbC and DsbG disulfide isomerases. Specifically, the V-shaped dimer of DsbP is inverted compared with EcDsbC and EcDsbG. In addition, the redox potential of DsbP (-161 mV) is more reducing than EcDsbC (-130 mV) and EcDsbG (-126 mV). Other catalytic properties of DsbP more closely resemble those of EcDsbG than EcDsbC. These catalytic differences are in part a consequence of the unusual active site motif of DsbP (CAVC); substitution to the EcDsbC-like (CGYC) motif converts the catalytic properties to those of EcDsbC. Structural comparison of the 12 independent subunit structures of DsbP that we determined revealed that conformational changes in the linker region contribute to mobility of the catalytic domain, providing mechanistic insight into DsbP function. In summary, our data reveal that the conserved plasmid-encoded DsbP protein is a bona fide disulfide isomerase and suggest that a dedicated oxidative folding enzyme is important for conjugative plasmid transfer.

  2. QM/MM Minimum Free Energy Path: Methodology and Application to Triosephosphate Isomerase.

    Science.gov (United States)

    Hu, Hao; Lu, Zhenyu; Yang, Weitao

    2007-03-01

    /MM free energy perturbation method. The free energy gradients with respect to the QM degrees of freedom are calculated from molecular dynamics simulations at given QM conformations. With the free energy and free energy gradients in hand, we further implement chain-of-conformation optimization algorithms in the search for the reaction path on the free energy surface without specifying a reaction coordinate. This method thus efficiently provides a unique minimum free energy path for solution and enzyme reactions, with structural and energetic properties being determined simultaneously. To further incorporate the dynamic contributions of the QM subsystem into the simulations, we develop the reaction path potential of Lu, et al.2 for the minimum free energy path. The combination of the methods developed here presents a comprehensive and accurate treatment for the simulation of reaction processes in solution and in enzymes with ab initio QM/MM methods. The method has been demonstrated on the first step of the reaction of the enzyme triosephosphate isomerase with good agreement with previous studies.

  3. HbIDI, SlIDI and EcIDI: A comparative study of isopentenyl diphosphate isomerase activity and structure.

    Science.gov (United States)

    Berthelot, Karine; Estevez, Yannick; Quiliano, Miguel; Baldera-Aguayo, Pedro A; Zimic, Mirko; Pribat, Anne; Bakleh, Marc-Elias; Teyssier, Emeline; Gallusci, Philippe; Gardrat, Christian; Lecomte, Sophie; Peruch, Frédéric

    2016-08-01

    In this study, we cloned, expressed and purified the isopentenyl diphosphate isomerases (IDIs) from two plants, Hevea brasiliensis and Solanum lycopersicum, and compared them to the already well characterized Escherichia coli IDI. Phylogenetic analysis showed high homology between the three enzymes. Their catalytic activity was investigated in vitro with recombinant purified enzymes and in vivo by complementation colorimetric tests. The three enzymes displayed consistent activities both in vitro and in vivo. In term of structure, studied by ATR-FTIR and molecular modeling, it is clear that both plant enzymes are more related to their human homologue than to E. coli IDI. But it is assumed that EcIDI represent the minimalistic part of the catalytic core, as both plant enzymes present a supplementary sequence forming an extra α-helice surrounding the catalytic site that could facilitate the biocatalysis. New potential biotechnological applications may be envisaged.

  4. The Unfolding and Refolding Reactions of Triosephosphate Isomerase from Trypanosoma Cruzi Follow Similar Pathways. Guanidinium Hydrochloride Studies

    Science.gov (United States)

    Vázquez-Contreras, Edgar; Pérez Hernández, Gerardo; Sánchez-Rebollar, Brenda Guadalupe; Chánez-Cárdenas, María Elena

    2005-04-01

    The unfolding and refolding reactions of Trypanosoma cruzi triosephosphate isomerase (TcTIM) was studied under equilibrium conditions at increasing guanidinium hydrochloride concentrations. The changes in activity intrinsic fluorescence and far-ultraviolet circular dichroism as a function of denaturant were used as a quaternary, tertiary and secondary structural probes respectively. The change in extrinsic ANS fluorescence intensity was also investigated. The results show that the transition between the homodimeric native enzyme to the unfolded monomers (unfolding), and its inverse reaction (refolding) are described by similar pathways and two equilibrium intermediates were detected in both reactions. The mild denaturant concentrations intermediate is active and contains significant amount of secondary and tertiary structures. The medium denaturant concentrations intermediate is inactive and able to bind the fluorescent dye. This intermediates are maybe related with those observed in the denaturation pattern of TIMs from other species; the results are discussed in this context.

  5. Inhibition of phosphomannose isomerase by fructose 1-phosphate: an explanation for defective N-glycosylation in hereditary fructose intolerance.

    Science.gov (United States)

    Jaeken, J; Pirard, M; Adamowicz, M; Pronicka, E; van Schaftingen, E

    1996-11-01

    Isoelectrofocusing of serum sialotransferrins from patients with untreated hereditary fructose intolerance (HFI) shows a cathodal shift similar to that in carbohydrate-deficient glycoprotein (CDG) syndrome type I and in untreated galactosemia. This report is on serum lysosomal enzyme abnormalities in untreated HFI that are identical to those found in CDG syndrome type I but different from those in untreated galactosemia. CDG syndrome type I is due to phosphomannomutase deficiency, a defect in the early glycosylation pathway. It was found that fructose 1-phosphate is a potent competitive inhibitor (Ki congruent to 40 microM) of phosphomannose isomerase (EC 5.3.1.8), the first enzyme of the N-glycosylation pathway thus explaining the N-glycosylation disturbances in HFI.

  6. BIOPHYSICS. Response to Comments on "Extreme electric fields power catalysis in the active site of ketosteroid isomerase".

    Science.gov (United States)

    Fried, Stephen D; Boxer, Steven G

    2015-08-28

    Natarajan et al. and Chen and Savidge comment that comparing the electric field in ketosteroid isomerase's (KSI's) active site to zero overestimates the catalytic effect of KSI's electric field because the reference reaction occurs in water, which itself exerts a sizable electrostatic field. To compensate, Natarajan et al. argue that additional catalytic weight arises from positioning of the general base, whereas Chen and Savidge propose a separate contribution from desolvation of the general base. We note that the former claim is not well supported by published results, and the latter claim is intriguing but lacks experimental basis. We also take the opportunity to clarify some of the more conceptually subtle aspects of electrostatic catalysis.

  7. Inhibition of enzyme activity of Rhipicephalus (Boophilus) microplus triosephosphate isomerase and BME26 cell growth by monoclonal antibodies.

    Science.gov (United States)

    Saramago, Luiz; Franceschi, Mariana; Logullo, Carlos; Masuda, Aoi; Vaz, Itabajara da Silva; Farias, Sandra Estrazulas; Moraes, Jorge

    2012-10-12

    In the present work, we produced two monoclonal antibodies (BrBm37 and BrBm38) and tested their action against the triosephosphate isomerase of Rhipicephalus (Boophilus) microplus (RmTIM). These antibodies recognize epitopes on both the native and recombinant forms of the protein. rRmTIM inhibition  by BrBm37 was up to 85% whereas that of BrBrm38 was 98%, depending on the antibody-enzyme ratio. RmTIM activity was lower in ovarian, gut, and fat body tissue extracts treated with BrBm37 or BrBm38 mAbs. The proliferation of the embryonic tick cell line (BME26) was inhibited by BrBm37 and BrBm38 mAbs. In summary, the results reveal that it is possible to interfere with the RmTIM function using antibodies, even in intact cells.

  8. Inhibition of Enzyme Activity of Rhipicephalus (Boophilus microplus Triosephosphate Isomerase and BME26 Cell Growth by Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Jorge Moraes

    2012-10-01

    Full Text Available In the present work, we produced two monoclonal antibodies (BrBm37 and BrBm38 and tested their action against the triosephosphate isomerase of Rhipicephalus (Boophilus microplus (RmTIM. These antibodies recognize epitopes on both the native and recombinant forms of the protein. rRmTIM inhibition  by BrBm37 was up to 85% whereas that of BrBrm38 was 98%, depending on the antibody-enzyme ratio. RmTIM activity was lower in ovarian, gut, and fat body tissue extracts treated with BrBm37 or BrBm38 mAbs. The proliferation of the embryonic tick cell line (BME26 was inhibited by BrBm37 and BrBm38 mAbs. In summary, the results reveal that it is possible to interfere with the RmTIM function using antibodies, even in intact cells.

  9. AtCXXS: atypical members of the Arabidopsis thaliana thioredoxin h family with a remarkably high disulfide isomerase activity.

    Science.gov (United States)

    Serrato, Antonio Jesús; Guilleminot, Jocelyne; Meyer, Yves; Vignols, Florence

    2008-07-01

    The Arabidopsis thaliana thioredoxin subgroup h III is composed of four members and includes the two monocysteinic (CXXS) thioredoxins encoded by the genome. We show that AtCXXS1 is the ortholog of monocysteinic thioredoxins present in all higher plants. In contrast, unicellular algae and the moss Physcomitrella patens do not encode monocysteinic thioredoxin. AtCXXS2, the second monocysteinic thioredoxin of Arabidopsis has no ortholog in any other higher plants. It probably appeared recently by duplications of a dicysteinic thioredoxin of the same subgroup h III. Both monocysteinic thioredoxins show a low disulfide reductase activity in vitro but are very efficient as disulfide isomerases in RNAse refolding tests. The possible interactions of these proteins with the glutathione glutaredoxin pathway are discussed on the basis of recent papers.

  10. A case study of proline isomerization in cell signaling.

    Science.gov (United States)

    Min, Lie; Fulton, D Bruce; Andreotti, Amy H

    2005-01-01

    Protein-mediated interactions and enzymatic function provide the foundation upon which cellular signaling cascades control all of the activities of a cell. Post-translational modifications such as phosphorylation or ubiquitiation are well known means for modulating protein activity within the cell. These chemical modifications create new recognition motifs on proteins or shift conformational preferences such that protein catalytic and binding functions are altered in response to external stimuli. Moreover, detection of such modifications is often straightforward by conventional biochemical methods leading investigators toward mechanistic models of cell signaling involving post-translational modifications such as phosphorylation/dephosphorylation. While there is little doubt that such modifications play a significant role in transmission of information throughout the cell, there are certainly other mechanisms at work that are not as well understood at this time. Of particular interest in the context of this review is the intrinsic conformational switch afforded to a polypeptide by peptidyl prolyl cis/trans isomerization. Proline isomerization is emerging as a critical component of certain cell signaling cascades. In addition to serving as a conformational switch that enables a protein to adopt functionally distinct states, proline isomerization may serve as a recognition element for the ubiquitous peptidyl prolyl isomerases. This overview takes a close look at one particular signaling protein, the T cell specific tyrosine kinase Itk, and examines the role of proline isomerization and the peptidyl prolyl isomerase cyclophilin A in mediating Itk function following T cell receptor engagement.

  11. The importance of hinge sequence for loop function and catalytic activity in the reaction catalyzed by triosephosphate isomerase.

    Science.gov (United States)

    Xiang, J; Sun, J; Sampson, N S

    2001-04-01

    We have determined the sequence requirements for the N-terminal protein hinge of the active-site lid of triosephosphate isomerase. The codons for the hinge (PVW) were replaced with a genetic library of all possible 8000 amino acid combinations. The most active of these 8000 mutants were selected using in vivo complementation of a triosephosphate isomerase-deficient strain of Escherichia coli, DF502. Approximately 0.3 % of the mutants complement DF502 with an activity that is between 10 and 70 % of wild-type activity. They all contain Pro at the first position. Furthermore, the sequences of these hinge mutants reveal that hydrophobic packing is very important for efficient formation of the enediol intermediate. However, the reduced catalytic activities observed are not due to increased rates of loop opening. To explore the relationship between the N-terminal and C-terminal hinges, three semi-active mutants from the N-terminal hinge selection experiment (PLH, PHS and PTF), and six active C-terminal hinge mutants from previous work (NSS, LWA, YSL, KTK, NPN, KVA) were combined to form 18 "double-hinge" mutants. The activities of these mutants suggest that the N-terminal and C-terminal hinge structures affect one another. It appears that specific side-chain interactions are important for forming a catalytically active enzyme, but not for preventing release of the unstable enediol intermediate from the active site of the enzyme. The independence of intermediate release on amino acid sequence is consistent with the absence of a "universal" hinge sequence in structurally related enzymes.

  12. Inhibition of d-xylose isomerase by polyols: atomic details by joint X-ray/neutron crystallography

    Energy Technology Data Exchange (ETDEWEB)

    Kovalevsky, Andrey, E-mail: ayk@lanl.gov [Los Alamos National Laboratory, PO Box 1663, MS M888, Los Alamos, NM 87545 (United States); Hanson, B. Leif [University of Toledo, 2801 West Bancroft Street, Toledo, OH 43606 (United States); Mason, Sax A. [Institut Laue–Langevin, 6 Rue Jules Horowitz, 38042 Grenoble (France); Forsyth, V. Trevor [Institut Laue–Langevin, 6 Rue Jules Horowitz, 38042 Grenoble (France); Keele University, Staffordshire (United Kingdom); Fisher, Zoe [Los Alamos National Laboratory, PO Box 1663, MS M888, Los Alamos, NM 87545 (United States); Mustyakimov, Marat [Los Alamos National Laboratory, PO Box 1663, MS M888, Los Alamos, NM 87545 (United States); Oak Ridge National Laboratory, PO Box 2008, MS 6475, Oak Ridge, TN 37831 (United States); Blakeley, Matthew P. [Institut Laue–Langevin, 6 Rue Jules Horowitz, 38042 Grenoble (France); Keen, David A. [Harwell Science and Innovation Campus, Didcot, Oxon OX11 0QX (United Kingdom); Langan, Paul [Oak Ridge National Laboratory, PO Box 2008, MS 6475, Oak Ridge, TN 37831 (United States); Los Alamos National Laboratory, PO Box 1663, MS M888, Los Alamos, NM 87545 (United States)

    2012-09-01

    A joint X-ray/neutron structure of d-xylose isomerase in complex with the inhibitor sorbitol was determined at room temperature at an acidic pH of 5.9. Protonation of the O5 O atom of the sugar was directly observed in the nuclear density maps. Under acidic conditions sorbitol gains a water-mediated interaction with the enzyme active site, which may explain the increased potency of the inhibitor at low pH. d-Xylose isomerase (XI) converts the aldo-sugars xylose and glucose to their keto analogs xylulose and fructose, but is strongly inhibited by the polyols xylitol and sorbitol, especially at acidic pH. In order to understand the atomic details of polyol binding to the XI active site, a 2.0 Å resolution room-temperature joint X-ray/neutron structure of XI in complex with Ni{sup 2+} cofactors and sorbitol inhibitor at pH 5.9 and a room-temperature X-ray structure of XI containing Mg{sup 2+} ions and xylitol at the physiological pH of 7.7 were obtained. The protonation of oxygen O5 of the inhibitor, which was found to be deprotonated and negatively charged in previous structures of XI complexed with linear glucose and xylulose, was directly observed. The Ni{sup 2+} ions occupying the catalytic metal site (M2) were found at two locations, while Mg{sup 2+} in M2 is very mobile and has a high B factor. Under acidic conditions sorbitol gains a water-mediated interaction that connects its O1 hydroxyl to Asp257. This contact is not found in structures at basic pH. The new interaction that is formed may improve the binding of the inhibitor, providing an explanation for the increased affinity of the polyols for XI at low pH.

  13. Xylose isomerase in substrate and inhibitor michaelis states: atomic resolution studies of a metal-mediated hydride shift.

    Science.gov (United States)

    Fenn, Timothy D; Ringe, Dagmar; Petsko, Gregory A

    2004-06-01

    Xylose isomerase (E.C. 5.3.1.5) catalyzes the interconversion of aldose and ketose sugars and has an absolute requirement for two divalent cations at its active site to drive the hydride transfer rates of sugar isomerization. Evidence suggests some degree of metal movement at the second metal site, although how this movement may affect catalysis is unknown. The 0.95 A resolution structure of the xylitol-inhibited enzyme presented here suggests three alternative positions for the second metal ion, only one of which appears positioned in a catalytically competent manner. To complete the reaction, an active site hydroxyl species appears appropriately positioned for hydrogen transfer, as evidenced by precise bonding distances. Conversely, the 0.98 A resolution structure of the enzyme with glucose bound in the alpha-pyranose state only shows one of the metal ion conformations at the second metal ion binding site, suggesting that the linear form of the sugar is required to promote the second and third metal ion conformations. The two structures suggest a strong degree of conformational flexibility at the active site, which seems required for catalysis and may explain the poor rate of turnover for this enzyme. Further, the pyranose structure implies that His53 may act as the initial acid responsible for ring opening of the sugar to the aldose form, an observation that has been difficult to establish in previous studies. The glucose ring also appears to display significant segmented disorder in a manner suggestive of ring opening, perhaps lending insight into means of enzyme destabilization of the ground state to promote catalysis. On the basis of these results, we propose a modified version of the bridged bimetallic mechanism for hydride transfer in the case of Streptomyces olivochromogenes xylose isomerase.

  14. The acid-tolerant L-arabinose isomerase from the mesophilic Shewanella sp. ANA-3 is highly active at low temperatures

    Directory of Open Access Journals (Sweden)

    Rhimi Moez

    2011-11-01

    Full Text Available Abstract Background L-arabinose isomerases catalyse the isomerization of L-arabinose into L-ribulose at insight biological systems. At industrial scale of this enzyme is used for the bioconversion of D-galactose into D-tagatose which has many applications in pharmaceutical and agro-food industries. The isomerization reaction is thermodynamically equilibrated, and therefore the bioconversion rates is shifted towards tagatose when the temperature is increased. Moreover, to prevent secondary reactions it will be of interest to operate at low pH. The profitability of this D-tagatose production process is mainly related to the use of lactose as cheaper raw material. In many dairy products it will be interesting to produce D-tagatose during storage. This requires an efficient L-arabinose isomerase acting at low temperature and pH values. Results The gene encoding the L-arabinose isomerase from Shewanella sp. ANA-3 was cloned and overexpressed in Escherichia coli. The purified protein has a tetrameric arrangement composed by four identical 55 kDa subunits. The biochemical characterization of this enzyme showed that it was distinguishable by its maximal activity at low temperatures comprised between 15-35°C. Interestingly, this biocatalyst preserves more than 85% of its activity in a broad range of temperatures from 4.0 to 45°C. Shewanella sp. ANA-3 L-arabinose isomerase was also optimally active at pH 5.5-6.5 and maintained over 80% of its activity at large pH values from 4.0 to 8.5. Furthermore, this enzyme exhibited a weak requirement for metallic ions for its activity evaluated at 0.6 mM Mn2+. Stability studies showed that this protein is highly stable mainly at low temperature and pH values. Remarkably, T268K mutation clearly enhances the enzyme stability at low pH values. Use of this L-arabinose isomerase for D-tagatose production allows the achievement of attractive bioconversion rates of 16% at 4°C and 34% at 35°C. Conclusions Here we

  15. Mitochondrial Thioredoxin System as a Modulator of Cyclophilin D Redox State

    OpenAIRE

    Alessandra Folda; Anna Citta; Valeria Scalcon; Tito Calì; Francesco Zonta; Guido Scutari; Alberto Bindoli; Maria Pia Rigobello

    2016-01-01

    The mitochondrial thioredoxin system (NADPH, thioredoxin reductase, thioredoxin) is a major redox regulator. Here we have investigated the redox correlation between this system and the mitochondrial enzyme cyclophilin D. The peptidyl prolyl cis-trans isomerase activity of cyclophilin D was stimulated by the thioredoxin system, while it was decreased by cyclosporin A and the thioredoxin reductase inhibitor auranofin. The redox state of cyclophilin D, thioredoxin 1 and 2 and peroxiredoxin 3 was...

  16. Community involvement

    Directory of Open Access Journals (Sweden)

    Editorial Office

    1979-09-01

    Full Text Available Community involvement is the main theme of Health Year. Governments have a responsibility for the health of their people, and in this country under the present 3-tier system of government, the responsibility for the rendering of health services is divided between central, provincial and local government. However, under our democratic system, all people have the right to, and it is indeed their duty, to participate individually and collectively in the planning and implementation of services to meet their health needs. Ultimately, through involvement of individuals, families and communities, greater self-reliance is achieved leading to greater responsibility being assumed by people for their own health.

  17. Triose phosphate isomerase from the coelacanth. An approach to the rapid determination of an amino acid sequence with small amounts of material.

    Science.gov (United States)

    Kolb, E; Harris, J I; Bridgen, J

    1974-02-01

    The preparation and purification of cyanogen bromide fragments from [(14)C]carboxymethylated coelacanth triose phosphate isomerase is presented. The automated sequencing of these fragments, the lysine-blocked tryptic peptides derived from them, and also of the intact protein, is described. Combination with results from manual sequence analysis has given the 247-residue amino acid sequence of coelacanth triose phosphate isomerase in 4 months, by using 100mg of enzyme. (Two small adjacent peptides were placed by homology with the rabbit enzyme.) Comparison of this sequence with that of the rabbit muscle enzyme shows that 207 (84%) of the residues are identical. This slow rate of evolutionary change (corresponding to two amino acid substitutions per 100 residues per 100 million years) is similar to that found for glyceraldehyde 3-phosphate dehydrogenase. The reliability of sequence information obtained by automated methods is discussed.

  18. SOD1 aggregation in astrocytes following ischemia/reperfusion injury: a role of NO-mediated S-nitrosylation of protein disulfide isomerase (PDI

    Directory of Open Access Journals (Sweden)

    Chen Xueping

    2012-10-01

    Full Text Available Abstract Background Ubiquitinated-protein aggregates are implicated in cerebral ischemia/reperfusion injury. The very presence of these ubiquitinated-protein aggregates is abnormal and seems to be disease-related. However, it is not clear what leads to aggregate formation and whether the aggregations represent a reaction to aggregate-mediated neurodegeneration. Methods To study the nitrosative stress-induced protein aggregation in cerebral ischemia/reperfusion injury, we used primary astrocyte cultures as a cell model, and systematically examined their iNOS expression and consequent NO generation following oxygen glucose deprivation and reperfusion. The expression of protein disulfide isomerase (PDI and copper-zinc superoxide dismutase (SOD1 were also examined, and the biochemical interaction between PDI and SOD1 was determined by immunoprecipitation. In addition, the levels of S-nitrosylated PDI in cultured astrocytes after oxygen glucose deprivation and reperfusion treatment were measured using the biotin-switch assay. The formation of ubiquitinated-protein aggregates was detected by immunoblot and immunofluorescence staining. Results Our data showed that the up-regulation of iNOS expression after oxygen glucose deprivation and reperfusion treatment led to excessive NO generation. Up-regulation of PDI and SOD1 was also identified in cultured astrocytes following oxygen glucose deprivation and reperfusion, and these two proteins were found to bind to each other. Furthermore, the increased nitrosative stress due to ischemia/reperfusion injury was highly associated with NO-induced S-nitrosylation of PDI, and this S-nitrosylation of PDI was correlated with the formation of ubiquitinated-protein aggregates; the levels of S-nitrosylated PDI increased in parallel with the formation of aggregates. When NO generation was pharmacologically inhibited by iNOS specific inhibitor 1400W, S-nitrosylation of PDI was significantly blocked. In addition, the

  19. Th1 stimulatory proteins of Leishmania donovani: comparative cellular and protective responses of rTriose phosphate isomerase, rProtein disulfide isomerase and rElongation factor-2 in combination with rHSP70 against visceral leishmaniasis.

    Science.gov (United States)

    Jaiswal, Anil Kumar; Khare, Prashant; Joshi, Sumit; Kushawaha, Pramod Kumar; Sundar, Shyam; Dube, Anuradha

    2014-01-01

    In visceral leishmaniasis, the recovery from the disease is always associated with the generation of Th1-type of cellular responses. Based on this, we have previously identified several Th1-stimulatory proteins of Leishmania donovani -triose phosphate isomerase (TPI), protein disulfide isomerase (PDI) and elongation factor-2 (EL-2) etc. including heat shock protein 70 (HSP70) which induced Th1-type of cellular responses in both cured Leishmania patients/hamsters. Since, HSPs, being the logical targets for vaccines aimed at augmenting cellular immunity and can be early targets in the immune response against intracellular pathogens; they could be exploited as vaccine/adjuvant to induce long-term immunity more effectively. Therefore, in this study, we checked whether HSP70 can further enhance the immunogenicity and protective responses of the above said Th1-stimulatory proteins. Since, in most of the studies, immunogenicity of HSP70 of L. donovani was assessed in native condition, herein we generated recombinant HSP70 and tested its potential to stimulate immune responses in lymphocytes of cured Leishmania infected hamsters as well as in the peripheral blood mononuclear cells (PBMCs) of cured patients of VL either individually or in combination with above mentioned recombinant proteins. rLdHSP70 alone elicited strong cellular responses along with remarkable up-regulation of IFN-γ and IL-12 cytokines and extremely lower level of IL-4 and IL-10. Among the various combinations, rLdHSP70 + rLdPDI emerged as superior one augmenting improved cellular responses followed by rLdHSP70 + rLdEL-2. These combinations were further evaluated for its protective potential wherein rLdHSP70 + rLdPDI again conferred utmost protection (∼80%) followed by rLdHSP70 + rLdEL-2 (∼75%) and generated a strong cellular immune response with significant increase in the levels of iNOS transcript as well as IFN-γ and IL-12 cytokines which was further supported by the high level of IgG2 antibody

  20. Determination of cis/trans phase of variations in the MC1R gene with allele-specific PCR and single base extension

    DEFF Research Database (Denmark)

    Mengel-From, Jonas; Børsting, Claus; Sanchez, Juan J;

    2008-01-01

    The MC1R gene encodes a protein with key regulatory functions in the melanin synthesis. A multiplex PCR and a multiplex single base extension protocol were established for genotyping six exonic MC1R variations highly penetrant for red hair (R), four exonic MC1R variations weakly penetrant for red...

  1. Dinamica molecolare ed NMR in soluzione: studio dell'equilibrio di isomerizzazione cis-trans del legame ammidico tyr-pro in peptidi a catena corta

    OpenAIRE

    Fenude, Emma; Fais, M; Muroni, M.

    2004-01-01

    Nella presente comunicazione verranno illustrati i risultati ottenuti dall’analisi conformazionale di peptidi bioattivi del tipo: Tyr-Pro Tyr-Pro-Phe Tyr-Pro-Phe-Pro βCM4 Tyr-Pro-Phe-Pro-NH2 βCM4 ammide Tyr-Pro-Phe-Pro-Gly βCM5 Tyr-Pro-Phe-Pro-Gly-Pro-Ile βCM7 Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro βCM8 Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn-Ser-Leu βCM11

  2. Capillary electrophoresis with capacitively coupled contactless conductivity detection for the determination of cis/trans isomers of octadec-9-enoic acid and other long chain fatty acids.

    Science.gov (United States)

    Wong, Yong Foo; Saad, Bahruddin; Makahleh, Ahmad

    2013-05-17

    A capillary electrophoresis (CE)-capacitively coupled contactless conductivity detection (C(4)D) method for the simultaneous separation of eleven underivatized fatty acids (FAs), namely, lauric, myristic, tridecanoic (internal standard), pentadecanoic, palmitic, stearic, oleic, elaidic, linoleic, linolenic and arachidic acids is described. The separation was carried out in normal polarity mode at 20 °C, 30 kV and using hydrodynamic injection (50 mbar for 1 s). The separation was achieved in a bare fused-silica capillary (70 cm × 75 μm i.d.) using a background electrolyte of methyl-β-cyclodextrin (~6 mM) and heptakis-(2,3,6-tri-O-methyl)-β-cyclodextrin (~8 mM) dissolved in a mixture of Na2HPO4/KH2PO4 (5 mM, pH 7.4):ACN:MeOH:n-octanol (3:4:2.5:0.5, v/v/v/v). C(4)D parameters were set at fixed amplitude of 100 V and frequency of 1000 kHz. The developed method was validated. Calibration curves of the ten FAs were well correlated (r(2)>0.99) within the range of 5-250 μg mL(-1) for lauric acid, and 3-250 μg mL(-1) for the other FAs. The method was simple and sensitive with detection limits (S/N=3) of 0.9-1.9 μg mL(-1) and good relative standard deviations of intra- and inter-day for migration times and peak areas (≤9.7%) were achieved. The method was applied to the determination of FAs in margarine samples. The proposed method offers distinct advantages over the GC and HPLC methods, especially in terms of simplicity (without derivatization) and sensitivity.

  3. Fatty acids attached to all-trans-astaxanthin alter its cis-trans equilibrium, and consequently its stability, upon light-accelerated autoxidation

    NARCIS (Netherlands)

    Bruijn, De Wouter J.C.; Weesepoel, Y.; Vincken, J.P.; Gruppen, H.

    2016-01-01

    Fatty acid esterification, common in naturally occurring astaxanthin, has been suggested to influence both colour stability and degradation of all-trans-astaxanthin. Therefore, astaxanthin stability was studied as influenced by monoesterification and diesterification with palmitate. Increased est

  4. Kinetics of Light-Induced cis-trans Isomerization of Four Piperines and their Levels in Ground Black Peppers as Determined by HPLC and LC/MS

    Science.gov (United States)

    The pungent compound piperine, a secondary metabolite present in black, white, and green pepper fruit, undergoes light-induced isomerizations. To facilitate studies in this area, an HPLC method has been developed for analysis and isolation of the following four possible piperine photo-induced isomer...

  5. Non-conjugated cis/trans 18:2 in Beef Fat are Mainly Δ-9 Desaturation Products of trans-18:1 Isomers.

    Science.gov (United States)

    Vahmani, P; Rolland, D C; Gzyl, K E; Dugan, M E R

    2016-12-01

    Human liver cells (HepG2) were cultured with individual trans (t) 18:1 including t6-, t12-, t13-, t14-, t15- and t16-18:1, and retention times of their Δ-9 desaturation products were determined using 100-m biscyanopropyl-polysiloxane and SLB-IL111 columns. Corresponding peaks were found in beef adipose tissues known to have different delta-9 desaturase activities. Further lines of evidence indicating the presence of Δ-9 desaturation products of t-18:1 isomers in beef fat were developed by analysis of fatty acid methyl esters (FAME) fractionated using Ag(+)-TLC, and by GC/MS. Some of the Δ-9 desaturation products of t-18:1 have been previously identified in ruminant fat (c9, t12- and c9, t13-18:2). Some of the Δ-9 desaturation products of t-18:1 (c9, t14- and c9, t15-18:2) have been previously tentatively identified as different fatty acids, and for the first time we provide evidence of the presence of c9, t16-18:2, and where t6, c9-18:2 may elute during analysis of FAME from beef fat.

  6. Effects of acetic acid on the kinetics of xylose fermentation by an engineered, xylose-isomerase-based Saccharomyces cerevisiae strain.

    Science.gov (United States)

    Bellissimi, Eleonora; van Dijken, Johannes P; Pronk, Jack T; van Maris, Antonius J A

    2009-05-01

    Acetic acid, an inhibitor released during hydrolysis of lignocellulosic feedstocks, has previously been shown to negatively affect the kinetics and stoichiometry of sugar fermentation by (engineered) Saccharomyces cerevisiae strains. This study investigates the effects of acetic acid on S. cerevisiae RWB 218, an engineered xylose-fermenting strain based on the Piromyces XylA (xylose isomerase) gene. Anaerobic batch cultures on synthetic medium supplemented with glucose-xylose mixtures were grown at pH 5 and 3.5, with and without addition of 3 g L(-1) acetic acid. In these cultures, consumption of the sugar mixtures followed a diauxic pattern. At pH 5, acetic acid addition caused increased glucose consumption rates, whereas specific xylose consumption rates were not significantly affected. In contrast, at pH 3.5 acetic acid had a strong and specific negative impact on xylose consumption rates, which, after glucose depletion, slowed down dramatically, leaving 50% of the xylose unused after 48 h of fermentation. Xylitol production was absent (fermentation in acetic -acid-stressed cultures at pH 3.5 could be restored by applying a continuous, limiting glucose feed, consistent with a key role of ATP regeneration in acetic acid tolerance.

  7. Ethylene responses in rice roots and coleoptiles are differentially regulated by a carotenoid isomerase-mediated abscisic acid pathway.

    Science.gov (United States)

    Yin, Cui-Cui; Ma, Biao; Collinge, Derek Phillip; Pogson, Barry James; He, Si-Jie; Xiong, Qing; Duan, Kai-Xuan; Chen, Hui; Yang, Chao; Lu, Xiang; Wang, Yi-Qin; Zhang, Wan-Ke; Chu, Cheng-Cai; Sun, Xiao-Hong; Fang, Shuang; Chu, Jin-Fang; Lu, Tie-Gang; Chen, Shou-Yi; Zhang, Jin-Song

    2015-04-01

    Ethylene and abscisic acid (ABA) act synergistically or antagonistically to regulate plant growth and development. ABA is derived from the carotenoid biosynthesis pathway. Here, we analyzed the interplay among ethylene, carotenoid biogenesis, and ABA in rice (Oryza sativa) using the rice ethylene response mutant mhz5, which displays a reduced ethylene response in roots but an enhanced ethylene response in coleoptiles. We found that MHZ5 encodes a carotenoid isomerase and that the mutation in mhz5 blocks carotenoid biosynthesis, reduces ABA accumulation, and promotes ethylene production in etiolated seedlings. ABA can largely rescue the ethylene response of the mhz5 mutant. Ethylene induces MHZ5 expression, the production of neoxanthin, an ABA biosynthesis precursor, and ABA accumulation in roots. MHZ5 overexpression results in enhanced ethylene sensitivity in roots and reduced ethylene sensitivity in coleoptiles. Mutation or overexpression of MHZ5 also alters the expression of ethylene-responsive genes. Genetic studies revealed that the MHZ5-mediated ABA pathway acts downstream of ethylene signaling to inhibit root growth. The MHZ5-mediated ABA pathway likely acts upstream but negatively regulates ethylene signaling to control coleoptile growth. Our study reveals novel interactions among ethylene, carotenogenesis, and ABA and provides insight into improvements in agronomic traits and adaptive growth through the manipulation of these pathways in rice.

  8. Metabolomic and (13)C-metabolic flux analysis of a xylose-consuming Saccharomyces cerevisiae strain expressing xylose isomerase.

    Science.gov (United States)

    Wasylenko, Thomas M; Stephanopoulos, Gregory

    2015-03-01

    Over the past two decades, significant progress has been made in the engineering of xylose-consuming Saccharomyces cerevisiae strains for production of lignocellulosic biofuels. However, the ethanol productivities achieved on xylose are still significantly lower than those observed on glucose for reasons that are not well understood. We have undertaken an analysis of central carbon metabolite pool sizes and metabolic fluxes on glucose and on xylose under aerobic and anaerobic conditions in a strain capable of rapid xylose assimilation via xylose isomerase in order to investigate factors that may limit the rate of xylose fermentation. We find that during xylose utilization the flux through the non-oxidative Pentose Phosphate Pathway (PPP) is high but the flux through the oxidative PPP is low, highlighting an advantage of the strain employed in this study. Furthermore, xylose fails to elicit the full carbon catabolite repression response that is characteristic of glucose fermentation in S. cerevisiae. We present indirect evidence that the incomplete activation of the fermentation program on xylose results in a bottleneck in lower glycolysis, leading to inefficient re-oxidation of NADH produced in glycolysis.

  9. Expression of. Arabidopsis tryptophan biosynthetic pathway genes: effect of the 5’ coding region of phosphoribosylanthranilate isomerase gene

    Institute of Scientific and Technical Information of China (English)

    何奕昆; 刘新仿; 李家洋

    1999-01-01

    There are three non-allelic isogenes encoding phosphoribosylanthranilate isomerase (PAI) in Arabidopsis thaliana. The expression plasmids were constructed by fusion of the GUS reporter gene to the three PAI promoters with or without the 5’ region encoding PAI N-terminal polypeptides and transferred into Arabidopsis plants by Agrobacterium tumefaciens. Analysis of GUS activity revealed that the PAI 5’ coding region was necessary for high expression of GUS activity. GUS activity in transgenic plants transformed with the expression plasmids containing the 5’ coding region of PAI1 or PAI3 was 60—100-fold higher than that without the corresponding 5’ region. However, the effect of 5’ coding region of PAI2 gene on the GUS activity was very small (only about 1 time difference). The GUS histochemical staining showed a similar result as revealed by GUS activity assay. It was expressed in the mesophyll cells and guard cells, but not in the epidermic cells, indicating that the N-terminal polypeptides encoded by t

  10. Glucose-6-phosphate isomerase is an endogenous inhibitor to myofibril-bound serine proteinase of crucian carp (Carassius auratus).

    Science.gov (United States)

    Sun, Le-Chang; Zhou, Li-Gen; Du, Cui-Hong; Cai, Qiu-Feng; Hara, Kenji; Su, Wen-Jin; Cao, Min-Jie

    2009-06-24

    Glucose-6-phosphate isomerase (GPI) was purified to homogeneity from the skeletal muscle of crucian carp ( Carassius auratus ) by ammonium sulfate fractionation, column chromatographies of Q-Sepharose, SP-Sepharose, and Superdex 200 with a yield of 8.0%, and purification folds of 468. The molecular mass of GPI was 120 kDa as estimated by gel filtration, while on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two subunits (55 and 65 kDa) were identified, suggesting that it is a heterodimer. Interestingly, GPI revealed specific inhibitory activity toward a myofibril-bound serine proteinase (MBSP) from crucian carp, while no inhibitory activity was identified toward other serine proteinases, such as white croaker MBSP and crucian carp trypsin. Kinetic analysis showed that GPI is a competitive inhibitor toward MBSP, and the K(i) was 0.32 microM. Our present results indicated that the multifunctional protein GPI is an endogenous inhibitor to MBSP and may play a significant role in the regulation of muscular protein metabolism in vivo.

  11. Characterization of a Mannose-6-Phosphate Isomerase from Bacillus amyloliquefaciens and Its Application in Fructose-6-Phosphate Production.

    Directory of Open Access Journals (Sweden)

    Sujan Sigdel

    Full Text Available The BaM6PI gene encoding a mannose-6-phosphate isomerase (M6PI, EC 5.3.1.8 was cloned from Bacillus amyloliquefaciens DSM7 and overexpressed in Escherichia coli. The enzyme activity of BaM6PI was optimal at pH and temperature of 7.5 and 70°C, respectively, with a kcat/Km of 13,900 s-1 mM-1 for mannose-6-phosphate (M6P. The purified BaM6PI demonstrated the highest catalytic efficiency of all characterized M6PIs. Although M6PIs have been characterized from several other sources, BaM6PI is distinguished from other M6PIs by its wide pH range and high catalytic efficiency for M6P. The binding orientation of the substrate M6P in the active site of BaM6PI shed light on the molecular basis of its unusually high activity. BaM6PI showed 97% substrate conversion from M6P to fructose-6-phosphate demonstrating the potential for using BaM6PI in industrial applications.

  12. Crystallization and preliminary X-ray crystallographic analysis of L-arabinose isomerase from thermophilic Geobacillus kaustophilus.

    Science.gov (United States)

    Cao, Thinh-Phat; Choi, Jin Myung; Lee, Sang-Jae; Lee, Yong-Jik; Lee, Sung-Keun; Jun, Youngsoo; Lee, Dong-Woo; Lee, Sung Haeng

    2014-01-01

    L-arabinose isomerase (AI), which catalyzes the isomerization of L-arabinose to L-ribulose, can also convert D-galactose to D-tagatose, a natural sugar replacer, which is of commercial interest in the food and healthcare industries. Intriguingly, mesophilic and thermophilic AIs showed different substrate preferences and metal requirements in catalysis and different thermostabilities. However, the catalytic mechanism of thermophilic AIs still remains unclear. Therefore, thermophilic Geobacillus kaustophilus AI (GKAI) was overexpressed, purified and crystallized, and a preliminary X-ray diffraction data set was obtained. Diffraction data were collected from a GKAI crystal to 2.70 Å resolution. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 224.12, b = 152.95, c = 91.28 Å, β = 103.61°. The asymmetric unit contained six molecules, with a calculated Matthews coefficient of 2.25 Å(3) Da(-1) and a solvent content of 45.39%. The three-dimensional structure determination of GKAI is currently in progress by molecular replacement and model building.

  13. Effect of pharmaceutical potential endocrine disruptor compounds on protein disulfide isomerase reductase activity using di-eosin-oxidized-glutathione.

    Directory of Open Access Journals (Sweden)

    Danièle Klett

    Full Text Available BACKGROUND: Protein Disulfide Isomerase (PDI in the endoplasmic reticulum of all cells catalyzes the rearrangement of disulfide bridges during folding of membrane and secreted proteins. As PDI is also known to bind various molecules including hormones such as estradiol and thyroxin, we considered the hypothesis that adverse effects of endocrine-disrupter compounds (EDC could be mediated through their interaction with PDI leading to defects in membrane or secreted proteins. METHODOLOGY/PRINCIPAL FINDINGS: Taking advantage of the recent description of the fluorescence self quenched substrate di-eosin-oxidized-glutathione (DiE-GSSG, we determined kinetically the effects of various potential pharmaceutical EDCs on the in-vitro reductase activity of bovine liver PDI by measuring the fluorescence of the reaction product (E-GSH. Our data show that estrogens (ethynylestradiol and bisphenol-A as well as indomethacin exert an inhibition whereas medroxyprogesteroneacetate and nortestosterone exert a potentiation of bovine PDI reductase activity. CONCLUSIONS: The present data indicate that the tested EDCs could not only affect endocrine target cells through nuclear receptors as previously shown, but could also affect these and all other cells by positively or negatively affecting PDI activity. The substrate DiE-GSSG has been demonstrated to be a convenient substrate to measure PDI reductase activity in the presence of various potential EDCs. It will certainly be usefull for the screening of potential effect of all kinds of chemicals on PDI reductase activity.

  14. In vivo control of redox potential during protein folding catalyzed by bacterial protein disulfide-isomerase (DsbA).

    Science.gov (United States)

    Wunderlich, M; Glockshuber, R

    1993-11-25

    The formation of disulfide bonds in Escherichia coli is catalyzed by periplasmic protein disulfide-isomerase (DsbA). When the alpha-amylase/trypsin inhibitor from Ragi, a protein containing five intramolecular disulfide bridges, is secreted into the periplasm of E. coli, large amounts of misfolded inhibitor with incomplete or incorrect disulfides are accumulated. Folding of the inhibitor in the periplasm is not improved when DsbA is coexpressed and cosecreted. However, an up to 14-fold increase in correctly folded inhibitor is observed by co-expression of DsbA in conjugation with the addition of reduced glutathione to the growth medium. This peptide acts as a disulfide-shuffling reagent and can pass the outer membrane of E. coli. Since the influence of DsbA on the folding yield of the inhibitor is reduced in the presence of oxidized glutathione, the in vivo function of DsbA appears to be dependent on the ratio between oxidizing and reducing thiol equivalents in the periplasm. The high stability of thiol reagents against air oxidation during growth of E. coli allows the investigation of oxidative protein folding in vivo under controlled, thiol-dependent redox conditions.

  15. Triosephosphate isomerase of Taenia solium (TTPI): phage display and antibodies as tools for finding target regions to inhibit catalytic activity.

    Science.gov (United States)

    Sanabria-Ayala, Víctor; Belmont, Iaraset; Abraham, Landa

    2015-01-01

    Previous studies demonstrated that antibodies against triosephosphate isomerase of Taenia solium (TTPI) can alter its enzymatic catalysis. In the present study, we used antibodies produced against the NH2-terminal region of TTPI (1/3NH2TTPI) and the phage display technology to find target regions to inhibit TTPI activity. As a first step, we obtained polyclonal antibodies against non-conserved regions from the 1/3NH2TTPI, which had an inhibitory effect of about 74 % on catalytic activity. Afterward, they were used to screen a library of phage-displayed dodecapeptides; as a result, 41 phage mimotope clones were isolated and grouped according to their amino acid sequence, finding the consensus A1 (VPTXPI), A2 (VPTXXI), B (LTPGQ), and D (DPLPR). Antibodies against selected phage mimotope clones were obtained by rabbit's immunization; these ones clearly recognized TTPI by both Western blot and ELISA. However, only the mimotope PDTS16 (DSVTPTSVMAVA) clone, which belongs to the VPTXXI consensus, raised antibodies capable of inhibiting the TTPI catalytic activity in 45 %. Anti-PDTS16 antibodies were confronted to several synthetic peptides that encompass the 1/3NH2TTPI, and they only recognized three, which share the motif FDTLQK belonging to the helix-α1 in TTPI. This suggests that this motif is the main part of the epitope recognized by anti-PDTS16 antibodies and revealed its importance for TTPI catalysis.

  16. Molecular Bases of Cyclic and Specific Disulfide Interchange between Human ERO1α Protein and Protein-disulfide Isomerase (PDI)*

    Science.gov (United States)

    Masui, Shoji; Vavassori, Stefano; Fagioli, Claudio; Sitia, Roberto; Inaba, Kenji

    2011-01-01

    In the endoplasmic reticulum (ER) of human cells, ERO1α and protein-disulfide isomerase (PDI) constitute one of the major electron flow pathways that catalyze oxidative folding of secretory proteins. Specific and limited PDI oxidation by ERO1α is essential to avoid ER hyperoxidation. To investigate how ERO1α oxidizes PDI selectively among more than 20 ER-resident PDI family member proteins, we performed docking simulations and systematic biochemical analyses. Our findings reveal that a protruding β-hairpin of ERO1α specifically interacts with the hydrophobic pocket present in the redox-inactive PDI b′-domain through the stacks between their aromatic residues, leading to preferred oxidation of the C-terminal PDI a′-domain. ERO1α associated preferentially with reduced PDI, explaining the stepwise disulfide shuttle mechanism, first from ERO1α to PDI and then from oxidized PDI to an unfolded polypeptide bound to its hydrophobic pocket. The interaction of ERO1α with ERp44, another PDI family member protein, was also analyzed. Notably, ERO1α-dependent PDI oxidation was inhibited by a hyperactive ERp44 mutant that lacks the C-terminal tail concealing the substrate-binding hydrophobic regions. The potential ability of ERp44 to inhibit ERO1α activity may suggest its physiological role in ER redox and protein homeostasis. PMID:21398518

  17. Quality properties and expression profiling of protein disulfide isomerase genes during grain development of three spring wheat near isogenic lines

    Directory of Open Access Journals (Sweden)

    Dong Liwei

    2016-01-01

    Full Text Available Three wheat glutenin near isogenic lines (NILs CB037A, CB037B and CB037C were used to investigate their quality properties and the transcriptional expression profiles of PDI gene family during grain development. Our purpose is to understand the relationships between the dynamic expression of different PDI genes and glutenin allelic compositions related to gluten quality. The results showed that glutenin allelic variations had no significant effects on main agronomic traits and yield performance, but resulted in clear gluten quality changes. CB037B with 5+10 subunits had higher glutenin macropolymer (GMP content and better breadmaking quality than CB037A with 2+12 while the lack of Glu-B3h encoding one abundant B-subunit in CB037C significantly reduced GMP content, dough strength and breadmaking quality. The dynamic expression patterns of eight protein disulfide isomerase (PDI genes during grain development detected by quantitative real-time polymerase chain reaction (qRT-PCR showed the close correlations between higher expression levels of PDI3-1, PDI5-1 and PDI8-1 and the presence of 5+10 subunits. Meanwhile, Glu-B3h silence resulted in significant decrease of expression levels of five PDI genes (PDI3-1, PDI5-1, PDI6-1, PDI7-2 and PDI8-1, suggesting the vital roles of certain PDI genes in glutenin and GMP synthesis and gluten quality formation.

  18. Development of a phosphomannose isomerase-based Agrobacterium-mediated transformation system for chickpea (Cicer arietinum L.).

    Science.gov (United States)

    Patil, Gunvant; Deokar, Amit; Jain, P K; Thengane, R J; Srinivasan, R

    2009-11-01

    To develop an alternative genetic transformation system that is not dependent on an antibiotic selection strategy, the phosphomannose isomerase gene (pmi) system was evaluated for producing transgenic plants of chickpea (Cicer arietinum L.). A shoot morphogenesis protocol based on the thidiazuron (TDZ)-induced shoot morphogenesis system was combined with Agrobacterium-mediated transformation of the pmi gene and selection of transgenic plants on mannose. Embryo axis explants of chickpea cv. C-235 were grown on a TDZ-supplemented medium for shoot proliferation. Embryo axis explants from which the first and second flush of shoots were removed were transformed using Agrobacterium carrying the pmi gene, and emerging shoots were allowed to regenerate on a zeatin-supplemented medium with an initial selection pressure of 20 g l(-1) mannose. Rooting was induced in the selected shoots on an indole-3-butyric acid (IBA)-supplemented medium with a selection pressure of 15 g l(-1) mannose. PCR with marker gene-specific primers and chlorophenol red (CPR) assay of the shoots indicated that shoots had been transformed. RT-PCR and Southern analysis of selected regenerated plants further confirmed integration of the transgene into the chickpea genome. These positive results suggest that the pmi/mannose selection system can be used to produce transgenic plants of chickpea that are free from antibiotic resistance marker genes.

  19. Successful recovery of transgenic cowpea (Vigna unguiculata) using the 6-phosphomannose isomerase gene as the selectable marker.

    Science.gov (United States)

    Bakshi, Souvika; Saha, Bedabrata; Roy, Nand Kishor; Mishra, Sagarika; Panda, Sanjib Kumar; Sahoo, Lingaraj

    2012-06-01

    A new method for obtaining transgenic cowpea was developed using positive selection based on the Escherichia coli 6-phosphomannose isomerase gene as the selectable marker and mannose as the selective agent. Only transformed cells were capable of utilizing mannose as a carbon source. Cotyledonary node explants from 4-day-old in vitro-germinated seedlings of cultivar Pusa Komal were inoculated with Agrobacterium tumefaciens strain EHA105 carrying the vector pNOV2819. Regenerating transformed shoots were selected on medium supplemented with a combination of 20 g/l mannose and 5 g/l sucrose as carbon source. The transformed shoots were rooted on medium devoid of mannose. Transformation efficiency based on PCR analysis of individual putative transformed shoots was 3.6%. Southern blot analysis on five randomly chosen PCR-positive plants confirmed the integration of the pmi transgene. Qualitative reverse transcription (qRT-PCR) analysis demonstrated the expression of pmi in T₀ transgenic plants. Chlorophenol red (CPR) assays confirmed the activity of PMI in transgenic plants, and the gene was transmitted to progeny in a Mendelian fashion. The transformation method presented here for cowpea using mannose selection is efficient and reproducible, and could be used to introduce a desirable gene(s) into cowpea for biotic and abiotic stress tolerance.

  20. mRNA and Protein levels of rat pancreas specific protein disulphide isomerase are downregulated during Hyperglycemia.

    Science.gov (United States)

    Gupta, Rajani; Bhar, Kaushik; Sen, Nandini; Bhowmick, Debajit; Mukhopadhyay, Satinath; Panda, Koustubh; Siddhanta, Anirban

    2016-02-01

    Diabetes (Type I and Type II) which affects nearly every organ in the body is a multi-factorial non-communicable disorder. Hyperglycemia is the most characteristic feature of this disease. Loss of beta cells is common in both types of diabetes whose detailed cellular and molecular mechanisms are yet to be elucidated. As this disease is complex, identification of specific biomarkers for its early detection, management and devising new therapies is challenging. Based on the fact that functionally defective proteins provide the biochemical basis for many diseases, in this study, we tried to identify differentially expressed proteins during hyperglycemia. For that, hyperglycemia was induced in overnight fasted rats by intra-peritoneal injection of streptozotocin (STZ). The pancreas was isolated from control and treated rats for subsequent analyses. The 2D-gel electrophoresis followed by MALDI-TOF-MS-MS analyses revealed several up- and down-regulated proteins in hyperglycemic rat pancreas including the downregulation of a pancreas specific isoform of protein disulphide isomerase a2 (Pdia2).This observation was validated by western blot. Quantitative PCR experiments showed that the level of Pdia2 mRNA is also proportionally reduced in hyperglycemic pancreas.

  1. Xanthobacter flavus employs a single triosephosphate isomerase for heterotrophic and autotrophic metabolism

    NARCIS (Netherlands)

    Meijer, WG; deBoer, P; vanKeulen, G

    1997-01-01

    The expression of the cbb and gap-pgk operons of Xanthobacter flavus encoding enzymes of the Calvin cycle is regulated by the transcriptional regulator CbbR. In order to identify other genes involved in the regulation of these operons, a mutant was isolated with a lowered activity of a fusion betwee

  2. Binding of 5-phospho-D-arabinonohydroxamate and 5-phospho-D-arabinonate inhibitors to zinc phosphomannose isomerase from Candida albicans studied by polarizable molecular mechanics and quantum mechanics.

    Science.gov (United States)

    Roux, Celine; Gresh, Nohad; Perera, Lalith E; Piquemal, Jean-Philip; Salmon, Laurent

    2007-04-15

    Type I phosphomannose isomerase (PMI) is a Zn-dependent metalloenzyme involved in the isomerization of D-fructose 6-phosphate to D-mannose 6-phosphate. One of our laboratories has recently designed and synthesized 5-phospho-D-arabinonohydroxamate (5PAH), an inhibitor endowed with a nanomolar affinity for PMI (Roux et al., Biochemistry 2004, 43, 2926). By contrast, the 5-phospho-D-arabinonate (5PAA), in which the hydroxamate moiety is replaced by a carboxylate one, is devoid of inhibitory potency. Subsequent biochemical studies showed that in its PMI complex, 5PAH binds Zn(II) through its hydroxamate moiety rather than through its phosphate. These results have stimulated the present theoretical investigation in which we resort to the SIBFA polarizable molecular mechanics procedure to unravel the structural and energetical aspects of 5PAH and 5PAA binding to a 164-residue model of PMI. Consistent with the experimental results, our theoretical studies indicate that the complexation of PMI by 5PAH is much more favorable than by 5PAA, and that in the 5PAH complex, Zn(II) ligation by hydroxamate is much more favorable than by phosphate. Validations by parallel quantum-chemical computations on model of the recognition site extracted from the PMI-inhibitor complexes, and totaling up to 140 atoms, showed the values of the SIBFA intermolecular interaction energies in such models to be able to reproduce the quantum-chemistry ones with relative errors complexed to the high-energy intermediate analogue inhibitor, which allows us to identify active site residues likely involved in the proton transfer between the two adjacent carbons of the substrates.

  3. Variation in the Subcellular Localization and Protein Folding Activity among Arabidopsis thaliana Homologs of Protein Disulfide Isomerase

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    Christen Y. L. Yuen

    2013-10-01

    Full Text Available Protein disulfide isomerases (PDIs catalyze the formation, breakage, and rearrangement of disulfide bonds to properly fold nascent polypeptides within the endoplasmic reticulum (ER. Classical animal and yeast PDIs possess two catalytic thioredoxin-like domains (a, a′ and two non-catalytic domains (b, b′, in the order a-b-b′-a′. The model plant, Arabidopsis thaliana, encodes 12 PDI-like proteins, six of which possess the classical PDI domain arrangement (AtPDI1 through AtPDI6. Three additional AtPDIs (AtPDI9, AtPDI10, AtPDI11 possess two thioredoxin domains, but without intervening b-b′ domains. C-terminal green fluorescent protein (GFP fusions to each of the nine dual-thioredoxin PDI homologs localized predominantly to the ER lumen when transiently expressed in protoplasts. Additionally, expression of AtPDI9:GFP-KDEL and AtPDI10: GFP-KDDL was associated with the formation of ER bodies. AtPDI9, AtPDI10, and AtPDI11 mediated the oxidative folding of alkaline phosphatase when heterologously expressed in the Escherichia coli protein folding mutant, dsbA−. However, only three classical AtPDIs (AtPDI2, AtPDI5, AtPDI6 functionally complemented dsbA−. Interestingly, chemical inducers of the ER unfolded protein response were previously shown to upregulate most of the AtPDIs that complemented dsbA−. The results indicate that Arabidopsis PDIs differ in their localization and protein folding activities to fulfill distinct molecular functions in the ER.

  4. Cloning, Expression, and Characterization of a Novel L-Arabinose Isomerase from the Psychrotolerant Bacterium Pseudoalteromonas haloplanktis.

    Science.gov (United States)

    Xu, Wei; Fan, Chen; Zhang, Tao; Jiang, Bo; Mu, Wanmeng

    2016-11-01

    L-Arabinose isomerase (L-AI, EC 5.3.1.4) catalyzes the isomerization between L-arabinose and L-ribulose, and most of the reported ones can also catalyze D-galactose to D-tagatose, except Bacillus subtilis L-AI. In this article, the L-AI from the psychrotolerant bacterium Pseudoalteromonas haloplanktis ATCC 14393 was characterized. The enzyme showed no substrate specificity toward D-galactose, which was similar to B. subtilis L-AI but distinguished from other reported L-AIs. The araA gene encoding the P. haloplanktis L-AI was cloned and overexpressed in E. coli BL21 (DE3). The recombinant enzyme was purified by one-step nickel affinity chromatography . The enzyme displayed the maximal activity at 40 °C and pH 8.0, and showed more than 75 % of maximal activity from pH 7.5-9.0. Metal ion Mn(2+) was required as optimum metal cofactor for activity simulation, but it did not play a significant role in thermostability improvement as reported previously. The Michaelis-Menten constant (K m), turnover number (k cat), and catalytic efficiency (k cat/K m) for substrate L-arabinose were measured to be 111.68 mM, 773.30/min, and 6.92/mM/min, respectively. The molecular docking results showed that the active site residues of P. haloplanktis L-AI could only immobilize L-arabinose and recognized it as substrate for isomerization.

  5. A zebrafish model of congenital disorders of glycosylation with phosphomannose isomerase deficiency reveals an early opportunity for corrective mannose supplementation

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    Jaime Chu

    2013-01-01

    Individuals with congenital disorders of glycosylation (CDG have recessive mutations in genes required for protein N-glycosylation, resulting in multi-systemic disease. Despite the well-characterized biochemical consequences in these individuals, the underlying cellular defects that contribute to CDG are not well understood. Synthesis of the lipid-linked oligosaccharide (LLO, which serves as the sugar donor for the N-glycosylation of secretory proteins, requires conversion of fructose-6-phosphate to mannose-6-phosphate via the phosphomannose isomerase (MPI enzyme. Individuals who are deficient in MPI present with bleeding, diarrhea, edema, gastrointestinal bleeding and liver fibrosis. MPI-CDG patients can be treated with oral mannose supplements, which is converted to mannose-6-phosphate through a minor complementary metabolic pathway, restoring protein glycosylation and ameliorating most symptoms, although liver disease continues to progress. Because Mpi deletion in mice causes early embryonic lethality and thus is difficult to study, we used zebrafish to establish a model of MPI-CDG. We used a morpholino to block mpi mRNA translation and established a concentration that consistently yielded 13% residual Mpi enzyme activity at 4 days post-fertilization (dpf, which is within the range of MPI activity detected in fibroblasts from MPI-CDG patients. Fluorophore-assisted carbohydrate electrophoresis detected decreased LLO and N-glycans in mpi morphants. These deficiencies resulted in 50% embryonic lethality by 4 dpf. Multi-systemic abnormalities, including small eyes, dysmorphic jaws, pericardial edema, a small liver and curled tails, occurred in 82% of the surviving larvae. Importantly, these phenotypes could be rescued with mannose supplementation. Thus, parallel processes in fish and humans contribute to the phenotypes caused by Mpi depletion. Interestingly, mannose was only effective if provided prior to 24 hpf. These data provide insight into treatment efficacy

  6. Molecular Characterization of Duplicate Cytosolic Phosphoglucose Isomerase Genes in Clarkia and Comparison to the Single Gene in Arabidopsis

    Science.gov (United States)

    Thomas, B. R.; Ford, V. S.; Pichersky, E.; Gottlieb, L. D.

    1993-01-01

    The nucleotide sequence of PgiC1-a which encodes a cytosolic isozyme of phosphoglucose isomerase (PGIC; EC 5.3.1.9) in Clarkia lewisii, a wildflower native to California, is described and compared to the previously published sequence of the duplicate PgiC2-a from the same genome. Both genes have the same structure of 23 exons and 22 introns located in identical positions, and they encode proteins of 569 amino acids. Exon and inferred protein sequences of the two genes are 96.4% and 97.2% identical, respectively. Intron sequences are 88.2% identical. The high nucleotide similarity of the two genes is consistent with previous genetic and biosystematic findings that suggest the duplication arose within Clarkia. A partial sequence of PgiC2-b was also obtained. It is 99.5% identical to PgiC2-a in exons and 99.7% in introns. The nucleotide sequence of the single PgiC from Arabidopsis thaliana was also determined for comparison to the Clarkia genes. The A. thaliana PgiC has 21 introns located at positions identical to those in Clarkia PgiC1 and PgiC2, but lacks the intron that divides Clarkia exons 21 and 22. The A. thaliana PGIC protein is shorter, with 560 amino acids, and differs by about 17% from the Clarkia PGICs. The PgiC in A. thaliana was mapped to a site 20 cM from restriction fragment length polymorphism marker 331 on chromosome 5. PMID:8293986

  7. Comparison of the xylose reductase-xylitol dehydrogenase and the xylose isomerase pathways for xylose fermentation by recombinant Saccharomyces cerevisiae

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    Hahn-Hägerdal Bärbel

    2007-02-01

    Full Text Available Abstract Background Two heterologous pathways have been used to construct recombinant xylose-fermenting Saccharomyces cerevisiae strains: i the xylose reductase (XR and xylitol dehydrogenase (XDH pathway and ii the xylose isomerase (XI pathway. In the present study, the Pichia stipitis XR-XDH pathway and the Piromyces XI pathway were compared in an isogenic strain background, using a laboratory host strain with genetic modifications known to improve xylose fermentation (overexpressed xylulokinase, overexpressed non-oxidative pentose phosphate pathway and deletion of the aldose reductase gene GRE3. The two isogenic strains and the industrial xylose-fermenting strain TMB 3400 were studied regarding their xylose fermentation capacity in defined mineral medium and in undetoxified lignocellulosic hydrolysate. Results In defined mineral medium, the xylose consumption rate, the specific ethanol productivity, and the final ethanol concentration were significantly higher in the XR- and XDH-carrying strain, whereas the highest ethanol yield was achieved with the strain carrying XI. While the laboratory strains only fermented a minor fraction of glucose in the undetoxified lignocellulose hydrolysate, the industrial strain TMB 3400 fermented nearly all the sugar available. Xylitol was formed by the XR-XDH-carrying strains only in mineral medium, whereas in lignocellulose hydrolysate no xylitol formation was detected. Conclusion Despite by-product formation, the XR-XDH xylose utilization pathway resulted in faster ethanol production than using the best presently reported XI pathway in the strain background investigated. The need for robust industrial yeast strains for fermentation of undetoxified spruce hydrolysates was also confirmed.

  8. Cloning of araA Gene Encoding L-Arabinose Isomerase from Marine Geobacillus stearothermophilus Isolated from Tanjung Api, Poso, Indonesia

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    DEWI FITRIANI

    2010-06-01

    Full Text Available L-arabinose isomerase is an enzyme converting D-galactose to D-tagatose. D-tagatose is a potential sweetener-sucrose substitute which has low calorie. This research was to clone and sequence araA gene from marine bacterial strain Geobacillus stearothermophilus isolated from Tanjung Api Poso Indonesia. The amplified araA gene consisted of 1494 bp nucleotides encoding 497 amino acids. DNA alignment analysis showed that the gene had high homology with that of G. stearothermophilus T6. The enzyme had optimum activity at high temperature and alkalin condition.

  9. Excision of transposable elements from the chalcone isomerase and dihydroflavonol 4-reductase genes may contribute to the variegation of the yellow-flowered carnation (Dianthus caryophyllus).

    Science.gov (United States)

    Itoh, Yoshio; Higeta, Daisuke; Suzuki, Akane; Yoshida, Hiroyuki; Ozeki, Yoshihiro

    2002-05-01

    In the "Rhapsody" cultivar of the carnation, which bears white flowers variegated with red flecks and sectors, a transposable element, dTdic1, belonging to the Ac/Ds superfamily, was found within the dihydroflavonol 4-reductase (DFR) gene. The red flecks and sectors of "Rhapsody" may be attributable to a reversion to DFR activity after the excision of dTdic1. The yellow color of the carnation petals is attributed to the synthesis and accumulation of chalcone 2'-glucoside. In several of the carnation cultivars that bear yellow flowers variegated with white flecks and sectors, both the chalcone isomerase (CHI) and DFR genes are disrupted by dTdic1.

  10. Determinação da estrutura cristalográfica por difração de raios-x da enzima glicose 6-fosfato isomerase humana

    OpenAIRE

    2001-01-01

    O trabalho realizado como parte do programa de mestrado em física aplicada sub-área biomolecular, teve como objeto de estudo a enzima glicose-6-fosfato isomerase de humanas (PGI-hum). Este trabalho envolveu principalmente três áreas de estudos: biologia molecular, bioquímica e cristalografia. A parte de biologia molecular refere-se a sub-clonagem do gene da PGI-hum a partir de uma biblioteca de cDNA de cérebro de feto humano - capítulo 2 - e a expressão deste gene em bactérias Escherichia col...

  11. Pin1 and neurodegeneration: a new player for prion disorders?

    Directory of Open Access Journals (Sweden)

    Elisa Isopi

    2015-07-01

    Full Text Available Pin1 is a peptidyl-prolyl isomerase that catalyzes the cis/trans conversion of phosphorylated proteins at serine or threonine residues which precede a proline. The peptidyl-prolyl isomerization induces a conformational change of the proteins involved in cell signaling process. Pin1 dysregulation has been associated with some neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and Huntington's disease. Proline-directed phosphorylation is a common regulator of these pathologies and a recent work showed that it is also involved in prion disorders. In fact, prion protein phosphorylation at the Ser-43-Pro motif induces prion protein conversion into a disease-associated form. Furthermore, phosphorylation at Ser-43-Pro has been observed to increase in the cerebral spinal fluid of sporadic Creutzfeldt-Jakob Disease patients. These findings provide new insights into the pathogenesis of prion disorders, suggesting Pin1 as a potential new player in the disease. In this paper, we review the mechanisms underlying Pin1 involvement in the aforementioned neurodegenerative pathologies focusing on the potential role of Pin1 in prion disorders.

  12. Proteomic analysis of Oesophagostomum dentatum (Nematoda during larval transition, and the effects of hydrolase inhibitors on development.

    Directory of Open Access Journals (Sweden)

    Martina Ondrovics

    Full Text Available In this study, in vitro drug testing was combined with proteomic and bioinformatic analyses to identify and characterize proteins involved in larval development of Oesophagostomum dentatum, an economically important parasitic nematode. Four hydrolase inhibitors ο-phenanthroline, sodium fluoride, iodoacetamide and 1,2-epoxy-3-(pnitrophenoxy-propane (EPNP significantly inhibited (≥90% larval development. Comparison of the proteomic profiles of the development-inhibited larvae with those of uninhibited control larvae using two-dimensional gel electrophoresis, and subsequent MALDI-TOF mass spectrometric analysis identified a down-regulation of 12 proteins inferred to be involved in various larval developmental processes, including post-embryonic development and growth. Furthermore, three proteins (i.e. intermediate filament protein B, tropomyosin and peptidyl-prolyl cis-trans isomerase inferred to be involved in the moulting process were down-regulated in moulting- and development-inhibited O. dentatum larvae. This first proteomic map of O. dentatum larvae provides insights in the protein profile of larval development in this parasitic nematode, and significantly improves our understanding of the fundamental biology of its development. The results and the approach used might assist in developing new interventions against parasitic nematodes by blocking or disrupting their key biological pathways.

  13. The sexually linked Mpi locus is presumably involved in imidothiazole resistance in Oesophagostomum dentatum parasites.

    Science.gov (United States)

    Snábel, V; DeMeeŵs, T; Várady, M; Nansen, P; Bjørn, H; Corba, J

    2000-06-01

    Information about genetic changes during the selection process could indicate mechanisms underlying the spread of resistance to anthelmintic drugs. For clarification of the role of the Mpi locus encoding mannose-phosphate isomerase enzyme in determining resistance, genotyping of Oesophagostomum dentatum strains was performed using an isoelectrofocusing technique. In levamisole- and pyrantel-selected strains the allele associated with resistance has probably been found. Significant values for genetic differentiation between treated and untreated strains of common origin were recorded by F(st) indices (theta = 0.078; P = 0.0008). The specific genomic makeup of a flubendazole-resistant strain, which did not correlate with that of the remaining isolates, might be ascribed to a different action of the anthelmintic or different environmental conditions under which resistance against this drug arose. The absence of heterozygotes in male populations indicated an XX/X0 system of sex determination for the Mpi locus, thus providing a greater potential for the development of resistance. A possible involvement of alleles linked with mannose-phosphate isomerase in alterations of membrane receptors that can be associated with resistance against imidothiazole-based drugs is discussed.

  14. Xylose isomerase overexpression along with engineering of the pentose phosphate pathway and evolutionary engineering enable rapid xylose utilization and ethanol production by Saccharomyces cerevisiae.

    Science.gov (United States)

    Zhou, Hang; Cheng, Jing-Sheng; Wang, Benjamin L; Fink, Gerald R; Stephanopoulos, Gregory

    2012-11-01

    Xylose is the main pentose and second most abundant sugar in lignocellulosic feedstocks. To improve xylose utilization, necessary for the cost-effective bioconversion of lignocellulose, several metabolic engineering approaches have been employed in the yeast Saccharomyces cerevisiae. In this study, we describe the rational metabolic engineering of a S. cerevisiae strain, including overexpression of the Piromyces xylose isomerase gene (XYLA), Pichia stipitis xylulose kinase (XYL3) and genes of the non-oxidative pentose phosphate pathway (PPP). This engineered strain (H131-A3) was used to initialize a three-stage process of evolutionary engineering, through first aerobic and anaerobic sequential batch cultivation followed by growth in a xylose-limited chemostat. The evolved strain H131-A3-AL(CS) displayed significantly increased anaerobic growth rate (0.203±0.006 h⁻¹) and xylose consumption rate (1.866 g g⁻¹ h⁻¹) along with high ethanol conversion yield (0.41 g/g). These figures exceed by a significant margin any other performance metrics on xylose utilization and ethanol production by S. cerevisiae reported to-date. Further inverse metabolic engineering based on functional complementation suggested that efficient xylose assimilation is attributed, in part, to the elevated expression level of xylose isomerase, which was accomplished through the multiple-copy integration of XYLA in the chromosome of the evolved strain.

  15. Raft-dependent endocytosis of autocrine motility factor/phosphoglucose isomerase: a potential drug delivery route for tumor cells.

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    Liliana D Kojic

    Full Text Available BACKGROUND: Autocrine motility factor/phosphoglucose isomerase (AMF/PGI is the extracellular ligand for the gp78/AMFR receptor overexpressed in a variety of human cancers. We showed previously that raft-dependent internalization of AMF/PGI is elevated in metastatic MDA-435 cells, but not metastatic, caveolin-1-expressing MDA-231 cells, relative to non-metastatic MCF7 and dysplastic MCF10A cells suggesting that it might represent a tumor cell-specific endocytic pathway. METHODOLOGY/PRINCIPAL FINDINGS: Similarly, using flow cytometry, we demonstrate that raft-dependent endocytosis of AMF/PGI is increased in metastatic HT29 cancer cells expressing low levels of caveolin-1 relative to metastatic, caveolin-1-expressing, HCT116 colon cells and non-metastatic Caco-2 cells. Therefore, we exploited the raft-dependent internalization of AMF/PGI as a potential tumor-cell specific targeting mechanism. We synthesized an AMF/PGI-paclitaxel conjugate and found it to be as efficient as free paclitaxel in inducing cytotoxicity and apoptosis in tumor cells that readily internalize AMF/PGI compared to tumor cells that poorly internalize AMF/PGI. Murine K1735-M1 and B16-F1 melanoma cells internalize FITC-conjugated AMF/PGI and are acutely sensitive to AMF/PGI-paclitaxel mediated cytotoxicity in vitro. Moreover, following in vivo intratumoral injection, FITC-conjugated AMF/PGI is internalized in K1735-M1 tumors. Intratumoral injection of AMF/PGI-paclitaxel induced significantly higher tumor regression compared to free paclitaxel, even in B16-F1 cells, known to be resistant to taxol treatment. Treatment with AMF/PGI-paclitaxel significantly prolonged the median survival time of tumor bearing mice. Free AMF/PGI exhibited a pro-survival role, reducing the cytotoxic effect of both AMF/PGI-paclitaxel and free paclitaxel suggesting that AMF/PGI-paclitaxel targets a pathway associated with resistance to chemotherapeutic agents. AMF/PGI-FITC uptake by normal murine spleen

  16. Overexpression of an isopentenyl diphosphate isomerase gene to enhance trans-polyisoprene production in Eucommia ulmoides Oliver

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    Chen Ren

    2012-10-01

    Full Text Available Abstract Background Natural rubber produced by plants, known as polyisoprene, is the most widely used isoprenoid polymer. Plant polyisoprenes can be classified into two types; cis-polyisoprene and trans-polyisoprene, depending on the type of polymerization of the isoprene unit. More than 2000 species of higher plants produce latex consisting of cis-polyisoprene. Hevea brasiliensis (rubber tree produces cis-polyisoprene, and is the key source of commercial rubber. In contrast, relatively few plant species produce trans-polyisoprene. Currently, trans-polyisoprene is mainly produced synthetically, and no plant species is used for its commercial production. Results To develop a plant-based system suitable for large-scale production of trans-polyisoprene, we selected a trans-polyisoprene-producing plant, Eucommia ulmoides Oliver, as the target for genetic transformation. A full-length cDNA (designated as EuIPI, Accession No. AB041629 encoding isopentenyl diphosphate isomerase (IPI was isolated from E. ulmoides. EuIPI consisted of 1028 bp with a 675-bp open reading frame encoding a protein with 224 amino acid residues. EuIPI shared high identity with other plant IPIs, and the recombinant protein expressed in Escherichia coli showed IPI enzymatic activity in vitro. EuIPI was introduced into E. ulmoides via Agrobacterium-mediated transformation. Transgenic lines of E. ulmoides overexpressing EuIPI showed increased EuIPI expression (up to 19-fold that of the wild-type and a 3- to 4-fold increase in the total content of trans-polyisoprenes, compared with the wild-type (non-transgenic root line control. Conclusions Increasing the expression level of EuIPI by overexpression increased accumulation of trans-polyisoprenes in transgenic E. ulmoides. IPI catalyzes the conversion of isopentenyl diphosphate to its highly electrophilic isomer, dimethylallyl diphosphate, which is the first step in the biosynthesis of all isoprenoids, including polyisoprene. Our

  17. Cyclophilin and Viruses: Cyclophilin as a Cofactor for Viral Infection and Possible Anti-Viral Target

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    Koichi Watashi

    2007-01-01

    Full Text Available Cyclophilin (CyP is a peptidyl prolyl cis/trans isomerase, catalyzing the cis-trans isomerization of proline residues in proteins. CyP plays key roles in several different aspects of cellular physiology including the immune response, transcription, mitochondrial function, cell death, and chemotaxis. In addition to these cellular events, a number of reports demonstrated that CyP plays a critical role in the life cycle of viruses, especially human immunodefi ciency virus (HIV and hepatitis C virus (HCV. These two viruses are signifi cant causes of morbidity and mortality worldwide, but current therapies are often insufficient. CyP may provide a novel therapeutic target for the management and/or cure of these diseases, in particular HCV.

  18. FKBP immunophilins and Alzheimer's disease: A chaperoned affair

    Indian Academy of Sciences (India)

    Weihuan Cao; Mary Konsolaki

    2011-08-01

    The FK506-binding protein (FKBP) family of immunophilins consists of proteins with a variety of protein–protein interaction domains and versatile cellular functions. Analysis of the functions of immunophilins has been the focus of studies in recent years and has led to the identification of various molecular pathways in which FKBPs play an active role. All FKBPs contain a domain with prolyl cis/trans isomerase (PPIase) activity. Binding of the immunosuppressant molecule FK506 to this domain inhibits their PPIase activity while mediating immune suppression through inhibition of calcineurin. The larger members, FKBP51 and FKBP52, interact with Hsp90 and exhibit chaperone activity that is shown to regulate steroid hormone signalling. From these studies it is clear that FKBP proteins are expressed ubiquitously but show relatively high levels of expression in the nervous system. Consistent with this expression, FKBPs have been implicated with both neuroprotection and neurodegeneration. This review will focus on recent studies involving FKBP immunophilins in Alzheimer’s-disease-related pathways.

  19. Dipentamethylene thiuram monosulfide is a novel inhibitor of Pin1.

    Science.gov (United States)

    Tatara, Yota; Lin, Yi-Chin; Bamba, Yoshimasa; Mori, Tadashi; Uchida, Takafumi

    2009-07-03

    Pin1 is involved in eukaryotic cell proliferation by changing the structure and function of phosphorylated proteins. PiB, the Pin1 specific inhibitor, blocks cancer cell proliferation. However, low solubility of PiB in DMSO has limited studies of its effectiveness. We screened for additional Pin1 inhibitors and identified the DMSO-soluble compound dipentamethylene thiuram monosulfide (DTM) that inhibits Pin1 activity with an EC50 value of 4.1 microM. Molecular modeling and enzyme kinetic analysis indicated that DTM competitively inhibits Pin1 activity, with a K(i) value of 0.05 microM. The K(D) value of DTM with Pin1 was determined to be 0.06 microM by SPR technology. Moreover, DTM specifically inhibited peptidyl-prolyl cis/trans isomerase activity in HeLa cells. FACS analysis showed that DTM induced G0 arrest of the HCT116 cells. Our results suggest that DTM has the potential to guide the development of novel antifungal and/or anticancer drugs.

  20. Human cyclophilin 33 (hCyP33) in T-cell binds specifically to poly(A)~+RNA (mRNA)

    Institute of Scientific and Technical Information of China (English)

    张万起; 袁直; 宓怀风; 元云峰; 何炳林; 王亦农

    2002-01-01

    Human cyclophilin 33 (hCyP33), found in 1996, consists of an RNA-binding domain in N-terminus, a cyclophilin domain in C-terminus and a connected part between the two domains. RNA-binding proteins concern functions, such as splicing, modification and transport, after transcription in eukaryotic cells. Cyclophilins (CyPs) possess enzymatic activity, namely peptidyl-proryl cis-trans isomerase (PPlase). They are involved in folding, transport and interaction of proteins. Cyclosporin A (CsA), an immunosuppressant used by organ transplantation, binds to CyPs and suppresses their enzymatic activity. However, up to now it is unknown that which cellular and physiological roles hCyP33, which possesses the above-mentioned both functions, plays. In this paper the binding specificity of hCyP33 to different cellular RNA is investigated by means of ion-exchange chromatography and affinity adsorption. The results show that it binds specifically to poly(A) tailed mRNA, namely poly(A)+RNA.

  1. In silico analysis of the cyclophilin repertoire of apicomplexan parasites

    Directory of Open Access Journals (Sweden)

    von Samson-Himmelstjerna Georg

    2009-06-01

    Full Text Available Abstract Background Cyclophilins (Cyps are peptidyl cis/trans isomerases implicated in diverse processes such as protein folding, signal transduction, and RNA processing. They are also candidate drug targets, in particular for the immunosuppressant cyclosporine A. In addition, cyclosporine is known to exhibit anti-parasitic effects on a wide range of organisms including several apicomplexa. In order to obtain new non-immunosuppressive drugs targeting apicomplexan cyclophilins, a profound knowledge of the cyclophilin repertoire of this phylum would be necessary. Results BLAST and maximum likelihood analyses identified 16 different cyclophilin subfamilies within the genomes of Cryptosporidium hominis, Toxoplasma gondii, Plasmodium falciparum, Theileria annulata, Theileria parva, and Babesia bovis. In addition to good statistical support from the phylogenetic analysis, these subfamilies are also confirmed by comparison of cyclophilin domain architecture. Within an individual genome, the number of different Cyp genes that could be deduced varies between 7–9 for Cryptosporidia and 14 for T. gondii. Many of the putative apicomplexan cyclophilins are predicted to be nuclear proteins, most of them presumably involved in RNA processing. Conclusion The genomes of apicomplexa harbor a cyclophilin repertoire that is at least as complex as that of most fungi. The identification of Cyp subfamilies that are specific for lower eukaryotes, apicomplexa, or even the genus Plasmodium is of particular interest since these subfamilies are not present in host cells and might therefore represent attractive drug targets.

  2. Soybean cyclophilin GmCYP1 interacts with an isoflavonoid regulator GmMYB176

    Science.gov (United States)

    Mainali, Hemanta Raj; Vadivel, Arun Kumaran Anguraj; Li, Xuyan; Gijzen, Mark; Dhaubhadel, Sangeeta

    2017-01-01

    Cyclophilins (CYPs) belong to the immunophilin superfamily with peptidyl-prolyl cis-trans isomerase (PPIase) activity. They catalyze the interconversion of the cis- and trans-rotamers of the peptidyl-prolyl amide bond of peptides. A yeast-two-hybrid screening using the isoflavonoid regulator GmMYB176 as bait identified GmCYP1 as one of the interacting proteins in soybean embryos. GmCYP1 localizes both in the nucleus and cytoplasm, and interacts in planta with GmMYB176, in the nucleus, and with SGF14l (a soybean 14-3-3 protein) in the nucleus and the cytoplasm. GmCYP1 contains a single cyclophilin-like domain and displays a high sequence identity with other plant CYPs that are known to have stress-specific function. Tissue-specific expression of GmCYP1 revealed higher expression in developing seeds compared to other vegetative tissues, suggesting their seed-specific role. Furthermore, GmCYP1 transcript level was reduced in response to stress. Since isoflavonoids are involved in plant stress resistance against biotic and abiotic factors, the interaction of GmCYP1 with the isoflavonoid regulators GmMYB176 and 14-3-3 protein suggests its role in defense in soybean. PMID:28074922

  3. Strigolactones, a novel carotenoid-derived plant hormone

    KAUST Repository

    Al-Babili, Salim

    2015-04-29

    Strigolactones (SLs) are carotenoid-derived plant hormones and signaling molecules. When released into the soil, SLs indicate the presence of a host to symbiotic fungi and root parasitic plants. In planta, they regulate several developmental processes that adapt plant architecture to nutrient availability. Highly branched/tillered mutants in Arabidopsis, pea, and rice have enabled the identification of four SL biosynthetic enzymes: a cis/trans-carotene isomerase, two carotenoid cleavage dioxygenases, and a cytochrome P450 (MAX1). In vitro and in vivo enzyme assays and analysis of mutants have shown that the pathway involves a combination of new reactions leading to carlactone, which is converted by a rice MAX1 homolog into an SL parent molecule with a tricyclic lactone moiety. In this review, we focus on SL biosynthesis, describe the hormonal and environmental factors that determine this process, and discuss SL transport and downstream signaling as well as the role of SLs in regulating plant development. ©2015 by Annual Reviews. All rights reserved.

  4. Dipentamethylene thiuram monosulfide is a novel inhibitor of Pin1

    Energy Technology Data Exchange (ETDEWEB)

    Tatara, Yota; Lin, Yi-Chin; Bamba, Yoshimasa; Mori, Tadashi [Molecular Enzymology, Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University, 1-1 Amamiya, Tsutsumidori, Aoba, Sendai, Miyagi 981-8555 (Japan); Uchida, Takafumi, E-mail: uchidat@biochem.tohoku.ac.jp [Molecular Enzymology, Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University, 1-1 Amamiya, Tsutsumidori, Aoba, Sendai, Miyagi 981-8555 (Japan)

    2009-07-03

    Pin1 is involved in eukaryotic cell proliferation by changing the structure and function of phosphorylated proteins. PiB, the Pin1 specific inhibitor, blocks cancer cell proliferation. However, low solubility of PiB in DMSO has limited studies of its effectiveness. We screened for additional Pin1 inhibitors and identified the DMSO-soluble compound dipentamethylene thiuram monosulfide (DTM) that inhibits Pin1 activity with an EC50 value of 4.1 {mu}M. Molecular modeling and enzyme kinetic analysis indicated that DTM competitively inhibits Pin1 activity, with a K{sub i} value of 0.05 {mu}M. The K{sub D} value of DTM with Pin1 was determined to be 0.06 {mu}M by SPR technology. Moreover, DTM specifically inhibited peptidyl-prolyl cis/trans isomerase activity in HeLa cells. FACS analysis showed that DTM induced G0 arrest of the HCT116 cells. Our results suggest that DTM has the potential to guide the development of novel antifungal and/or anticancer drugs.

  5. Human cyclophilin 33 (hCyP33) in T-cell binds specifically to poly(A)+RNA (mRNA)

    Institute of Scientific and Technical Information of China (English)

    张万起; 元云峰; 王亦农; 袁直; 宓怀风; 何炳林

    2002-01-01

    Human cyclophilin 33 (hCyP33), found in 1996, consists of an RNA-binding domain in N-terminus, a cyclophilin domain in C-terminus and a connected part between the two domains. RNA-binding proteins concern functions, such as splicing, modification and transport, after transcription in eukaryotic cells. Cyclophilins (CyPs) possess enzymatic activity, namely peptidyl-proryl cis-trans isomerase (PPIase). They are involved in folding, transport and interaction of proteins. Cyclosporin A (CsA), an immunosuppressant used by organ transplantation, binds to CyPs and suppresses their enzymatic activity. However, up to now it is unknown that which cellular and physiological roles hCyP33, which possesses the above-mentioned both functions, plays. In this paper the binding specificity of hCyP33 to different cellular RNA is investigated by means of ion-exchange chromatography and affinity adsorption. The results show that it binds specifically to poly(A) tailed mRNA, namely poly(A)+RNA.

  6. Nuclear translocation of β-catenin during mesenchymal stem cells differentiation into hepatocytes is associated with a tumoral phenotype.

    Science.gov (United States)

    Herencia, Carmen; Martínez-Moreno, Julio M; Herrera, Concepción; Corrales, Fernando; Santiago-Mora, Raquel; Espejo, Isabel; Barco, Monserrat; Almadén, Yolanda; de la Mata, Manuel; Rodríguez-Ariza, Antonio; Muñoz-Castañeda, Juan R

    2012-01-01

    Wnt/β-catenin pathway controls biochemical processes related to cell differentiation. In committed cells the alteration of this pathway has been associated with tumors as hepatocellular carcinoma or hepatoblastoma. The present study evaluated the role of Wnt/β-catenin activation during human mesenchymal stem cells differentiation into hepatocytes. The differentiation to hepatocytes was achieved by the addition of two different conditioned media. In one of them, β-catenin nuclear translocation, up-regulation of genes related to the Wnt/β-catenin pathway, such as Lrp5 and Fzd3, as well as the oncogenes c-myc and p53 were observed. While in the other protocol there was a Wnt/β-catenin inactivation. Hepatocytes with nuclear translocation of β-catenin also had abnormal cellular proliferation, and expressed membrane proteins involved in hepatocellular carcinoma, metastatic behavior and cancer stem cells. Further, these cells had also increased auto-renewal capability as shown in spheroids formation assay. Comparison of both differentiation protocols by 2D-DIGE proteomic analysis revealed differential expression of 11 proteins with altered expression in hepatocellular carcinoma. Cathepsin B and D, adenine phosphoribosyltransferase, triosephosphate isomerase, inorganic pyrophosphatase, peptidyl-prolyl cis-trans isomerase A or lactate dehydrogenase β-chain were up-regulated only with the protocol associated with Wnt signaling activation while other proteins involved in tumor suppression, such as transgelin or tropomyosin β-chain were down-regulated in this protocol. In conclusion, our results suggest that activation of the Wnt/β-catenin pathway during human mesenchymal stem cells differentiation into hepatocytes is associated with a tumoral phenotype.

  7. Computational Identification of Amino-Acid Mutations that Further Improve the Activity of a Chalcone–Flavonone Isomerase from Glycine max

    Science.gov (United States)

    Yuan, Hui; Wu, Jiaqi; Wang, Xiaoqiang; Chen, Jiakuan; Zhong, Yang; Huang, Qiang; Nan, Peng

    2017-01-01

    Protein design for improving enzymatic activity remains a challenge in biochemistry, especially to identify target amino-acid sites for mutagenesis and to design beneficial mutations for those sites. Here, we employ a computational approach that combines multiple sequence alignment, positive selection detection, and molecular docking to identify and design beneficial amino-acid mutations that further improve the intramolecular-cyclization activity of a chalcone–flavonone isomerase from Glycine max (GmCHI). By this approach, two GmCHI mutants with higher activities were predicted and verified. The results demonstrate that this approach could determine the beneficial amino-acid mutations for improving the enzymatic activity, and may find more applications in engineering of enzymes. PMID:28286513

  8. Characterization of a recombinant L-fucose isomerase from Caldicellulosiruptor saccharolyticus that isomerizes L-fucose, D-arabinose, D-altrose, and L-galactose.

    Science.gov (United States)

    Ju, Yo-Han; Oh, Deok-Kun

    2010-02-01

    A recombinant L-fucose isomerase from Caldicellulosiruptor saccharolyticus was purified as a single 68 kDa band with an activity of 76 U mg(-1). The molecular mass of the native enzyme was 204 kDa as a trimer. The maximum activity for L-fucose isomerization was at pH 7 and 75 degrees C in the presence of 1 mM Mn(2+). Its half-life at 70 degrees C was 6.1 h. For aldose substrates, the enzyme displayed activity in decreasing order for L-fucose, with a k (cat) of 11,910 min(-1) and a K (m) of 140 mM, D-arabinose, D-altrose, and L-galactose. These aldoses were converted to the ketoses L-fuculose, D-ribulose, D-psicose, and L-tagatose, respectively, with 24, 24, 85, 55% conversion yields after 3 h.

  9. The role of the NAD-dependent glutamate dehydrogenase in restoring growth on glucose of a Saccharomyces cerevisiae phosphoglucose isomerase mutant.

    Science.gov (United States)

    Boles, E; Lehnert, W; Zimmermann, F K

    1993-10-01

    Phosphoglucose isomerase pgi1-deletion mutants of Saccharomyces cerevisiae cannot grow on glucose as the sole carbon source and are even inhibited by glucose. These growth defects could be suppressed by an over-expression on a multi-copy plasmid of the structural gene GDH2 coding for the NAD-dependent glutamate dehydrogenase. GDH2 codes for a protein with 1092 amino acids which is located on chromosome XII and shows high sequence similarity to the Neurospora crassa NAD-glutamate dehydrogenase. Suppression of the pgi1 deletion by over-expression of GDH2 was abolished in strains with a deletion of the glucose-6-phosphate dehydrogenase gene ZWF1 or gene GDH1 coding for the NADPH-dependent glutamate dehydrogenase. Moreover, this suppression required functional mitochondria. It is proposed that the growth defect of pgi1 deletion mutants on glucose is due to a rapid depletion of NADP which is needed as a cofactor in the oxidative reactions of the pentose phosphate pathway. Over-expression of the NAD-dependent glutamate dehydrogenase leads to a very efficient conversion of glutamate with NADH generation to 2-oxoglutarate which can be converted back to glutamate by the NADPH-dependent glutamate dehydrogenase with the consumption of NADPH. Consequently, over-expression of the NAD-dependent glutamate dehydrogenase causes a substrate cycling between 2-oxoglutarate and glutamate which restores NADP from NADPH through the coupled conversion of NAD to NADH which can be oxidized in the mitochondria. Furthermore, the requirement for an increase in NADPH consumption for the suppression of the phosphoglucose isomerase defect could be met by addition of oxidizing agents which are known to reduce the level of NADPH.

  10. Research Progress of Glucose Phosphate Isomerase%葡萄糖-6-磷酸异构酶研究进展

    Institute of Scientific and Technical Information of China (English)

    韩龙; 杜翠红

    2012-01-01

    葡萄糖-6-磷酸异构酶(Glucose phosphate isomerase,GPI)是一类多功能蛋白质,在糖代谢的糖酵解中催化葡萄糖-6-磷酸和果糖-6-磷酸之间的可逆反应,同时它还具有其他重要生理生化功能.人体GPI的缺乏可导致非球型血红细胞贫血症以及神经功能的紊乱,GPI还具有细胞因子的活性,并与类风湿关节炎的发生有密切关系.此外在渔业研究方面有研究表明,鱼体GPI具有特异抑制鱼体自身肌原纤维结合型丝氨酸蛋白酶活性的功能等.文章GPI的功能、空间结构、酶学性质及克隆表达等方面简要介绍了目前研究的一些情况.%Glucose phosphate isomerase is a multifunctional protein. It catalyzes the reversible isomerization between glucose-6-phosphate and fructose- 6-phosphate as a part of the glycolytic pathway. In addition, it has some other biological functions. For example,the lack of GPI from human can result in nonspherocytic red blood cell hemolytic anemia and nervous disorder. GPI also has cytokine activity and is closely related to the incidence of rheumatoid arthritis. In fisheries research, GPI also has been found to have specific inhibitory activity toward myofibril-bound serine proteinases in fish. This article briefly introduces the overview of the GPI research in the side of the functional roles of GPI,the spatial structure,enzymatic properties,cloning and expression.

  11. A method for the production of D-tagatose using a recombinant Pichia pastoris strain secreting β-D-galactosidase from Arthrobacter chlorophenolicus and a recombinant L-arabinose isomerase from Arthrobacter sp. 22c

    Directory of Open Access Journals (Sweden)

    Wanarska Marta

    2012-08-01

    Full Text Available Abstract Background D-Tagatose is a natural monosaccharide which can be used as a low-calorie sugar substitute in food, beverages and pharmaceutical products. It is also currently being tested as an anti-diabetic and obesity control drug. D-Tagatose is a rare sugar, but it can be manufactured by the chemical or enzymatic isomerization of D-galactose obtained by a β-D-galactosidase-catalyzed hydrolysis of milk sugar lactose and the separation of D-glucose and D-galactose. L-Arabinose isomerases catalyze in vitro the conversion of D-galactose to D-tagatose and are the most promising enzymes for the large-scale production of D-tagatose. Results In this study, the araA gene from psychrotolerant Antarctic bacterium Arthrobacter sp. 22c was isolated, cloned and expressed in Escherichia coli. The active form of recombinant Arthrobacter sp. 22c L-arabinose isomerase consists of six subunits with a combined molecular weight of approximately 335 kDa. The maximum activity of this enzyme towards D-galactose was determined as occurring at 52°C; however, it exhibited over 60% of maximum activity at 30°C. The recombinant Arthrobacter sp. 22c L-arabinose isomerase was optimally active at a broad pH range of 5 to 9. This enzyme is not dependent on divalent metal ions, since it was only marginally activated by Mg2+, Mn2+ or Ca2+ and slightly inhibited by Co2+ or Ni2+. The bioconversion yield of D-galactose to D-tagatose by the purified L-arabinose isomerase reached 30% after 36 h at 50°C. In this study, a recombinant Pichia pastoris yeast strain secreting β-D-galactosidase Arthrobacter chlorophenolicus was also constructed. During cultivation of this strain in a whey permeate, lactose was hydrolyzed and D-glucose was metabolized, whereas D-galactose was accumulated in the medium. Moreover, cultivation of the P. pastoris strain secreting β-D-galactosidase in a whey permeate supplemented with Arthrobacter sp. 22c L-arabinose isomerase resulted in a 90% yield

  12. Who and What Does Involvement Involve?

    DEFF Research Database (Denmark)

    Hansen, Jeppe Oute; Petersen, Anders; Huniche, Lotte

    2015-01-01

    This article gives an account of aspects of a multi-sited field study of involvement of relatives in Danish psychiatry. By following metaphors of involvement across three sites of the psychiatric systema family site, a clinical site and a policy sitethe first author (J.O.) investigated how...

  13. 固定化葡萄糖异构酶活化条件对其酶活的影响%Influence of activation conditions on the enzyme activity of immobilized glucose isomerase

    Institute of Scientific and Technical Information of China (English)

    胡弢; 周雪艳; 赵国群

    2012-01-01

    Activation was need before immobilized glucose isomerase was used in order to make it have the best catalytic ability.The influences of activation conditions on the enzyme activity of GENSWEETTM IGI-SA immobilized glucose isomerase were studied including concentration of glucose syrup,temperature,pH,activation time and metal ions.The optimal activation condition was as follows:Glucose syrup 60%,temperature 55℃,pH 7.5 and activation time 4h.Under this condition,the enzyme activity of immobilized glucose isomerase was 815U/g,which was 40% higher than one under normal activation condition.Mg2+,Co2+,Mn2+and Zn2+were not necessary to be added into glucose syrup when immobilized glucose isomerase was activated.%固定化葡萄糖异构酶在使用前需先进行活化,从而使酶发挥其最佳催化功效。本文从糖液浓度、温度、pH、活化时间和金属离子五个方面研究了活化条件对GENSWEETTMIGI-SA固定化葡萄糖异构酶酶活的影响。该酶的最适活化条件为:葡萄糖液浓度60%、温度55℃、pH7.5、时间4h。经此条件活化之后,其酶活达815U/g,与常规活化条件相比,酶活提高了40%以上。固定化葡萄糖异构酶活化时不宜加入Mg2+、Co2+、Mn2+和Zn2+。

  14. Prolyl isomerase Pin1 is highly expressed in Her2-positive breast cancer and regulates erbB2 protein stability

    Directory of Open Access Journals (Sweden)

    Lu Kun

    2008-12-01

    Full Text Available Abstract Overexpression of HER-2/Neu occurs in about 25–30% of breast cancer patients and is indicative of poor prognosis. While Her2/Neu overexpression is primarily a result of erbB2 amplification, it has recently been recognized that erbB2 levels are also regulated on the protein level. However, factors that regulate Her2/Neu protein stability are less well understood. The prolyl isomerase Pin1 catalyzes the isomerization of specific pSer/Thr-Pro motifs that have been phosphorylated in response to mitogenic signaling. We have previously reported that Pin1-catalyzed post-phosphorylational modification of signal transduction modulates the oncogenic pathways downstream from c-neu. The goal of this study was to examine the expression of prolyl isomerase Pin1 in human Her2+ breast cancer, and to study if Pin1 affects the expression of Her2/Neu itself. Methods Immunohistochemistry for Her2 and Pin1 were performed on two hundred twenty-three human breast cancers, with 59% of the specimen from primary cancers and 41% from metastatic sites. Pin1 inhibition was achieved using siRNA in Her2+ breast cancer cell lines, and its effects were studied using cell viability assays, immunoblotting and immunofluorescence. Results Sixty-four samples (28.7% stained positive for Her2 (IHC 3+, and 54% (122/223 of all breast cancers stained positive for Pin1. Of the Her2-positive cancers 40 (62.5% were also Pin1-positive, based on strong nuclear or nuclear and cytoplasmic staining. Inhibition of Pin1 via RNAi resulted in significant suppression of Her2-positive tumor cell growth in BT474, SKBR3 and AU565 cells. Pin1 inhibition greatly increased the sensitivity of Her2-positive breast cancer cells to the mTOR inhibitor Rapamycin, while it did not increase their sensitivity to Trastuzumab, suggesting that Pin1 might act on Her2 signaling. We found that Pin1 interacted with the protein complex that contains ubiquitinated erbB2 and that Pin1 inhibition accelerated erbB2

  15. Differential expression of neuroleukin in osseous tissues and its involvement in mineralization during osteoblast differentiation

    Science.gov (United States)

    Zhi, J.; Sommerfeldt, D. W.; Rubin, C. T.; Hadjiargyrou, M.

    2001-01-01

    Osteoblast differentiation is a multistep process that involves critical spatial and temporal regulation of cellular processes marked by the presence of a large number of differentially expressed molecules. To identify key functional molecules, we used differential messenger RNA (mRNA) display and compared RNA populations isolated from the defined transition phases (proliferation, matrix formation, and mineralization) of the MC3T3-E1 osteoblast-like cell line. Using this approach, a complementary DNA (cDNA) fragment was isolated and identified as neuroleukin (NLK), a multifunctional cytokine also known as autocrine motility factor (AMF), phosphoglucose isomerase (PGI; phosphohexose isomerase [PHI]), and maturation factor (MF). Northern analysis showed NLK temporal expression during MC3T3-E1 cell differentiation with a 3.5-fold increase during matrix formation and mineralization. Immunocytochemical studies revealed the presence of NLK in MC3T3-E1 cells as well as in the surrounding matrix, consistent with a secreted molecule. In contrast, the NLK receptor protein was detected primarily on the cell membrane. In subsequent studies, a high level of NLK expression was identified in osteoblasts and superficial articular chondrocytes in bone of 1-, 4-, and 8-month-old normal mice, as well as in fibroblasts, proliferating chondrocytes, and osteoblasts within a fracture callus. However, NLK was not evident in hypertrophic chondrocytes or osteocytes. In addition, treatment of MC3T3 cells with 6-phosphogluconic acid (6PGA; a NLK inhibitor) resulted in diminishing alkaline phosphatase (ALP) activity and mineralization in MC3T3-E1 cells, especially during the matrix formation stage of differentiating cells. Taken together, these data show specific expression of NLK in discrete populations of bone and cartilage cells and suggest a possible role for this secreted protein in bone development and regeneration.

  16. Posttranslocation Chaperone PrsA2 Regulates the Maturation and Secretion of Listeria monocytogenes Proprotein Virulence Factors ▿

    OpenAIRE

    Forster, Brian M.; Zemansky, Jason; Portnoy, Daniel A.; Marquis, Hélène

    2011-01-01

    PrsA2 is a conserved posttranslocation chaperone and a peptidyl prolyl cis-trans isomerase (PPIase) that contributes to the virulence of the Gram-positive intracellular pathogen Listeria monocytogenes. One of the phenotypes associated with a prsA2 mutant is decreased activity of the broad-range phospholipase C (PC-PLC). PC-PLC is made as a proenzyme whose maturation is mediated by a metalloprotease (Mpl). The proforms of PC-PLC and Mpl accumulate at the membrane-cell wall interface until a de...

  17. AcEST: BP919238 [AcEST

    Lifescience Database Archive (English)

    Full Text Available 15|NKTR_MOUSE NK-tumor recognition protein OS=Mus musculu... 34 0.39 sp|Q6FTW5|GC...4 tr|Q7ZYY1|Q7ZYY1_DANRE Peptidyl-prolyl cis-trans isomerase OS=Da... 40 0.044 tr|Q4DVE3|Q4DVE3_TRYCR Mucin-associated surface...0286299 OS=Di... 34 0.39 sp|Q5XG24|RNPS1_XENLA RNA-binding protein with serine-rich domai... 34 0.39 sp|P304

  18. 以Pin1为靶点的肿瘤反义基因治疗研究进展%Progress on Pin1-targeted antisense gene therapy for cancer

    Institute of Scientific and Technical Information of China (English)

    孙环星; 邵荣光

    2007-01-01

    @@ 肽基脯氨酰顺反式异构酶1(peptidyl-prolyl cis/trans isomerase 1,Pin1)是进化上非常保守的肽基脯氨酰异构酶(peptidyl-prolylisomerase,PPIase)家族的成员,属微小菌素蛋白[1],最初报道于1996年,在分离与NIMA(never in mitosis geneA)相互作用的蛋白时得到[2].

  19. Computational insights into the suicide inhibition of Plasmodium falciparum Fk506-binding protein 35.

    Science.gov (United States)

    MacDonald, Corey A; Boyd, Russell J

    2015-08-15

    Malaria is a parasite affecting millions of people worldwide. With the risk of malarial resistance reaching catastrophic levels, novel methods into the inhibition of this disease need to be prioritized. The exploitation of active site differences between parasitic and human peptidyl-prolyl cis/trans isomerases can be used for suicide inhibition, effectively poisoning the parasite without affecting the patient. This method of inhibition was explored using Plasmodium falciparum and Homo sapiens Fk506-binding proteins as templates for quantum mechanics/molecular mechanics calculations. Modification of the natural substrate has shown suicide inhibition is a valid approach for novel anti-malarials with little risk for parasitic resistance.

  20. 变异链球菌核糖-5-磷酸异构酶A的表达、纯化与鉴定%Expression, purification and characterization of ribose 5-phosphate isomerase A from Streptococcus mutans

    Institute of Scientific and Technical Information of China (English)

    武文琦; 丛旭珍; 殷爱红; 胡家; 翟方丽; 李慎涛

    2012-01-01

    目的 在大肠杆菌中高效表达变异链球菌核糖-5-磷酸异构酶A( ribose 5-phosphate isomerase A,rpiA ),并对表达产物进行纯化和鉴定.方法 根据GenBank中变异链球菌UA159株基因组rpiA的DNA编码序列,设计PCR引物,扩增变异链球菌核糖-5-磷酸异构酶A的DNA编码序列,将其克隆至pGEX-6p-1载体中,构建重组质粒,将测序正确的重组质粒转化入大肠杆菌BL21 (DE3)中,用异丙基-β-D-硫代吡喃半乳糖苷(isopropyl β-D-1-thiogalactopyranoside,IPTG)诱导表达;对培养温度、IPTG用量、诱导时间等条件进行了优化;用亲和层析、离子交换层析纯化目标蛋白;用SDS-PAGE和质谱对目标蛋白进行鉴定.结果 变异链球菌核糖-5-磷酸异构酶A在大肠杆菌中高效、可溶性表达,经质谱鉴定及SDS-PAGE分析,表达产物为变异链球菌核糖-5-磷酸异构酶A蛋白.经过纯化,得到纯度高达95%的变异链球菌核糖-5-磷酸异构酶A.结论 成功地在大肠杆菌中高效表达了变异链球菌核糖-5-磷酸异构酶A蛋白,并建立了纯化工艺,得到高纯度的重组蛋白,为进一步研究变异链球菌属核糖-5-磷酸异构酶蛋白的生物学活性及功能奠定了基础.%Objective To express, purify and characterize the ribose 5-phosphate isomerase A(rpiA) from Streptococcus muians. Methods A DNA fragment encoding S. mutans ribose 5-phosphate isomerase A was amplified by PCR using the genomic DNA of Streptococcus mutans UA159 as a template. The PCR product was cloned into vector pGEX-6p-l. The construct carrying the coding DNA sequence of rpiA fused with GST was transformed into E. coli BL21 (DE3) , and the fusion protein was expressed by induction with IPTG. The recombinant protein was purified by affinity chromatography and ion exchange chromatography. The purified target protein was identified by SDS-PAGE and MALDI-TOF MS. Results S. mutans ribose 5-phosphate isomerase A was successfully expressed in E. coli in

  1. Co-expression of D-glucose isomerase and D-psicose 3-epimerase: development of an efficient one-step production of D-psicose.

    Science.gov (United States)

    Men, Yan; Zhu, Yueming; Zeng, Yan; Izumori, Ken; Sun, Yuanxia; Ma, Yanhe

    2014-10-01

    D-Psicose has been attracting attention in recent years because of its alimentary activities and is used as an ingredient in a range of foods and dietary supplements. To develop a one-step enzymatic process of D-psicose production, thermoactive D-glucose isomerase and the D-psicose 3-epimerase obtained from Bacillus sp. and Ruminococcus sp., respectively, were successfully co-expressed in Escherichia coli BL21 strain. The substrate of one-step enzymatic process was D-glucose. The co-expression system exhibited maximum activity at 65 °C and pH 7.0. Mg(2+) could enhance the output of D-psicose by 2.32 fold to 1.6 g/L from 10 g/L of D-glucose. When using high-fructose corn syrup (HFCS) as substrate, 135 g/L D-psicose was produced under optimum conditions. The mass ratio of D-glucose, D-fructose, and D-psicose was almost 3.0:2.7:1.0, when the reaction reached equilibrium after an 8h incubation time. This co-expression system approaching to produce D-psicose has potential application in food and beverage products, especially softdrinks.

  2. Ethylene Responses in Rice Roots and Coleoptiles Are Differentially Regulated by a Carotenoid Isomerase-Mediated Abscisic Acid Pathway[OPEN

    Science.gov (United States)

    Yin, Cui-Cui; Ma, Biao; Collinge, Derek Phillip; Pogson, Barry James; He, Si-Jie; Xiong, Qing; Duan, Kai-Xuan; Chen, Hui; Yang, Chao; Lu, Xiang; Wang, Yi-Qin; Zhang, Wan-Ke; Chu, Cheng-Cai; Sun, Xiao-Hong; Fang, Shuang; Chu, Jin-Fang; Lu, Tie-Gang; Chen, Shou-Yi; Zhang, Jin-Song

    2015-01-01

    Ethylene and abscisic acid (ABA) act synergistically or antagonistically to regulate plant growth and development. ABA is derived from the carotenoid biosynthesis pathway. Here, we analyzed the interplay among ethylene, carotenoid biogenesis, and ABA in rice (Oryza sativa) using the rice ethylene response mutant mhz5, which displays a reduced ethylene response in roots but an enhanced ethylene response in coleoptiles. We found that MHZ5 encodes a carotenoid isomerase and that the mutation in mhz5 blocks carotenoid biosynthesis, reduces ABA accumulation, and promotes ethylene production in etiolated seedlings. ABA can largely rescue the ethylene response of the mhz5 mutant. Ethylene induces MHZ5 expression, the production of neoxanthin, an ABA biosynthesis precursor, and ABA accumulation in roots. MHZ5 overexpression results in enhanced ethylene sensitivity in roots and reduced ethylene sensitivity in coleoptiles. Mutation or overexpression of MHZ5 also alters the expression of ethylene-responsive genes. Genetic studies revealed that the MHZ5-mediated ABA pathway acts downstream of ethylene signaling to inhibit root growth. The MHZ5-mediated ABA pathway likely acts upstream but negatively regulates ethylene signaling to control coleoptile growth. Our study reveals novel interactions among ethylene, carotenogenesis, and ABA and provides insight into improvements in agronomic traits and adaptive growth through the manipulation of these pathways in rice. PMID:25841037

  3. Performance of Glutamate Dehydrogenase and Triose Phosphate Isomerase Genes in the Analysis of Genotypic Variability of Isolates of Giardia duodenalis from Livestocks

    Directory of Open Access Journals (Sweden)

    Natália M. N. Fava

    2013-01-01

    Full Text Available Giardia duodenalis is a small intestinal protozoan parasite of several terrestrial vertebrates. This work aims to assess the genotypic variability of Giardia duodenalis isolates from cattle, sheep and pigs in the Southeast of Brazil, by comparing the standard characterization between glutamate dehydrogenase (gdh and triose phosphate isomerase (tpi primers. Fecal samples from the three groups of animals were analyzed using the zinc sulphate centrifugal flotation technique. Out of 59 positive samples, 30 were from cattle, 26 from sheep and 3 from pigs. Cyst pellets were stored and submitted to PCR and nested-PCR reactions with gdh and tpi primers. Fragment amplification of gdh and tpi genes was observed in 25 (42.4% and 36 (61.0% samples, respectively. Regarding the sequencing, 24 sequences were obtained with gdh and 20 with tpi. For both genes, there was a prevalence of E specific species assemblage, although some isolates have been identified as A and B, by the tpi sequencing. This has also shown a larger number of heterogeneous sequences, which have been attribute to mixed infections between assemblages B and E. The largest variability of inter-assemblage associated to the frequency of heterogeneity provided by tpi sequencing reinforces the polymorphic nature of this gene and makes it an excellent target for studies on molecular epidemiology.

  4. Construction of a xylose-metabolizing yeast by genome integration of xylose isomerase gene and investigation of the effect of xylitol on fermentation.

    Science.gov (United States)

    Tanino, Takanori; Hotta, Atsushi; Ito, Tomonori; Ishii, Jun; Yamada, Ryosuke; Hasunuma, Tomohisa; Ogino, Chiaki; Ohmura, Naoto; Ohshima, Takayuki; Kondo, Akihiko

    2010-11-01

    A yeast with the xylose isomerase (XI) pathway was constructed by the multicopy integration of XI overexpression cassettes into the genome of the Saccharomyces cerevisiae MT8-1 strain. The resulting yeast strain successfully produced ethanol from both xylose as the sole carbon source and a mixed sugar, consisting of xylose and glucose, without any adaptation procedure. Ethanol yields in the fermentation from xylose and mixed sugar were 61.9% and 62.2% of the theoretical carbon recovery, respectively. Knockout of GRE3, a gene encoding nonspecific aldose reductase, of the host yeast strain improved the fermentation profile. Not only specific ethanol production rates but also xylose consumption rates was improved more than twice that of xylose-metabolizing yeast with the XI pathway using GRE3 active yeast as the host strain. In addition, it was demonstrated that xylitol in the medium exhibits a concentration-dependent inhibition effect on the ethanol production from xylose with the yeast harboring the XI-based xylose metabolic pathway. From our findings, the combination of XI-pathway integration and GRE3 knockout could be result in a consolidated xylose assimilation pathway and increased ethanol productivity.

  5. Molecular cloning and transgenic characterization of the genes encoding chalcone synthase and chalcone isomerase from the Tibetan herbal plant Mirabilis himalaica.

    Science.gov (United States)

    Lan, Xiaozhong; Quan, Hong; Xia, Xinli; Yin, Weilun; Zheng, Weilie

    2016-05-01

    Mirabilis himalaica is an endangered medicinal plant species in the Tibetan Plateau. The two genes respectively encoding chalcone synthase (MhCHS) and chalcone isomerase (MhCHI) were isolated and characterized from M. himalaica. The sequence analysis revealed that the two genes were similar with their corresponding homologous genes in other plants. The tissue profiles showed that both MhCHS and MhCHI had higher expression levels in roots than in stems and leaves. Transgenic hairy root cultures respectively with overexpressing MhCHS and MhCHI were established. The genomic PCR detection confirmed the authority of transgenic hairy root lines, in which either MhCHS or MhCHI expression levels were much higher than that in non-transgenic hairy root line. Finally, the HPLC detection results demonstrated that the rotenoid contents in MhCHS/MhCHI-transformed hairy root lines were enhanced. This study provided two candidate genes that could be used to genetic engineering rotenoid biosynthesis in M. himalaica and an alternative method to produce rotenoid using transgenic hairy root cultures.

  6. Genetic interactions between the ESS1 prolyl-isomerase and the RSP5 ubiquitin ligase reveal opposing effects on RNA polymerase II function.

    Science.gov (United States)

    Wu, X; Chang, A; Sudol, M; Hanes, S D

    2001-12-01

    Transcription of protein-coding genes by RNA polymerase II (pol II) is a highly coordinated process that requires the stepwise association of distinct protein complexes with the C-terminal domain (CTD) of Rpbl, the largest subunit of RNA pol II. Interaction of these complexes with the CTD might be subject to regulation by proteins such as Ess1 and Rsp5. Ess1, a prolyl-isomerase, binds the CTD and is thought to play a positive role in pol II transcription by generating conformational isomers of the CTD. Rsp5, a ubiquitin ligase, binds the CTD and is thought to play a negative role in transcription by mediating Rpbl ubiquitination and degradation. In this paper, we demonstrate that ESS1 and RSP5 interact genetically and that these interactions occur via RPBI. We show that over-expression of RSP5 enhances the growth defect of ess1ts cells and this effect is reversed by introducing extra copies of RPB1. Over-expression of RSP5 also mimics the sensitivity of ess1ts mutant cells to the toxicity of plasmids carrying dominant-negative CTD mutations, whereas mutations in RSP5 suppress this effect. Using a modified two-hybrid assay, we also demonstrate that Essl and Rsp5 compete directly for binding to the CTD. The results suggest a model in which Essl and Rsp5 act opposingly on pol II function to control the level of pol II available for transcription.

  7. Improvement and characterization of a hyperthermophilic glucose isomerase from Thermoanaerobacter ethanolicus and its application in production of high fructose corn syrup.

    Science.gov (United States)

    Liu, Zhi-Qiang; Zheng, Wei; Huang, Jian-Feng; Jin, Li-Qun; Jia, Dong-Xu; Zhou, Hai-Yan; Xu, Jian-Miao; Liao, Cheng-Jun; Cheng, Xin-Ping; Mao, Bao-Xing; Zheng, Yu-Guo

    2015-08-01

    High fructose corn syrup (HFCS) is an alternative of liquid sweetener to sucrose that is isomerized by commercial glucose isomerase (GI). One-step production of 55 % HFCS by thermostable GI has been drawn more and more attentions. In this study, a new hyperthermophilic GI from Thermoanaerobacter ethanolicus CCSD1 (TEGI) was identified by genome mining, and then a 1317 bp fragment encoding the TEGI was synthesized and expressed in Escherichia coli BL21(DE3). To improve the activity of TEGI, two amino acid residues, Trp139 and Val186, around the active site and substrate-binding pocket based on the structural analysis and molecular docking were selected for site-directed mutagenesis. The specific activity of mutant TEGI-W139F/V186T was 2.3-fold and the value of k cat/K m was 1.86-fold as compared to the wild type TEGI, respectively. Thermostability of mutant TEGI-W139F/V186T at 90 °C for 24 h showed 1.21-fold extension than that of wild type TEGI. During the isomerization of glucose to fructose, the yield of fructose could maintain above 55.4 % by mutant TEGI-W139F/V186T as compared to 53.8 % by wild type TEGI at 90 °C. This study paved foundation for the production of 55 % HFCS using the thermostable TEGI.

  8. Connecting Active-Site Loop Conformations and Catalysis in Triosephosphate Isomerase: Insights from a Rare Variation at Residue 96 in the Plasmodial Enzyme.

    Science.gov (United States)

    Pareek, Vidhi; Samanta, Moumita; Joshi, Niranjan V; Balaram, Hemalatha; Murthy, Mathur R N; Balaram, Padmanabhan

    2016-04-01

    Despite extensive research into triosephosphate isomerases (TIMs), there exists a gap in understanding of the remarkable conjunction between catalytic loop-6 (residues 166-176) movement and the conformational flip of Glu165 (catalytic base) upon substrate binding that primes the active site for efficient catalysis. The overwhelming occurrence of serine at position 96 (98% of the 6277 unique TIM sequences), spatially proximal to E165 and the loop-6 residues, raises questions about its role in catalysis. Notably, Plasmodium falciparum TIM has an extremely rare residue--phenylalanine--at this position whereas, curiously, the mutant F96S was catalytically defective. We have obtained insights into the influence of residue 96 on the loop-6 conformational flip and E165 positioning by combining kinetic and structural studies on the PfTIM F96 mutants F96Y, F96A, F96S/S73A, and F96S/L167V with sequence conservation analysis and comparative analysis of the available apo and holo structures of the enzyme from diverse organisms.

  9. Genetical control and linkage relationships of isozyme markers in sugar beet (B. vulgaris L.) : 1. Isocitrate dehydrogenase, adenylate kinase, phosphoglucomutase, glucose phosphate isomerase and cathodal peroxidase.

    Science.gov (United States)

    Smed, E; Van Geyt, J P; Oleo, M

    1989-07-01

    Five isozyme systems were genetically investigated. The different separation techniques, the developmental expression and the use as marker system in sugar beet genetics and breeding is discussed. Isocitrate dehydrogenase was controlled by two genes. The gene products form inter- as well as intralocus dimers, even with the gene products of the Icd gene in B. procumbens and B. patellaris. Adenylate kinase was controlled by one gene. Three different allelic forms were detected, which were active as monomeric proteins. Glucose phosphate isomerase showed two zones of activity. One zone was polymorphic. Three allelic variants, active as dimers, were found. Phosphoglucomutase also showed two major zones of activity. One zone was polymorphic and coded for monomeric enzymes. Two allelic forms were found in the accessions studied. The cathodal peroxidase system was controlled by two independent genes, of which only one was polymorphic. The gene products are active as monomers. Linkage was found between red hypocotyl color (R) and Icd 2. Pgm 1, Gpi 2, Ak 1 and the Icd 2-R linkage group segregated independently.

  10. [Screening of food-grade microorganisms for biotransformation of D-tagatose and cloning and expression of L-arabinose isomerase].

    Science.gov (United States)

    Men, Yan; Zhu, Yueming; Guan, Yuping; Zhang, Tongcun; Izumori, Ken; Sun, Yuanxia

    2012-05-01

    L-Arabinose isomerase (L-AI) is an intracellular enzyme that catalyzes the reversible isomerization of D-galactose and D-tagatose. Given the widespread use of D-tagatose in the food industry, food-grade microorganisms and the derivation of L-AI for the production of D-tagatose is gaining increased attention. In the current study, food-grade strains from different foods that can convert D-galactose to D-tagatose were screened. According to physiological, biochemical, and 16S rDNA gene analyses, the selected strain was found to share 99% identity with Pediococcus pentosaceus, and was named as Pediococcus pentosaceus PC-5. The araA gene encoding L-AI from Pediococcus pentosaceus PC-5 was cloned and overexpressed in E. coli BL21. The yield of D-tagatose using D-galactose as the substrate catalyzed by the crude enzyme in the presence of Mn2+ was found to be 33% at 40 degrees C.

  11. Kinetic analysis of the mechanism and specificity of protein-disulfide isomerase using fluorescence-quenched peptides

    DEFF Research Database (Denmark)

    Westphal, V; Spetzler, J C; Meldal, M;

    1998-01-01

    to conventional PDI assays involving larger polypeptides, the starting material for this assay is homogenous. It is furthermore simple and highly sensitive (requires less than 0.5 microgram of PDI/assay) and thus opens the possibility for quantitative determination of PDI activity and specificity....

  12. Doctors' involvement in torture

    DEFF Research Database (Denmark)

    Sonntag, Jesper

    2008-01-01

    Doctors from both non-democratic and democratic countries are involved in torture. The majority of doctors involved in torture are doctors at risk. Doctors at risk might compromise their ethical duty towards patients for the following possible reasons: individual factors (such as career, economic...

  13. Structure, expression profile and phylogenetic inference of chalcone isomerase-like genes from the narrow-leafed lupin (Lupinus angustifolius L. genome

    Directory of Open Access Journals (Sweden)

    Łucja ePrzysiecka

    2015-04-01

    Full Text Available Lupins, like other legumes, have a unique biosynthesis scheme of 5-deoxy-type flavonoids and isoflavonoids. A key enzyme in this pathway is chalcone isomerase (CHI, a member of CHI-fold protein family, encompassing subfamilies of CHI1, CHI2, CHI-like (CHIL, and fatty acid-binding (FAP proteins. Here, two Lupinus angustifolius (narrow-leafed lupin CHILs, LangCHIL1 and LangCHIL2, were identified and characterized using DNA fingerprinting, cytogenetic and linkage mapping, sequencing and expression profiling. Clones carrying CHIL sequences were assembled into two contigs. Full gene sequences were obtained from these contigs, and mapped in two L. angustifolius linkage groups by gene-specific markers. Bacterial artificial chromosome fluorescence in situ hybridization approach confirmed the localization of two LangCHIL genes in distinct chromosomes. The expression profiles of both LangCHIL isoforms were very similar. The highest level of transcription was in the roots of the third week of plant growth; thereafter, expression declined. The expression of both LangCHIL genes in leaves and stems was similar and low. Comparative mapping to reference legume genome sequences revealed strong syntenic links; however, LangCHIL2 contig had a much more conserved structure than LangCHIL1. LangCHIL2 is assumed to be an ancestor gene, whereas LangCHIL1 probably appeared as a result of duplication. As both copies are transcriptionally active, questions arise concerning their hypothetical functional divergence. Screening of the narrow-leafed lupin genome and transcriptome with CHI-fold protein sequences, followed by Bayesian inference of phylogeny and cross-genera synteny survey, identified representatives of all but one (CHI1 main subfamilies. They are as follows: two copies of CHI2, FAPa2 and CHIL, and single copies of FAPb and FAPa1. Duplicated genes are remnants of whole genome duplication which is assumed to have occurred after the divergence of Lupinus, Arachis

  14. In Silico Identification of Protein Disulfide Isomerase Gene Families in the De Novo Assembled Transcriptomes of Four Different Species of the Genus Conus.

    Science.gov (United States)

    Figueroa-Montiel, Andrea; Ramos, Marco A; Mares, Rosa E; Dueñas, Salvador; Pimienta, Genaro; Ortiz, Ernesto; Possani, Lourival D; Licea-Navarro, Alexei F

    2016-01-01

    Small peptides isolated from the venom of the marine snails belonging to the genus Conus have been largely studied because of their therapeutic value. These peptides can be classified in two groups. The largest one is composed by peptides rich in disulfide bonds, and referred to as conotoxins. Despite the importance of conotoxins given their pharmacology value, little is known about the protein disulfide isomerase (PDI) enzymes that are required to catalyze their correct folding. To discover the PDIs that may participate in the folding and structural maturation of conotoxins, the transcriptomes of the venom duct of four different species of Conus from the peninsula of Baja California (Mexico) were assembled. Complementary DNA (cDNA) libraries were constructed for each species and sequenced using a Genome Analyzer Illumina platform. The raw RNA-seq data was converted into transcript sequences using Trinity, a de novo assembler that allows the grouping of reads into contigs without a reference genome. An N50 value of 605 was established as a reference for future assemblies of Conus transcriptomes using this software. Transdecoder was used to extract likely coding sequences from Trinity transcripts, and PDI-specific sequence motif "APWCGHCK" was used to capture potential PDIs. An in silico analysis was performed to characterize the group of PDI protein sequences encoded by the duct-transcriptome of each species. The computational approach entailed a structural homology characterization, based on the presence of functional Thioredoxin-like domains. Four different PDI families were characterized, which are constituted by a total of 41 different gene sequences. The sequences had an average of 65% identity with other PDIs. Using MODELLER 9.14, the homology-based three-dimensional structure prediction of a subset of the sequences reported, showed the expected thioredoxin fold which was confirmed by a "simulated annealing" method.

  15. In Silico Identification of Protein Disulfide Isomerase Gene Families in the De Novo Assembled Transcriptomes of Four Different Species of the Genus Conus.

    Directory of Open Access Journals (Sweden)

    Andrea Figueroa-Montiel

    Full Text Available Small peptides isolated from the venom of the marine snails belonging to the genus Conus have been largely studied because of their therapeutic value. These peptides can be classified in two groups. The largest one is composed by peptides rich in disulfide bonds, and referred to as conotoxins. Despite the importance of conotoxins given their pharmacology value, little is known about the protein disulfide isomerase (PDI enzymes that are required to catalyze their correct folding. To discover the PDIs that may participate in the folding and structural maturation of conotoxins, the transcriptomes of the venom duct of four different species of Conus from the peninsula of Baja California (Mexico were assembled. Complementary DNA (cDNA libraries were constructed for each species and sequenced using a Genome Analyzer Illumina platform. The raw RNA-seq data was converted into transcript sequences using Trinity, a de novo assembler that allows the grouping of reads into contigs without a reference genome. An N50 value of 605 was established as a reference for future assemblies of Conus transcriptomes using this software. Transdecoder was used to extract likely coding sequences from Trinity transcripts, and PDI-specific sequence motif "APWCGHCK" was used to capture potential PDIs. An in silico analysis was performed to characterize the group of PDI protein sequences encoded by the duct-transcriptome of each species. The computational approach entailed a structural homology characterization, based on the presence of functional Thioredoxin-like domains. Four different PDI families were characterized, which are constituted by a total of 41 different gene sequences. The sequences had an average of 65% identity with other PDIs. Using MODELLER 9.14, the homology-based three-dimensional structure prediction of a subset of the sequences reported, showed the expected thioredoxin fold which was confirmed by a "simulated annealing" method.

  16. A ribosomal misincorporation of Lys for Arg in human triosephosphate isomerase expressed in Escherichia coli gives rise to two protein populations.

    Directory of Open Access Journals (Sweden)

    Beatriz Aguirre

    Full Text Available We previously observed that human homodimeric triosephosphate isomerase (HsTIM expressed in Escherichia coli and purified to apparent homogeneity exhibits two significantly different thermal transitions. A detailed exploration of the phenomenon showed that the preparations contain two proteins; one has the expected theoretical mass, while the mass of the other is 28 Da lower. The two proteins were separated by size exclusion chromatography in 3 M urea. Both proteins correspond to HsTIM as shown by Tandem Mass Spectrometry (LC/ESI-MS/MS. The two proteins were present in nearly equimolar amounts under certain growth conditions. They were catalytically active, but differed in molecular mass, thermostability, susceptibility to urea and proteinase K. An analysis of the nucleotides in the human TIM gene revealed the presence of six codons that are not commonly used in E. coli. We examined if they were related to the formation of the two proteins. We found that expression of the enzyme in a strain that contains extra copies of genes that encode for tRNAs that frequently limit translation of heterologous proteins (Arg, Ile, Leu, as well as silent mutations of two consecutive rare Arg codons (positions 98 and 99, led to the exclusive production of the more stable protein. Further analysis by LC/ESI-MS/MS showed that the 28 Da mass difference is due to the substitution of a Lys for an Arg residue at position 99. Overall, our work shows that two proteins with different biochemical and biophysical properties that coexist in the same cell environment are translated from the same nucleotide sequence frame.

  17. Prokaryotic soluble overexpression and purification of bioactive human growth hormone by fusion to thioredoxin, maltose binding protein, and protein disulfide isomerase.

    Directory of Open Access Journals (Sweden)

    Minh Tan Nguyen

    Full Text Available Human growth hormone (hGH is synthesized by somatotroph cells of the anterior pituitary gland and induces cell proliferation and growth. This protein has been approved for the treatment of various conditions, including hGH deficiency, chronic renal failure, and Turner syndrome. Efficient production of hGH in Escherichia coli (E. coli has proven difficult because the E. coli-expressed hormone tends to aggregate and form inclusion bodies, resulting in poor solubility. In this study, seven N-terminal fusion partners, hexahistidine (His6, thioredoxin (Trx, glutathione S-transferase (GST, maltose-binding protein (MBP, N-utilization substance protein A (NusA, protein disulfide bond isomerase (PDI, and the b'a' domain of PDI (PDIb'a', were tested for soluble overexpression of codon-optimized hGH in E. coli. We found that MBP and hPDI tags significantly increased the solubility of the hormone. In addition, lowering the expression temperature to 18°C also dramatically increased the solubility of all the fusion proteins. We purified hGH from MBP-, PDIb'a'-, or Trx-tagged hGH expressed at 18°C in E. coli using simple chromatographic techniques and compared the final purity, yield, and activity of hGH to assess the impact of each partner protein. Purified hGH was highly pure on silver-stained gel and contained very low levels of endotoxin. On average, ∼37 mg, ∼12 mg, and ∼7 mg of hGH were obtained from 500 mL-cell cultures of Trx-hGH, MBP-hGH, and PDIb'a'-hGH, respectively. Subsequently, hGH was analyzed using mass spectroscopy to confirm the presence of two intra-molecular disulfide bonds. The bioactivity of purified hGHs was demonstrated using Nb2-11 cell.

  18. Molecular characterization of a thermostable L-fucose isomerase from Dictyoglomus turgidum that isomerizes L-fucose and D-arabinose.

    Science.gov (United States)

    Hong, Seung-Hye; Lim, Yu-Ri; Kim, Yeong-Su; Oh, Deok-Kun

    2012-09-01

    A recombinant thermostable l-fucose isomerase from Dictyoglomus turgidum was purified with a specific activity of 93 U/mg by heat treatment and His-trap affinity chromatography. The native enzyme existed as a 410 kDa hexamer. The maximum activity for l-fucose isomerization was observed at pH 7.0 and 80 °C with a half-life of 5 h in the presence of 1 mM Mn(2+) that was present one molecular per monomer. The isomerization activity of the enzyme with aldose substrates was highest for l-fucose (with a k(cat) of 15,500 min(-1) and a K(m) of 72 mM), followed by d-arabinose, d-altrose, and l-galactose. The 15 putative active-site residues within 5 Å of the substrate l-fucose in the homology model were individually replaced with other amino acids. The analysis of metal-binding capacities of these alanine-substituted variants revealed that Glu349, Asp373, and His539 were metal-binding residues, and His539 was the most influential residue for metal binding. The activities of all variants at 349 and 373 positions except for a dramatically decreased k(cat) of D373A were completely abolished, suggesting that Glu349 and Asp373 were catalytic residues. Alanine substitutions at Val131, Met197, Ile199, Gln314, Ser405, Tyr451, and Asn538 resulted in substantial increases in K(m), suggesting that these amino acids are substrate-binding residues. Alanine substitutions at Arg30, Trp102, Asn404, Phe452, and Trp510 resulted in decreases in k(cat), but had little effect on K(m).

  19. The peptidyl-prolyl isomerase Pin1 up-regulation and proapoptotic function in dopaminergic neurons: relevance to the pathogenesis of Parkinson disease.

    Science.gov (United States)

    Ghosh, Anamitra; Saminathan, Hariharan; Kanthasamy, Arthi; Anantharam, Vellareddy; Jin, Huajun; Sondarva, Gautam; Harischandra, Dilshan S; Qian, Ziqing; Rana, Ajay; Kanthasamy, Anumantha G

    2013-07-26

    Parkinson disease (PD) is a chronic neurodegenerative disease characterized by a slow and progressive degeneration of dopaminergic neurons in substantia nigra. The pathophysiological mechanisms underlying PD remain unclear. Pin1, a major peptidyl-prolyl isomerase, has recently been associated with certain diseases. Notably, Ryo et al. (Ryo, A., Togo, T., Nakai, T., Hirai, A., Nishi, M., Yamaguchi, A., Suzuki, K., Hirayasu, Y., Kobayashi, H., Perrem, K., Liou, Y. C., and Aoki, I. (2006) J. Biol. Chem. 281, 4117-4125) implicated Pin1 in PD pathology. Therefore, we sought to systematically characterize the role of Pin1 in PD using cell culture and animal models. To our surprise we observed a dramatic up-regulation of Pin1 mRNA and protein levels in dopaminergic MN9D neuronal cells treated with the parkinsonian toxicant 1-methyl-4-phenylpyridinium (MPP(+)) as well as in the substantia nigra of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. Notably, a marked expression of Pin1 was also observed in the substantia nigra of human PD brains along with a high co-localization of Pin1 within dopaminergic neurons. In functional studies, siRNA-mediated knockdown of Pin1 almost completely prevented MPP(+)-induced caspase-3 activation and DNA fragmentation, indicating that Pin1 plays a proapoptotic role. Interestingly, multiple pharmacological Pin1 inhibitors, including juglone, attenuated MPP(+)-induced Pin1 up-regulation, α-synuclein aggregation, caspase-3 activation, and cell death. Furthermore, juglone treatment in the MPTP mouse model of PD suppressed Pin1 levels and improved locomotor deficits, dopamine depletion, and nigral dopaminergic neuronal loss. Collectively, our findings demonstrate for the first time that Pin1 is up-regulated in PD and has a pathophysiological role in the nigrostriatal dopaminergic system and suggest that modulation of Pin1 levels may be a useful translational therapeutic strategy in PD.

  20. The adaptive evolution divergence of triosephosphate isomerases between parasitic and free-living flatworms and the discovery of a potential universal target against flatworm parasites.

    Science.gov (United States)

    Chen, Bing; Wen, Jian-Fan

    2011-08-01

    Triosephosphate isomerase (TIM) is an important drug target or vaccine candidate for pathogenetic organisms such as schistosomes. Parasitic and free-living flatworms shared their last common ancestor but diverged from each other for adapting to parasitic and free-living lives afterwards, respectively. Therefore, adaptive evolution divergence must have occurred between them. Here, for the first time, TIMs were identified from three free-living planarian flatworms, namely Dugesia japonica, Dugesia ryukyuensis, and Schmidtea mediterranea. When these were compared with parasitic flatworms and other organisms, the following results were obtained: (1) planarian TIM genes each contain only one intron, while parasitic flatworm genes each contain other four introns, which are usually present in common metazoans, suggesting planarian-specific intron loss must have occurred; (2) planarian TIM protein sequences are more similar to those of vertebrates rather than to their parasitic relatives or other invertebrates. This implies that relatively rapid evolution occurred in parasitic flatworm TIMs; (3) All the investigated parasitic flatworm TIMs contain a unique tripeptide insert (SXD/E), which may imply its insertion importance to the adaptation of parasitic life. Moreover, our homology modeling results showed the insert region was largely surface-exposed and predicted to be of a B cell epitope location. Finally, the insert is located within one of the three regions previously suggested to be promising immunogenic epitopes in Schistosoma mansoni TIM. Therefore, this unique insert might be significant to developing new effective vaccines or specific drugs against all parasitic flatworm diseases such as schistosomiasis and taeniosis/cysticercosis.

  1. Spectroscopic investigation of new water soluble Mn(II)(2) and Mg(II)(2) complexes for the substrate binding models of xylose/glucose isomerases.

    Science.gov (United States)

    Patra, Ayan; Bera, Manindranath

    2014-01-30

    In methanol, the reaction of stoichiometric amounts of Mn(OAc)(2)·4H(2)O and the ligand H(3)hpnbpda [H(3)hpnbpda=N,N'-bis(2-pyridylmethyl)-2-hydroxy-1,3-propanediamine-N,N'-diacetic acid] in the presence of NaOH, afforded a new water soluble dinuclear manganese(II) complex, [Mn2(hpnbpda)(μ-OAc)] (1). Similarly, the reaction of Mg(OAc)(2)·4H(2)O and the ligand H3hpnbpda in the presence of NaOH, in methanol, yielded a new water soluble dinuclear magnesium(II) complex, [Mg2(hpnbpda)(μ-OAc)(H2O)2] (2). DFT calculations have been performed for the structural optimization of complexes 1 and 2. The DFT optimized structure of complex 1 shows that two manganese(II) centers are in a distorted square pyramidal geometry, whereas the DFT optimized structure of complex 2 reveals that two magnesium(II) centers adopt a six-coordinate distorted octahedral geometry. To understand the mode of substrate binding and the mechanistic details of the active site metals in xylose/glucose isomerases (XGI), we have investigated the binding interactions of biologically important monosaccharides d-glucose and d-xylose with complexes 1 and 2, in aqueous alkaline solution by a combined approach of FTIR, UV-vis, fluorescence, and (13)C NMR spectroscopic techniques. Fluorescence spectra show the binding-induced gradual decrease in emission of complexes 1 and 2 accompanied by a significant blue shift upon increasing the concentration of sugar substrates. The binding modes of d-glucose and d-xylose with complex 2 are indicated by their characteristic coordination induced shift (CIS) values in (13)C NMR spectra for C1 and C2 carbon atoms.

  2. The Activity and Localization of 3β-hydroxysteroid Dehydrogenase/Δ5-Δ4 Isomerase and Release of Androstenedione and Progesterone by Uterine Tissues During Early Pregnancy and the Estrous Cycle in Pigs

    OpenAIRE

    Wojciechowicz, Bartosz; KOTWICA, Genowefa; Kolakowska, Justyna; Franczak, Anita

    2012-01-01

    Abstract Steroid hormones are produced by the porcine uterus. We hypothesized that the uterus in pigs possesses active 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD) responsible for progesterone and androstenedione production, that uterine steroids may supplement the amount of steroid hormones produced by embryos and corpus luteum and that these steroids are necessary for maintenance of pregnancy. In this study, we examined 1) endometrial and myometrial expression of 3β-HSD mRNA, 2)...

  3. Protein Disulfide Isomerase Chaperone ERP-57 Decreases Plasma Membrane Expression of the Human GnRH Receptor

    Science.gov (United States)

    Yánez, Rodrigo Ayala; Conn, P. Michael

    2012-01-01

    Retention of misfolded proteins by the endoplasmic reticulum (ER) is a quality control mechanism involving the participation of endogenous chaperones such as calnexin (CANX) which interact and restrict plasma membrane expression of gonadotropin releasing hormone receptor (GnRHR), a G protein coupled receptor. CANX also interacts with ERP-57, a thiol oxidoreductase chaperone present in the ER. CANX along with ERP-57, promotes the formation of disulfide bond bridges in nascent proteins. The human GnRH receptor (hGnRHR) is stabilized by two disulfide bond bridges (Cys14-Cys200 and Cys114-Cys196), that, when broken, its expression at plasma membrane decreases. To determine if the presence of chaperones CANX and ERP-57 exert an influence over membrane routing and second messenger activation, we assessed the effect of various mutants including those with broken bridges (Cys→Ala) along with the wild type hGnRHR. The effect of chaperones on mutants was insignificant, whereas the overexpression of ERP-57 led to a wild type hGnRHR retention which was further enhanced by cotransfection with CANX cDNA disclosing receptor retention by ERP-57 augmented by CANX, suggesting a quality control mechanism. PMID:20029959

  4. Consumer Involvement in Rehabilitation

    Science.gov (United States)

    Daniels, Susan

    1976-01-01

    With the emphasis on consumer involvement in the Rehabilitation Act of 1973, changes in the counseling relationship must occur. This article discusses new interaction patterns for consumer and counselor. (Author)

  5. Clinical studies involving probiotics

    OpenAIRE

    Degnan, Fred H

    2012-01-01

    Researchers from a diverse array of scientific disciplines have focused and continue to focus on opportunities and areas for responsible clinical research involving the possible beneficial health effects of “probiotics.” Investigators and researchers should be aware that not all clinical research involving probiotics reasonably falls within the requirements of the “investigational new drug” (IND) rubric administered and enforced by the US Food and Drug Administration. In determining whether a...

  6. Pancreatic Involvement in Melioidosis

    Directory of Open Access Journals (Sweden)

    Vui Heng Chong

    2010-07-01

    Full Text Available Context Melioidosis is endemic to tropical regions and, despite the common occurrence of intra-abdominal abscesses, pancreatic involvement in melioidosis has not previously been reported. Objective We report our experience with pancreatic melioidosis. Patients All 65 patients treated for melioidosis who had computed tomography (CT scans were identified from prospective databases and were retrospectively reviewed. Main outcome measures A detailed review of cases with pancreas involvement was carried out. Results There were four cases (three males and one female; median age 29.5 years, range: 25-48 years with pancreatic melioidosis, giving a prevalence of 6.2%. All had predisposing conditions (two had poorly controlled diabetes mellitus and two had thalassemia for melioidosis. Fever (100%, anorexia (100%, weight loss (100%, rigor (75% and abdominal pain (75% were the most common symptoms at presentation and the median duration of symptoms before presentation was six weeks (range: 2-8 weeks. All pancreatic abscesses were detected on CT scan. Multiple foci involvement was common (3 to 6 sites: blood (4 patients, liver (3 patients, psoas muscle (2 patients, spleen (2 patients, infected ascites (2 patients and lung (1 patient. Pancreatic involvement ranged from multi-focal micro-abscesses to focal large abscesses and involved all parts of the pancreas (body 100%, head 75% and tail 50%. Associated pancreatic findings included splenic vein thrombosis, peripancreatic inflammation and peripancreatic fat streaking. All the pancreatic abscesses were resolved with antibiotics without requiring pancreatic abscess drainage (including one patient who died from disseminated melioidosis. Conclusion Pancreatic involvement typically occurs as part of multi-organ involvement and commonly manifests as multifoci micro-abscesses. Associated pancreatic abnormalities were also common. All responded to treatment without requiring drainage

  7. [Pulmonary involvements of sarcoidosis].

    Science.gov (United States)

    Ohmichi, M; Hiraga, Y; Hirasawa, M

    1990-01-01

    We reported about intrathoracic changes and prognosis of 686 patients with sarcoidosis diagnosed in our hospital between 1963 and 1988. We evaluated CT findings in 135 patients with sarcoidosis and found pulmonary involvements in 81. We analyzed CT findings according to the classification by Tuengerthal which classified radiographic findings combining ILO classification of pneumoconiosis and characteristic findings of bronchovascular sheath with sarcoidosis. The CT findings were as follows: small opacities (44 out of 81 cases, 54.3%), large opacities (37 cases, 46.7%). Additional findings were as follows: peribronchial marking (42 cases, 51.9%), contraction (17 cases, 21.0%), pleural involvement (9 cases, 11.1%), bulla (5 cases, 6.2%). The characteristic CT findings of serious sarcoidosis were extasis of bronchus, thickening of the bronchial wall, unclearness of vascular shadow, atelectasis and thickening of pleura. Concerning the prognosis of pulmonary involvement, according to age, patients younger than 30 years old at initial diagnosis were better than those of 30 years and over in terms of disappearance of pulmonary involvements. According to stage, patients of stage I and stage II were better than those of stage III. Among the patients we were able to observe chest X-ray findings during five years according to the character of shadow, ill-defined shadow of small opacities and rounded shadows of large opacities had a higher disappearance rate of pulmonary involvements than irregular shadows of large opacities, atelectasis and contraction.

  8. Determinación de cis-/trans-Estilbenos (Resveratrol y Piceido), Triptófano y Metabolitos en productos apícolas por cromatografía de líquidos

    OpenAIRE

    Soto Sarria, María Ernestina

    2012-01-01

    Es de sobra conocido el papel fundamental que las abejas ejercen no solamente en lo que atañe a la producción de la colmena sino también como polinizadores tanto de plantas cultivadas como de especies silvestres, por lo que todo aquello que les afecte negativamente tiene una tremenda repercusión sobre la Agricultura y Medio Ambiente. En la última década se ha observado una drástica reducción en el número de individuos y la pérdida definitiva de numerosas colmenas, situación que en el último a...

  9. 3 Beta-hydroxy-delta 5-steroid dehydrogenase/3-keto-delta 5-steroid isomerase from bovine adrenals: mechanism of inhibition by 3-oxo-4-aza steroids and kinetic mechanism of the dehydrogenase.

    Science.gov (United States)

    Brandt, M; Levy, M A

    1989-01-10

    Several 3-oxo-4-aza steroids (1) have been identified as inhibitors of the 3 beta-hydroxy-delta 5-steroid dehydrogenase/3-keto-delta 5-steroid isomerase catalyzed conversion of pregnenolone to progesterone. By kinetically decoupling the two enzyme activities isolated from bovine adrenal cortex, it has been demonstrated that inhibition by 1 occurs through interference of both activities. A preferred ordered association of substrates to the 3 beta-hydroxy-delta 5-steroid dehydrogenase in which the cofactor binds prior to steroid was determined by isotope exchange at equilibrium. With this result, the dead-end inhibition patterns of 1 with the dehydrogenase were interpreted to originate from a preferred association of inhibitor within an enzyme ternate containing NADH; this proposal is supported by data from multiple inhibition analysis indicating synergistic binding of NADH and 1. Similarly, inhibition of the 3-keto-delta 5-steroid isomerase by the 3-oxo-4-aza steroids was enhanced in the presence of the positive effector NADH. On the basis of pH profiles upon Vm, Vm/Km, and 1/Ki for both enzyme activities, inhibition is proposed to result from the structural similarity of 1 to intermediate states formed upon enzyme catalysis.

  10. Involve physicians in marketing.

    Science.gov (United States)

    Randolph, G T; Baker, K M; Laubach, C A

    1984-01-01

    Many everyday problems in medical group practice can be attacked by a marketing approach. To be successful, however, this kind of approach must have the full support of those involved, especially the physicians, since they are the principal providers of healthcare services. When marketing is presented in a broad context, including elements such as patient mix, population distribution, and research, physicians are more likely to be interested and supportive. The members of Geisinger Medical Center's Department of Cardiovascular Medicine addressed their patient appointment backlog problem with a marketing approach. Their method is chronicled here and serves as a fine example of how physician involvement in marketing can lead to a positive outcome.

  11. The effects of disruption of phosphoglucose isomerase gene on carbon utilisation and cellulase production in Trichoderma reesei Rut-C30

    Directory of Open Access Journals (Sweden)

    Pakula Tiina

    2011-05-01

    Full Text Available Abstract Background Cellulase and hemicellulase genes in the fungus Trichoderma reesei are repressed by glucose and induced by lactose. Regulation of the cellulase genes is mediated by the repressor CRE1 and the activator XYR1. T. reesei strain Rut-C30 is a hypercellulolytic mutant, obtained from the natural strain QM6a, that has a truncated version of the catabolite repressor gene, cre1. It has been previously shown that bacterial mutants lacking phosphoglucose isomerase (PGI produce more nucleotide precursors and amino acids. PGI catalyzes the second step of glycolysis, the formation of fructose-6-P from glucose-6-P. Results We deleted the gene pgi1, encoding PGI, in the T. reesei strain Rut-C30 and we introduced the cre1 gene in a Δpgi1 mutant. Both Δpgi1 and cre1+Δpgi1 mutants showed a pellet-like and growth as well as morphological alterations compared with Rut-C30. None of the mutants grew in media with fructose, galactose, xylose, glycerol or lactose but they grew in media with glucose, with fructose and glucose, with galactose and fructose or with lactose and fructose. No growth was observed in media with xylose and glucose. On glucose, Δpgi1 and cre1+Δpgi1 mutants showed higher cellulase activity than Rut-C30 and QM6a, respectively. But in media with lactose, none of the mutants improved the production of the reference strains. The increase in the activity did not correlate with the expression of mRNA of the xylanase regulator gene, xyr1. Δpgi1 mutants were also affected in the extracellular β-galactosidase activity. Levels of mRNA of the glucose 6-phosphate dehydrogenase did not increase in Δpgi1 during growth on glucose. Conclusions The ability to grow in media with glucose as the sole carbon source indicated that Trichoderma Δpgi1 mutants were able to use the pentose phosphate pathway. But, they did not increase the expression of gpdh. Morphological characteristics were the result of the pgi1 deletion. Deletion of pgi1 in

  12. Codon optimization of xylA gene for recombinant glucose isomerase production in Pichia pastoris and fed-batch feeding strategies to fine-tune bioreactor performance.

    Science.gov (United States)

    Ata, Özge; Boy, Erdem; Güneş, Hande; Çalık, Pınar

    2015-05-01

    The objectives of this work are the optimization of the codons of xylA gene from Thermus thermophilus to enhance the production of recombinant glucose isomerase (rGI) in P. pastoris and to investigate the effects of feeding strategies on rGI production. Codons of xylA gene from T. thermophilus were optimized, ca. 30 % of the codons were replaced with those with higher frequencies according to the codon usage bias of P. pastoris, codon optimization resulted in a 2.4-fold higher rGI activity. To fine-tune bioreactor performance, fed-batch bioreactor feeding strategies were designed as continuous exponential methanol feeding with pre-calculated feeding rate based on the pre-determined specific growth rate, and fed-batch methanol-stat feeding. Six feeding strategies were designed, as follows: (S1) continuous exponential methanol- and pulse- sorbitol feeding; (S2) continuous exponential methanol- and peptone- feeding; (S3) continuous exponential methanol- and pulse- mannitol feeding; (S4) continuous exponential methanol- and peptone- feeding and pulse-mannitol feeding; (S5) methanol-stat feeding by keeping methanol concentration at 5 g L(-1); and, (S6) methanol-stat feeding by keeping methanol concentration at 5 g L(-1) and pulse-mannitol feeding. The highest cell and rGI activity was attained as 117 g L(-1) at t = 66 h and 32530 U L(-1) at t = 53 h, in strategy-S5. The use of the co-substrate mannitol does not increase the rGI activity in methanol-stat feeding, where 4.1-fold lower rGI activity was obtained in strategy-S6. The overall cell yield on total substrate was determined at t = 53 h as 0.21 g g(-1) in S5 strategy.

  13. Phosphoglucose isomerase genotype affects running speed and heat shock protein expression after exposure to extreme temperatures in a montane willow beetle.

    Science.gov (United States)

    Rank, Nathan E; Bruce, Douglas A; McMillan, David M; Barclay, Colleen; Dahlhoff, Elizabeth P

    2007-03-01

    Eastern Sierra Nevada populations of the willow beetle Chrysomela aeneicollis commonly experience stressfully high and low environmental temperatures that may influence survival and reproduction. Allele frequencies at the enzyme locus phosphoglucose isomerase (PGI) vary across a climatic latitudinal gradient in these populations, with PGI allele 1 being most common in cooler regions and PGI allele 4 in warmer ones. PGI genotypes differ in heat and cold tolerance and in expression of a 70 kDa heat shock protein. Here we examine genetic, behavioral and environmental factors affecting a performance character, running speed, for willow beetles, and assess effects of consecutive cold and heat exposure on running speed and expression of Hsp70 in the laboratory. In nature, running speed depends on air temperature and is higher for males than females. Mating beetles ran faster than single beetles, and differences among PGI genotypes in male running speed depended on the presence of females. In the laboratory, exposure to cold reduced subsequent running speed, but the amount of this reduction depended on PGI genotype and previous thermal history. Effects of exposure to heat also depended on life history stage and PGI genotype. Adults possessing allele 1 ran fastest after a single exposure to stressful temperature, whereas those possessing allele 4 ran faster after repeated exposure. Larvae possessing allele 4 ran fastest after a single stressful exposure, but running speed generally declined after a second exposure to stressful temperature. The ranking of PGI genotypes after the second exposure depended on whether a larva had been exposed to cold or heat. Effects of temperature on Hsp70 expression also varied among PGI genotypes and depended on type of exposure, especially for adults (single heat exposure, two cold exposures: PGI 1-1>1-4>4-4; other multiple extreme exposures: 4-4>1-4>1-1). There was no consistent association between alleles at other polymorphic enzyme loci

  14. Phosphoglucose isomerase/autocrine motility factor mediates epithelial-mesenchymal transition regulated by miR-200 in breast cancer cells.

    Science.gov (United States)

    Ahmad, Aamir; Aboukameel, Amro; Kong, Dejuan; Wang, Zhiwei; Sethi, Seema; Chen, Wei; Sarkar, Fazlul H; Raz, Avraham

    2011-05-01

    Phosphoglucose isomerase/autocrine motility factor (PGI/AMF) plays an important role in glycolysis and gluconeogenesis and is associated with invasion and metastasis of cancer cells. We have previously shown its role in the induction of epithelial-mesenchymal transition (EMT) in breast cancer cells, which led to increased aggressiveness; however, the molecular mechanism by which PGI/AMF regulates EMT is not known. Here we show, for the first time, that PGI/AMF overexpression led to an increase in the DNA-binding activity of NF-κB, which, in turn, led to increased expression of ZEB1/ZEB2. The microRNA-200s (miR-200s) miR-200a, miR-200b, and miR-200c are known to negatively regulate the expression of ZEB1/ZEB2, and we found that the expression of miR-200s was lost in PGI/AMF overexpressing MCF-10A cells and in highly invasive MDA-MB-231 cells, which was consistent with increased expression of ZEB1/ZEB2. Moreover, silencing of PGI/AMF expression in MDA-MB-231 cells led to overexpression of miR-200s, which was associated with reversal of EMT phenotype (i.e., mesenchymal-epithelial transition), and these findings were consistent with alterations in the relative expression of epithelial (E-cadherin) and mesenchymal (vimentin, ZEB1, ZEB2) markers and decreased aggressiveness as judged by clonogenic, motility, and invasion assays. Moreover, either reexpression of miR-200 or silencing of PGI/AMF suppressed pulmonary metastases of MDA-MB-231 cells in vivo, and anti-miR-200 treatment in vivo resulted in increased metastases. Collectively, these results suggest a role of miR-200s in PGI/AMF-induced EMT and thus approaches for upregulation of miR-200s could be a novel therapeutic strategy for the treatment of highly invasive breast cancer.

  15. Structural modeling and docking studies of ribose 5-phosphate isomerase from Leishmania major and Homo sapiens: a comparative analysis for Leishmaniasis treatment.

    Science.gov (United States)

    Capriles, Priscila V S Z; Baptista, Luiz Phillippe R; Guedes, Isabella A; Guimarães, Ana Carolina R; Custódio, Fabio L; Alves-Ferreira, Marcelo; Dardenne, Laurent E

    2015-02-01

    Leishmaniases are caused by protozoa of the genus Leishmania and are considered the second-highest cause of death worldwide by parasitic infection. The drugs available for treatment in humans are becoming ineffective mainly due to parasite resistance; therefore, it is extremely important to develop a new chemotherapy against these parasites. A crucial aspect of drug design development is the identification and characterization of novel molecular targets. In this work, through an in silico comparative analysis between the genomes of Leishmania major and Homo sapiens, the enzyme ribose 5-phosphate isomerase (R5PI) was indicated as a promising molecular target. R5PI is an important enzyme that acts in the pentose phosphate pathway and catalyzes the interconversion of d-ribose-5-phosphate (R5P) and d-ribulose-5-phosphate (5RP). R5PI activity is found in two analogous groups of enzymes called RpiA (found in H. sapiens) and RpiB (found in L. major). Here, we present the first report of the three-dimensional (3D) structures and active sites of RpiB from L. major (LmRpiB) and RpiA from H. sapiens (HsRpiA). Three-dimensional models were constructed by applying a hybrid methodology that combines comparative and ab initio modeling techniques, and the active site was characterized based on docking studies of the substrates R5P (furanose and ring-opened forms) and 5RP. Our comparative analyses show that these proteins are structural analogs and that distinct residues participate in the interconversion of R5P and 5RP. We propose two distinct reaction mechanisms for the reversible isomerization of R5P to 5RP, which is catalyzed by LmRpiB and HsRpiA. We expect that the present results will be important in guiding future molecular modeling studies to develop new drugs that are specially designed to inhibit the parasitic form of the enzyme without significant effects on the human analog.

  16. Soluble prokaryotic overexpression and purification of bioactive human granulocyte colony-stimulating factor by maltose binding protein and protein disulfide isomerase.

    Directory of Open Access Journals (Sweden)

    Bich Hang Do

    Full Text Available Human granulocyte colony-stimulating factor (hGCSF, a neutrophil-promoting cytokine, is an effective therapeutic agent for neutropenia patients who have undergone several cancer treatments. Efficient production of hGCSF using E. coli is challenging because the hormone tends to aggregate and forms inclusion bodies. This study examined the ability of seven different N-terminal fusion tags to increase expression of soluble hGCSF in E. coli. Four tag proteins, namely maltose-binding protein (MBP, N-utilization substance protein A, protein disulfide isomerase (PDI, and the b'a' domain of PDI (PDIb'a', increased the solubility of hGCSF under normal conditions. Lowering the expression temperature from 30°C to 18°C also increased the solubility of thioredoxin-tagged and glutathione S-transferase-tagged hGCSF. By contrast, hexahistidine-tagged hGCSF was insoluble at both temperatures. Simple conventional chromatographic methods were used to purify hGCSF from the overexpressed PDIb'a'-hGCSF and MBP-hGCSF proteins. In total, 11.3 mg or 10.2 mg of pure hGCSF were obtained from 500 mL cultures of E. coli expressing PDIb'a'-hGCSF or MBP-hGCSF, respectively. SDS-PAGE analysis and silver staining confirmed high purity of the isolated hGCSF proteins, and the endotoxin levels were less than 0.05 EU/µg of protein. Subsequently, the bioactivity of the purified hGCSF proteins similar to that of the commercially available hGCSF was confirmed using the mouse M-NFS-60 myelogenous leukemia cell line. The EC50s of the cell proliferation dose-response curves for hGCSF proteins purified from MBP-hGCSF and PDIb'a'-hGCSF were 2.83±0.31 pM, and 3.38±0.41 pM, respectively. In summary, this study describes an efficient method for the soluble overexpression and purification of bioactive hGCSF in E. coli.

  17. Soluble prokaryotic overexpression and purification of bioactive human granulocyte colony-stimulating factor by maltose binding protein and protein disulfide isomerase.

    Science.gov (United States)

    Do, Bich Hang; Ryu, Han-Bong; Hoang, Phuong; Koo, Bon-Kyung; Choe, Han

    2014-01-01

    Human granulocyte colony-stimulating factor (hGCSF), a neutrophil-promoting cytokine, is an effective therapeutic agent for neutropenia patients who have undergone several cancer treatments. Efficient production of hGCSF using E. coli is challenging because the hormone tends to aggregate and forms inclusion bodies. This study examined the ability of seven different N-terminal fusion tags to increase expression of soluble hGCSF in E. coli. Four tag proteins, namely maltose-binding protein (MBP), N-utilization substance protein A, protein disulfide isomerase (PDI), and the b'a' domain of PDI (PDIb'a'), increased the solubility of hGCSF under normal conditions. Lowering the expression temperature from 30°C to 18°C also increased the solubility of thioredoxin-tagged and glutathione S-transferase-tagged hGCSF. By contrast, hexahistidine-tagged hGCSF was insoluble at both temperatures. Simple conventional chromatographic methods were used to purify hGCSF from the overexpressed PDIb'a'-hGCSF and MBP-hGCSF proteins. In total, 11.3 mg or 10.2 mg of pure hGCSF were obtained from 500 mL cultures of E. coli expressing PDIb'a'-hGCSF or MBP-hGCSF, respectively. SDS-PAGE analysis and silver staining confirmed high purity of the isolated hGCSF proteins, and the endotoxin levels were less than 0.05 EU/µg of protein. Subsequently, the bioactivity of the purified hGCSF proteins similar to that of the commercially available hGCSF was confirmed using the mouse M-NFS-60 myelogenous leukemia cell line. The EC50s of the cell proliferation dose-response curves for hGCSF proteins purified from MBP-hGCSF and PDIb'a'-hGCSF were 2.83±0.31 pM, and 3.38±0.41 pM, respectively. In summary, this study describes an efficient method for the soluble overexpression and purification of bioactive hGCSF in E. coli.

  18. Unprovability results involving braids

    CERN Document Server

    Carlucci, Lorenzo; Weiermann, Andreas

    2007-01-01

    We construct long sequences of braids that are descending with respect to the standard order of braids (``Dehornoy order''), and we deduce that, contrary to all usual algebraic properties of braids, certain simple combinatorial statements involving the braid order are true, but not provable in the subsystems ISigma1 or ISigma2 of the standard Peano system.

  19. Conformational plasticity and dynamics in the generic protein folding catalyst SlyD unraveled by single-molecule FRET.

    Science.gov (United States)

    Kahra, Dana; Kovermann, Michael; Löw, Christian; Hirschfeld, Verena; Haupt, Caroline; Balbach, Jochen; Hübner, Christian Gerhard

    2011-08-26

    The relation between conformational dynamics and chemistry in enzyme catalysis recently has received increasing attention. While, in the past, the mechanochemical coupling was mainly attributed to molecular motors, nowadays, it seems that this linkage is far more general. Single-molecule fluorescence methods are perfectly suited to directly evidence conformational flexibility and dynamics. By labeling the enzyme SlyD, a member of peptidyl-prolyl cis-trans isomerases of the FK506 binding protein type with an inserted chaperone domain, with donor and acceptor fluorophores for single-molecule fluorescence resonance energy transfer, we directly monitor conformational flexibility and conformational dynamics between the chaperone domain and the FK506 binding protein domain. We find a broad distribution of distances between the labels with two main maxima, which we attribute to an open conformation and to a closed conformation of the enzyme. Correlation analysis demonstrates that the conformations exchange on a rate in the 100 Hz range. With the aid from Monte Carlo simulations, we show that there must be conformational flexibility beyond the two main conformational states. Interestingly, neither the conformational distribution nor the dynamics is significantly altered upon binding of substrates or other known binding partners. Based on these experimental findings, we propose a model where the conformational dynamics is used to search the conformation enabling the chemical step, which also explains the remarkable substrate promiscuity connected with a high efficiency of this class of peptidyl-prolyl cis-trans isomerases.

  20. Differential proteome and gene expression for testis of mice exposed to carbon ion radiation

    Science.gov (United States)

    Zhang, Hong; Li, Hongyan

    Objective To investigate the effect and mechanism of high linear energy transfer (LET) carbon ion irradiation (CIR) on reproduction in the testis of male Swiss Webster mice, and assess the risk associated with space environment. Methods Male mice underwent whole-body irradiation with CIR (0.5, 1 and 4Gy), and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF) analysis was used to determine the alteration in protein expression in 2-DE (two-dimensional gel electrophoresis) gels of testes caused by irradiation after 7, 14 days. Results 15 differentially expressed proteins, such as glucose-regulated protein(GRP78), aconitate hydratase-mitochondrial precursor (ACO), pyruvate kinase isozymes M1/M2 (PKM1/M2), glutathione-S-transferaseA3 (GSTA3), glutathione S-transferase Pi 1 (GSTP1), Cu/Zn super-oxide dismutase (SOD1), Peptidyl-prolyl cis-trans isomerase (Pin1) and Heat shock 70 kDa protein 4L (HSPa4L), were identified and these proteins were mainly involved in energy supply, the endoplasmic reticulum, cell proliferation, cell cycle, antioxidant capacity and mitochondrial respiration, which play important roles in the inhibition of testicular function in response to CIR. Furthermore, we confirmed the relationship between transcription of mRNA and the abundance of proteins. Conclusion The findings of the present study demonstrated that these proteins may lead to new insights into the molecular mechanism of CIR toxicity, and suggested that the gene expression response to CIR involves diverse regulatory mechanisms from transcription of mRNA to the formation of functional proteins. These data also may provide a scientific basis for protecting astronauts and space traveler’s health and safety.

  1. Identification and functional characterization of the putative polysaccharide biosynthesis protein (CapD) of Enterococcus faecium U0317.

    Science.gov (United States)

    Ali, Liaqat; Spiess, Meike; Wobser, Dominique; Rodriguez, Marta; Blum, Hubert E; Sakιnç, Türkân

    2016-01-01

    Most bacterial species produce capsular polysaccharides that contribute to disease pathogenesis through evasion of the host innate immune system and are also involved in inhibiting leukocyte killing. In the present study, we identified a gene in Enterococcus faecium U0317 with homologies to the polysaccharide biosynthesis protein CapD that is made up of 336 amino acids and putatively catalyzes N-linked glycosylation. A capD deletion mutant was constructed and complemented by homologous recombination that was confirmed by PCR and sequencing. The mutant revealed different growth behavior and morphological changes compared to wild-type by scanning electron microscopy, also the capD mutant showed a strong hydrophobicity and that was reversed in the reconstituted mutant. For further characterization and functional analyses, in-vitro cell culture and in-vivo a mouse infection models were used. Antibodies directed against alpha lipotechoic acid (αLTA) and the peptidyl-prolyl cis-trans isomerase (αPpiC), effectively mediated the opsonophagocytic killing in the capD knock-out mutant, while this activity was not observed in the wild-type and reconstituted mutant. By comparison more than 2-fold decrease was seen in mutant colonization and adherence to both T24 and Caco2 cells. However, a significant higher bacterial colonization was observed in capD mutant during bacteremia in the animal model, while virulence in a mouse UTI (urinary tract infection) model, there were no obvious differences. Further studies are needed to elucidate the function of capsular polysaccharide synthesis gene clusters and its involvement in the disease pathogenesis with the aim to develop targeted therapies to treat multidrug-resistant E. faecium infections.

  2. Involved Node Radiation Therapy

    DEFF Research Database (Denmark)

    Maraldo, Maja V; Aznar, Marianne C; Vogelius, Ivan R

    2012-01-01

    PURPOSE: The involved node radiation therapy (INRT) strategy was introduced for patients with Hodgkin lymphoma (HL) to reduce the risk of late effects. With INRT, only the originally involved lymph nodes are irradiated. We present treatment outcome in a retrospective analysis using this strategy...... to 36 Gy). Patients attended regular follow-up visits until 5 years after therapy. RESULTS: The 4-year freedom from disease progression was 96.4% (95% confidence interval: 92.4%-100.4%), median follow-up of 50 months (range: 4-71 months). Three relapses occurred: 2 within the previous radiation field......, and 1 in a previously uninvolved region. The 4-year overall survival was 94% (95% confidence interval: 88.8%-99.1%), median follow-up of 58 months (range: 4-91 months). Early radiation therapy toxicity was limited to grade 1 (23.4%) and grade 2 (13.8%). During follow-up, 8 patients died, none from HL, 7...

  3. Microorganisms involved in MIC

    Energy Technology Data Exchange (ETDEWEB)

    Sorensen, K. [Danish Technological Institute (Denmark)

    2011-07-01

    Microbiologically influenced corrosion (MIC) is a widespread problem that is difficult to detect and assess because of its complex mechanism. This paper presents the involvement of microorganisms in MIC. Some of the mechanisms that cause MIC include hydrogen consumption, production of acids, anode-cathode formation and electron shuttling. A classic bio-corrosive microorganism in the oil and gas industry is sulphate-reducing prokaryotes (SRP). Methanogens also increase corrosion rates in metals. Some of the phylogenetic orders detected while studying SRP and methanogens are archaeoglobales, clostridiales, methanosarcinales and methanothermococcus. There were some implications, such as growth of SRP not being correlated with growth of methanogens; methanogens were included in MIC risk assessment. A few examples are used to display how microorganisms are involved in topside corrosion and microbial community in producing wells. From the study, it can be concluded that, MIC risk assessment includes system data and empirical knowledge of the distribution and number of microorganisms in the system.

  4. Involvement Without Participation?

    DEFF Research Database (Denmark)

    Olsén, Peter

    2012-01-01

    The article presents a case study of a knowledge-intensive company that launched a 2-year project to improve their psychosocial working environment. All parties agreed on the project, and the methods used aimed to promote the involvement of the employees. Surprisingly, the psychosocial working...... and participation. In order to develop a more sustainable and viable psychosocial working environment, a broader and more democratic notion of organisational learning and managing is proposed....

  5. Getting involved in research.

    Science.gov (United States)

    Banner, Davina; Grant, Lyle G

    2011-01-01

    The need for quality nursing research to promote evidence-based practice and optimize patient care is well recognized. This is particularly pertinent in cardiovascular nursing, where cardiovascular disease continues to be the leading cause of morbidity and mortality worldwide (World Health Organization, 2007). Across the spectrum of academic, clinical, and health care administration nursing roles, research remains fundamental to bridging theory, practice, and education (LoBiondo-Wood, Haber, Cameron, & Singh, 2009). Despite recognition of the importance of nursing research, the gap between research and practice continues to be an ongoing issue (Funk, Tornquist, & Champagne, 1995; Pettengill, Gillies, & Clark, 1994; Rizzuto, Bostrom, Suterm, & Chenitz, 1994; Rolfe, 1998). Nurses are appropriately situated to contribute to research that improves clinical outcomes and health service delivery. However, the majority of nurses in clinical practice do not have a significant research component structured into their nursing role. In this research column, the authors outline the importance of nurses being engaged in research and present some different levels of involvement that nurses may assume. A continuum of nursing research involvement includes asking researchable questions, being a savvy consumer of research evidence, finding your own level of research involvement, and aspiring to lead.

  6. Involvement in Physical Activity

    Directory of Open Access Journals (Sweden)

    James Gavin

    2013-04-01

    Full Text Available A total of 1,096 adolescents participated in 123 focus groups regarding the perceived outcomes of their involvement in sports and physical activity (PA. The groups, segmented by grade level, sex, and school types, were conducted in both public and private high schools in Montreal, Quebec. We sought to understand, through the participants’ own words, their perception of the outcome matrix of involvement in sports and PA. Focus group questions emphasized changes that adolescents associated with such engagement. In particular, participants were asked how sports and PA might influence behaviors, emotional states, personal characteristics, and other outcomes. Twelve themes were identified in the responses: Positive Health and Physical Changes (18.5%, Activity-Related Positive Emotions (15.6%, and Personal Learning (11.3% were most prevalent in the discussions. A cluster of deeper personal changes thematically described as Self-Identity, Autonomy, and Positive Character Development accounted for another 16.5% of the responses. Relatively few commentaries emphasized negative effects (7.1%. Converting the proportions of qualitative data into a quantitative index allowed us to analyze potential differences in emphasis according to sex, age, and school type. Though a few significant findings emerged, the larger pattern was of a uniform perceptual map across the variables for this adolescent sample. Implications drawn from this investigation highlight the need to clearly articulate concrete pathways to positive nonphysical changes (e.g., mood states, autonomy, positive character development from engagements in sports and PA.

  7. The proteome response to amyloid protein expression in vivo.

    Directory of Open Access Journals (Sweden)

    Ricardo A Gomes

    Full Text Available Protein misfolding disorders such as Alzheimer, Parkinson and transthyretin amyloidosis are characterized by the formation of protein amyloid deposits. Although the nature and location of the aggregated proteins varies between different diseases, they all share similar molecular pathways of protein unfolding, aggregation and amyloid deposition. Most effects of these proteins are likely to occur at the proteome level, a virtually unexplored reality. To investigate the effects of an amyloid protein expression on the cellular proteome, we created a yeast expression system using human transthyretin (TTR as a model amyloidogenic protein. We used Saccharomyces cerevisiae, a living test tube, to express native TTR (non-amyloidogenic and the amyloidogenic TTR variant L55P, the later forming aggregates when expressed in yeast. Differential proteome changes were quantitatively analyzed by 2D-differential in gel electrophoresis (2D-DIGE. We show that the expression of the amyloidogenic TTR-L55P causes a metabolic shift towards energy production, increased superoxide dismutase expression as well as of several molecular chaperones involved in protein refolding. Among these chaperones, members of the HSP70 family and the peptidyl-prolyl-cis-trans isomerase (PPIase were identified. The latter is highly relevant considering that it was previously found to be a TTR interacting partner in the plasma of ATTR patients but not in healthy or asymptomatic subjects. The small ubiquitin-like modifier (SUMO expression is also increased. Our findings suggest that refolding and degradation pathways are activated, causing an increased demand of energetic resources, thus the metabolic shift. Additionally, oxidative stress appears to be a consequence of the amyloidogenic process, posing an enhanced threat to cell survival.

  8. Housekeeping and tissue-specific genes in mouse tissues

    Directory of Open Access Journals (Sweden)

    St-Amand Jonny

    2007-05-01

    Full Text Available Abstract Background This study aims to characterize the housekeeping and tissue-specific genes in 15 mouse tissues by using the serial analysis of gene expression (SAGE strategy which indicates the relative level of expression for each transcript matched to the tag. Results Here, we identified constantly expressed housekeeping genes, such as eukaryotic translation elongation factor 2, which is expressed in all tissues without significant difference in expression levels. Moreover, most of these genes were not regulated by experimental conditions such as steroid hormones, adrenalectomy and gonadectomy. In addition, we report previously postulated housekeeping genes such as peptidyl-prolyl cis-trans isomerase A, glyceraldehyde-3-phosphate dehydrogenase and beta-actin, which are expressed in all the tissues, but with significant difference in their expression levels. We have also identified genes uniquely detected in each of the 15 tissues and other tissues from public databases. Conclusion These identified housekeeping genes could represent appropriate controls for RT-PCR and northern blot when comparing the expression levels of genes in several tissues. The results reveal several tissue-specific genes highly expressed in testis and pituitary gland. Furthermore, the main function of tissue-specific genes expressed in liver, lung and bone is the cell defence, whereas several keratins involved in cell structure function are exclusively detected in skin and vagina. The results from this study can be used for example to target a tissue for agent delivering by using the promoter of tissue-specific genes. Moreover, this study could be used as basis for further researches on physiology and pathology of these tissues.

  9. Multiple redox and non-redox interactions define 2-Cys peroxiredoxin as a regulatory hub in the chloroplast.

    Science.gov (United States)

    Muthuramalingam, Meenakumari; Seidel, Thorsten; Laxa, Miriam; Nunes de Miranda, Susana M; Gärtner, Florian; Ströher, Elke; Kandlbinder, Andrea; Dietz, Karl-Josef

    2009-11-01

    In plants, the highly abundant 2-cysteine peroxiredoxin (2-CysPrx) is associated with the chloroplast and involved in protecting photosynthesis. This work addresses the multiple interactions of the 2-CysPrx in the chloroplast, which depend on its redox state. Transcript co-regulation analysis showed a strong linkage to the peptidyl-prolyl-cis/trans isomerase Cyclophilin 20-3 (Cyp20-3) and other components of the photosynthetic apparatus. Co-expression in protoplasts and quantification of fluorescence resonance energy transfer (FRET) efficiency in vivo confirmed protein interactions of 2-CysPrx with Cyp20-3 as well as NADPH-dependent thioredoxin reductase C (NTRC), while thioredoxin x (Trx-x) did not form complexes that could enable FRET. Likewise, changes in FRET of fluorescently labeled 2-CysPrx in vitro and in vivo proved redox dependent dynamics of 2-CysPrx. Addition of Cyp20-3 to an in vitro peroxidase assay with 2-CysPrx had no significant effect on peroxide reduction. Also, in the presence of NTRC, addition of Cyp20-3 did not further enhance peroxide reduction. In addition, 2-CysPrx functioned as chaperone and inhibited aggregation of citrate synthase during heat treatment. This activity was partly inhibited by Cyp20-3. As a new interaction partner of decameric 2-CysPrx, photosystem II could be identified after chloroplast fractionation and in pull-down assays after reconstitution. In summary, the data indicate a dynamic function of plant 2-CysPrx as redox sensor, chaperone, and regulator in the chloroplast with diverse functions beyond its role as thiol peroxidase.

  10. Three-dimensional structures of virulence proteins of Legionella establish targets for new antibacterials

    Institute of Scientific and Technical Information of China (English)

    Guido Hansen; Rolf Hilgenfeld

    2012-01-01

    Legionella pneumophila,the causative agent of Legionnaires' disease,has been recognized as a major health problem responsible for an estimated number of 15 000-30 000 cases of severe pneumonia per year in Germany alone.Despite of the high clinical relevance,many aspects of the intracellular life cycle of Legionella,especially details on interactions with host cells,are not well understood.Structural information on virulence proteins helps unravel basal pathogenicity mechanisms and is a prerequisite for the rational development of effective drug molecules.Here we discuss structures of three important virulence proteins ofLegionella that have been determined in our laboratory.The structure of the macrophage infectivity potentiator (Mip) protein of Legionella pneumophila is the first of a novel subgroup within the family of FK506-binding protein (FKBP) peptidyl-prolyl cis/trans isomerases.On the basis of the Mip structure,promising antibacterial agents are being designed.Recently,structures of two equally exciting Legionella proteins have been reported.The ferrous iron transport protein FeoB is a transmembrane protein responsible for Fe2+ aquisition after entry of the pathogen into the host cell.The structure of the cytoplasmic domain of FeoB provides insights into the family of prokaryotic G proteins and allows a detailed comparison with structures of related FeoBs.Furthermore,the characterization of DegQ,a periplasmatic chaperone-protease involved in protein quality control represents an intriguing example of how enzymatic activity is regulated by oligomerization as well as by an intrinsic loop activation cascade,depending on subtle conformational rearrangements.

  11. Psychrophily and Catalysis

    Directory of Open Access Journals (Sweden)

    Charles Gerday

    2013-04-01

    Full Text Available Polar and other low temperature environments are characterized by a low content in energy and this factor has a strong incidence on living organisms which populate these rather common habitats. Indeed, low temperatures have a negative effect on ectothermic populations since they can affect their growth, reaction rates of biochemical reactions, membrane permeability, diffusion rates, action potentials, protein folding, nucleic acids dynamics and other temperature-dependent biochemical processes. Since the discovery that these ecosystems, contrary to what was initially expected, sustain a rather high density and broad diversity of living organisms, increasing efforts have been dedicated to the understanding of the molecular mechanisms involved in their successful adaptation to apparently unfavorable physical conditions. The first question that comes to mind is: How do these organisms compensate for the exponential decrease of reaction rate when temperature is lowered? As most of the chemical reactions that occur in living organisms are catalyzed by enzymes, the kinetic and thermodynamic properties of cold-adapted enzymes have been investigated. Presently, many crystallographic structures of these enzymes have been elucidated and allowed for a rather clear view of their adaptation to cold. They are characterized by a high specific activity at low and moderate temperatures and a rather low thermal stability, which induces a high flexibility that prevents the freezing effect of low temperatures on structure dynamics. These enzymes also display a low activation enthalpy that renders them less dependent on temperature fluctuations. This is accompanied by a larger negative value of the activation entropy, thus giving evidence of a more disordered ground state. Appropriate folding kinetics is apparently secured through a large expression of trigger factors and peptidyl–prolyl cis/trans-isomerases.

  12. Application of SAIL phenylalanine and tyrosine with alternative isotope-labeling patterns for protein structure determination

    Energy Technology Data Exchange (ETDEWEB)

    Takeda, Mitsuhiro [Nagoya University, Structural Biology Research Center, Graduate School of Science (Japan); Ono, Akira M.; Terauchi, Tsutomu [SAIL Technologies Co., Inc. (Japan); Kainosho, Masatsune, E-mail: kainosho@nmr.chem.metro-u.ac.j [Nagoya University, Structural Biology Research Center, Graduate School of Science (Japan)

    2010-01-15

    The extensive collection of NOE constraint data involving the aromatic ring signals is essential for accurate protein structure determination, although it is often hampered in practice by the pervasive signal overlapping and tight spin couplings for aromatic rings. We have prepared various types of stereo-array isotope labeled phenylalanines ({epsilon}- and {zeta}-SAIL Phe) and tyrosine ({epsilon}-SAIL Tyr) to overcome these problems (Torizawa et al. 2005), and proven that these SAIL amino acids provide dramatic spectral simplification and sensitivity enhancement for the aromatic ring NMR signals. In addition to these SAIL aromatic amino acids, we recently synthesized {delta}-SAIL Phe and {delta}-SAIL Tyr, which allow us to observe and assign {delta}-{sup 13}C/{sup 1}H signals very efficiently. Each of the various types of SAIL Phe and SAIL Tyr yields well-resolved resonances for the {delta}-, {epsilon}- or {zeta}-{sup 13}C/{sup 1}H signals, respectively, which can readily be assigned by simple and robust pulse sequences. Since the {delta}-, {epsilon}-, and {zeta}-proton signals of Phe/Tyr residues give rise to complementary NOE constraints, the concomitant use of various types of SAIL-Phe and SAIL-Tyr would generate more accurate protein structures, as compared to those obtained by using conventional uniformly {sup 13}C, {sup 15}N-double labeled proteins. We illustrated this with the case of an 18.2 kDa protein, Escherichia coli peptidyl-prolyl cis-trans isomerase b (EPPIb), and concluded that the combined use of {zeta}-SAIL Phe and {epsilon}-SAIL Tyr would be practically the best choice for protein structural determinations.

  13. Between Involvement and Detachment

    DEFF Research Database (Denmark)

    Thomasen, Gry

    a traditional reading of de Gaulle’s policies, and feared that if Gaullist thinking spread among the European allies, it would merit to a return to traditional European power politics. The analysis shows that, by 1964 the administration believed, according to this study, that NATO’s principle of integration......Between Involvement and Detachment takes grasp with the Johnson administration’s (1963-1969) perceptions of and responses to the Western European realignments. Arguing that the Johnson administration set out to maintain the American unilateralist position in the transatlantic relation, not just......, essentially, were detached as America rejected the European reason of state. The Western European realignments were recorded in the Johnson administration with de Gaulle’s critique of US hegemony in Western Europe in the early 1960s. The thesis argues that the administration to a large extent had...

  14. Effectiveness of citizen involvement

    Energy Technology Data Exchange (ETDEWEB)

    Robinson, L. [Prince William Sound Regional Citizen' s Advisory Council, Anchorage, AK (United States)

    2006-07-01

    This paper reviewed the rise of citizen involvement in industry that affects their community. Following the Exxon Valdez oil spill (EVOS) in 1989, the Oil Pollution Act of 1990 provided funding by industry for a citizens group to provide oversite of the Alyeska Pipeline Service Agency terminal and associated tankers. That role is currently filled by the Prince William Sound Regional Citizen's Advisory Council, a volunteer organization that represents communities that were affected by the EVOS. The history of the Prince William Sound Regional Citizen's Advisory Council was discussed along with its structure, funding and overview of projects and research into safer transportation of oil, better oil spill response capabilities and improved environmental protection practices. Some of the successes involving citizen input include the requirement that all tankers going into Prince William Sound be double hull by 2015; a world-class system of tugs escorting tankers in Prince William Sound; installation of an ice-detection radar on a small island near the site of the EVOS; a guidebook for communities affected by man-made disasters; identification of nearshore locations that should be the first to be protected in the case of another spill; and, the installation of a system to capture crude oil vapors when tankers take on cargo. Other projects underway include the study of invasive species that can be transported in the ballast water of tankers, efficacy of dispersants, soil contamination at the tanker loading site, emissions of hazardous air pollutants from ballast water treatment processes, and continual review of emergency response plans. In the 17 years since the formation of the Prince William Sound Regional Citizen's Advisory Council, it has been shown that communication and transparency are the keys to solving complacency, which is believed to have been a contributing factor to the EVOS. 3 refs.

  15. 葡萄糖异构酶及其在高果糖浆生产中的应用%Glucose Isomerase and Its Application in Production of High Fructose Corn Syrup

    Institute of Scientific and Technical Information of China (English)

    刘成龙; 周海岩; 柳志强; 郑裕国

    2014-01-01

    葡萄糖异构酶(Glucose isomerase,GI)能催化D-葡萄糖的异构化反应,生成D-果糖,是目前工业上制备高果糖浆(HFCS)的关键酶之一.本文对GI的来源、分类、高级结构特征和催化机制进行了介绍,并从GI催化功能的改善、基因工程菌的构建和固定化三个方面对GI在HFCS生产中应用的关键技术和策略进行分析.

  16. A survey for isoenzymes of glucosephosphate isomerase, phosphoglucomutase, glucose-6-phosphate dehydrogenase and 6-Phosphogluconate dehydrogenase in C3-, C 4-and crassulacean-acid-metabolism plants, and green algae.

    Science.gov (United States)

    Herbert, M; Burkhard, C; Schnarrenberger, C

    1979-01-01

    Two isoenzymes each of glucosephosphate isomerase (EC 5.3.1.9), phosphoglucomutase (EC 2.7.5.1), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.43) were separated by (NH4)2SO4 gradient solubilization and DEAE-cellulose ion-exchange chromatography from green leaves of the C3-plants spinach (Spinacia oleracea L.), tobacco (Nicotiana tabacum L.) and wheat (Triticum aestivum L.), of the Crassulacean-acid-metabolism plants Crassula lycopodioides Lam., Bryophyllum calycinum Salisb. and Sedum rubrotinctum R.T. Clausen, and from the green algae Chlorella vulgaris and Chlamydomonas reinhardii. After isolation of cell organelles from spinach leaves by isopyenic centrifugation in sucrose gradients one of two isoenzymes of each of the four enzymes was found to be associated with whole chloroplasts while the other was restricted to the soluble cell fraction, implying the same intracellular distribution of these isoenzymes also in the other species.Among C4-plants, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were found in only one form in corn (Zea mays L.), sugar cane (Saccharum officinarum L.) and Coix lacrymajobi L., but as two isoenzymes in Atriplex spongiosa L. and Portulaca oleracea L. In corn, the two dehydrogenases were mainly associated with isolated mesophyll protoplasts while in Atriplex spongiosa they were of similar specific activity in both mesophyll protoplasts and bundle-sheath strands. In all five C4-plants three isoenzymes of glucosephosphate isomerase and phosphoglucomutase were found. In corn two were localized in the bundle-sheath strands and the third one in the mesophyll protoplasts. The amount of activity of the enzymes was similar in each of the two cell fractions. Apparently, C4 plants have isoenzymes not only in two cell compartments, but also in physiologically closely linked cell types such as mesophyll and bundle-sheath cells.

  17. [Nail involvement in leprosy].

    Science.gov (United States)

    Belinchón Romero, I; Ramos Rincón, J M; Reyes Rabell, F

    2012-05-01

    Leprosy, a disease caused by Mycobacterium leprae, primarily affects the skin and nerves, but the nails are also involved in as many as 3 out of 4 patients .The factors that trigger nail changes in leprosy are numerous and include repeated trauma, neuropathy, vascular impairment, infections, lepra reactions, and the drugs used to manage the disease. The changes most often reported include subungual hematomas, onycholysis, onychauxis, onychogryphosis, pterygium unguis, and onychoheterotopia, most of which can be attributed to nerve damage and trauma. Furthermore, the acro-osteolysis that occurs in the advanced stages of the disease may present with brachyonychia, racquet nails, or even anonychia. Infections of the nail bed leading to paronychia and onychomycosis should also be taken into account in leprosy. Other typical changes include longitudinal striae, pitting, macrolunula, Terry nails, leukonychia, hapalonychia, and Beau lines. In this review, we describe the principal nail changes associated with leprosy. These changes, which are highly varied and diverse in origin, are in fact a reflection of the significant morbidity caused by M. leprae infection.

  18. Multidrug toxicity involving sumatriptan.

    Science.gov (United States)

    Knittel, Jessica L; Vorce, Shawn P; Levine, Barry; Hughes, Rhome L; Bosy, Thomas Z

    2015-01-01

    A multidrug fatality involving sumatriptan is reported. Sumatriptan is a tryptamine derivative that acts at 5-HT(1B/1D) receptors and is used for the treatment of migraines. The decedent was a 21-year-old white female found dead in bed by her spouse. No signs of physical trauma were observed and a large number of prescription medications were discovered at the scene. Toxicological analysis of the central blood revealed sumatriptan at a concentration of 1.03 mg/L. Following therapeutic dosing guidelines, sumatriptan concentrations do not exceed 0.095 mg/L. Sumatriptan was isolated by solid-phase extraction and analyzed using liquid chromatography-tandem mass spectrometry in multiple reaction monitoring mode. A tissue distribution study was completed with the following concentrations measured: 0.61 mg/L in femoral blood, 0.56 mg/L in iliac blood, 5.01 mg/L in urine, 0.51 mg/kg in liver, 3.66 mg/kg in kidney, 0.09 mg/kg in heart, 0.32 mg/kg in spleen, 0.01 mg/kg in brain, 15.99 mg/kg in lung and 78.54 mg/45 mL in the stomach contents. Carisoprodol, meprobamate, fluoxetine, doxylamine, orphenadrine, dextromethorphan and hydroxyzine were also present in the blood at the following concentrations: 3.35, 2.36, 0.63, 0.19, 0.06, 0.55 and 0.16 mg/L. The medical examiner ruled the cause of death as acute mixed drug toxicity and the manner of death as accident.

  19. Identification and expression analysis of candidate genes involved in carotenoid biosynthesis in chickpea seeds

    Directory of Open Access Journals (Sweden)

    Mohammad Kazem Rezaei

    2016-12-01

    Full Text Available Plant carotenoids have a key role in preventing various diseases in human because of their antioxidant and provitamin A properties. Chickpea is a good source of carotenoid among legumes and its diverse germplasm and genome accessibility makes it a good model for carotenogenesis studies. The structure, location and copy numbers of genes involved in carotenoid biosynthesis were retrieved from the chickpea genome. The majority of the single nucleotide polymorphism (SNPs within these genes across five diverse chickpea cultivars was synonymous mutation. We examined the expression of the carotenogenesis genes and their association with carotenoid concentration at different seed development stages of five chickpea cultivars. Total carotenoid concentration ranged from 22 μg g-1 in yellow cotyledon kabuli to 44 μg g-1 in green cotyledon desi at 32 days post anthesis (DPA. The majority of carotenoids in chickpea seeds consists of lutein and zeaxanthin. The expression of the selected 19 genes involved in carotenoid biosynthesis pathway showed common pattern across five cultivars with higher expression at 8 and/or 16 DPA then dropped considerably at 24 and 32 DPA. Almost all genes were up-regulated in CDC Jade cultivar. Correlation analysis between gene expression and carotenoid concentration showed that the genes involved in the primary step of carotenoid biosynthesis pathway including carotenoid desaturase and isomerase positively correlated with various carotenoid components in chickpea seeds. A negative correlation was found between hydroxylation activity and provitamin A concentration in the seeds. The highest provitamin A concentration including β-carotene and β-cryptoxanthin were found in green cotyledon chickpea cultivars.

  20. The Azospirillum brasilense Sp7 noeJ and noeL genes are involved in extracellular polysaccharide biosynthesis.

    Science.gov (United States)

    Lerner, Anat; Castro-Sowinski, Susana; Valverde, Angel; Lerner, Hadas; Dror, Rachel; Okon, Yaacov; Burdman, Saul

    2009-12-01

    Azospirillum brasilense is a plant root-colonizing bacterium that exerts beneficial effects on the growth of many agricultural crops. Extracellular polysaccharides of the bacterium play an important role in its interactions with plant roots. The pRhico plasmid of A. brasilense Sp7, also named p90, carries several genes involved in synthesis and export of cell surface polysaccharides. We generated two Sp7 mutants impaired in two pRhico-located genes, noeJ and noeL, encoding mannose-6-phosphate isomerase and GDP-mannose 4,6-dehydratase, respectively. Our results demonstrate that in A. brasilense Sp7, noeJ and noeL are involved in lipopolysaccharide and exopolysaccharide synthesis. noeJ and noeL mutant strains were significantly altered in their outer membrane and cytoplasmic/periplasmic protein profiles relative to the wild-type strain. Moreover, both noeJ and noeL mutations significantly affected the bacterial responses to several stresses and antimicrobial compounds. Disruption of noeL, but not noeJ, affected the ability of the A. brasilense Sp7 to form biofilms. The pleiotropic alterations observed in the mutants could be due, at least partially, to their altered lipopolysaccharides and exopolysaccharides relative to the wild-type.

  1. Combined overexpression of genes involved in pentose phosphate pathway enables enhanced D-xylose utilization by Clostridium acetobutylicum.

    Science.gov (United States)

    Jin, Lin; Zhang, Hui; Chen, Liwen; Yang, Chen; Yang, Sheng; Jiang, Weihong; Gu, Yang

    2014-03-10

    D-Xylose utilization by Clostridium acetobutylicum, an important industrial microorganism used in ABE (Acetone, Butanol and Ethanol) production, has attracted increasing interests. We demonstrated previously that co-overexpression of genes, encoding d-xylose symporter, D-xylose isomerase and xylulokinase, improved D-xylose utilization by C. acetobutylicum (Xiao, H., et al., 2011. Applied and Environmental Microbiology 77, 7886-7895). Here, we further identified genes involved in PPP (Pentose Phosphate Pathway) in C. acetobutylicum and evaluated their contribution to d-xylose utilization. Among all the candidate genes, the CAC1347, CAC1348, CAC1730 and CAC2880 were validated to encode genes tal, tkl, rpe and rpi, four key genes involved in PPP, respectively. The following combined overexpression of these genes conferred a significantly improved xylose-utilizing ability to the recombinant strain, reaching a solvent titer 42% higher than that of the wild-type strain. This finding offers a useful strategy to optimize d-xylose utilization by C. acetobutylicum.

  2. Bradykinin and its gly sup 6 analogue are substrates of cyclophilin: A fluorine-19 magnetization transfer study

    Energy Technology Data Exchange (ETDEWEB)

    London, R.E.; Davis, D.G. (NIEHS, NC (USA)); Vavrek, R.J.; Stewart, J.M. (Univ. of Colorado Health Sciences Center, Denver, CO (USA)); Handschumacher, R.E. (Yale Univ., New Haven, CT (USA))

    1990-11-01

    Fluorine-19 magnetization transfer experiments have been used to determine the rates of cis/trans isomerization about the X-Pro{sup 7} peptide bond in (p-fluoro-Phe{sup 8})bradykinin and its Gly{sup 6} analogue. The measurements were carried out both prior to and after the addition of cyclophilin, which has recently been shown to have peptidyl-proline cis/trans isomerase activity and is the apparent target enzyme of the immunosuppressive agent cyclosporin A. Magnetization transfer measurements over the temperature range 40-75 {degree}C in the absence of enzyme give activation energies of 22.8 and 23.0 kcal/mol for (p-fluoro-Phe{sup 8})bradykinin and its Gly{sup 6} analogue, respectively. The values for the uncatalyzed cis {r arrow} trans rate constant, k{sub c}, are determined by extrapolation to be 4.8 {times} 10{sup {minus}2} and 2.1 {times} 10{sup {minus}2} s{sup {minus}1} for the two peptides at 25 {degree}C. The enzyme-catalyzed enhancement of the cis/trans interconversion rate was proportional to added cyclophilin concentration and was strongly sequence specific, with bradykinin a much better substrate than (Gly{sup 6})bradykinin. At a peptide concentration of 2.2 mM, the catalytic activity expressed as k{sub c} per micromolar cyclophilin was determined to be 1.2 s{sup {minus}1}/{mu}M for (p-fluoro-Phe{sup 8})bradykinin and 0.13 s{sup {minus}1}/{mu}M for the Gly{sup 6}analogue. The increased cis {r arrow} trans interconversion rates were strongly inhibited by cyclosporin A and the 6-(methylalanine) derivative, which bind to cyclophilin, but not by the 1-(tetrahydrofurfuryl) derivative of cyclosporin that binds weakly.

  3. The intraspecific difference of the triose phosphate isomerase (tim) gene from Giardia lamblia%蓝氏贾第鞭毛虫磷酸丙糖异构酶基因种内差异研究

    Institute of Scientific and Technical Information of China (English)

    卢思奇; 李继红; 张亚平; 文建凡; 王凤云

    2002-01-01

    目的探讨蓝氏贾第鞭毛虫(Giardia lamblia)磷酸丙糖异构酶基因种内差异.方法提取虫体总DNA,对所有虫株磷酸丙糖异构酶(tim)基因部分片段进行PCR扩增.测定序列后,用简约法和NJ法构建系统树进行系统发育分析.结果共有124个位点存在变异(占所有测定序列中的23%),且大多数为发生在密码子的同义突变. 两种构树方法所得二树的分枝结构相似,均将受试的16株蓝氏贾第虫分为明显的两组.结论宿主及地理因素对蓝氏贾第虫群体的遗传多样性影响不大.在DNA分子进化水平上,自然选择的影响十分显著.可将tim基因作为蓝氏贾第虫群体遗传结构一个十分有效的遗传标记.%Objective To investigate the intraspecific difference of the triose phosphate isomerase(ti m) gene from Giardia lamblia (G. lamblia).Methods Total genomic DNA of G. lamblia was extracted and partial fragments of the triose phosphate isomerase (tim) gene were amplified by polymerase chain reactio n (PCR). All nucleotide sequences were analyzed by using a phylogenetic analysi s, which was constructed with parsimony and Neighbor-joining (N-J) methods. Results A total of 124 variable sites (23% of all sequences detected) was defined, most of which were found at the silent sites of codons. Two similar phylogenetic tre es were constructed, subdividing 16 Giardia isolates into two groups. Conclusion The genetic diversity of G. lamblia appeared to be little affected by facto rs of both host and geography, while natural-selection played an important role in DNA molecular evolution level of the tim gene. The tim gene may be consider ed a very useful genetic marker of the population genetic structure of G. lam blia.

  4. Sacroiliac joint involvement in psoriasis.

    Science.gov (United States)

    Kaçar, Cahit; Sezer, Ilhan; Kocabaş, Hilal; Cay, Hasan Fatih; Cevikol, Can; Alpsoy, Erkan; Melikoğlu, Meltem Alkan; Akman, Ayşe

    2010-07-01

    Psoriasis is a skin disorder that is associated with arthritis. Sacroiliac joint involvement is considered to be less frequent than the other types of psoriatic arthritis. Additionally, the psoriatic sacroiliitis is considered to be asymmetric in general. We aimed to define the frequency and type of sacroiliac involvement in patients with psoriasis. Patients with psoriasis were included the study. Characteristics of skin, nail and articular involvement were noted. Psoriasis area and severity index was calculated. Antero-posterior pelvic X-rays were obtained and graded by two rheumatologists and a radiologist independently. One hundred and thirty-three patients were included. Thirty-seven of patients (27%) have articular involvement symptomatically. The sacroiliac joint involvement was observed in 34 (26%) of patients. More than one-half of sacroiliac involvement was bilateral while less than one-half was in symptomatic patients regarding sacroiliitis. Fifty-seven percentages of all patients have psoriatic nail involvement. Sacroiliac joint involvement did not show any significant association with psoriatic nail involvement or the severity of skin disease. We found higher frequency of sacroiliac joint involvement and bilateral sacroiliitis in patients with psoriasis. This is in contrast to present information about the association of psoriasis and sacroiliitis. These findings need confirmation by further studies and with more sophisticated techniques such as magnetic resonance imaging.

  5. Rearrangements in ground and excited states

    CERN Document Server

    de Mayo, Paul

    1980-01-01

    Rearrangements in Ground and Excited States, Volume 3 presents essays on the chemical generation of excited states; the cis-trans isomerization of olefins; and the photochemical rearrangements in trienes. The book also includes essays on the zimmerman rearrangements; the photochemical rearrangements of enones; the photochemical rearrangements of conjugated cyclic dienones; and the rearrangements of the benzene ring. Essays on the photo rearrangements via biradicals of simple carbonyl compounds; the photochemical rearrangements involving three-membered rings or five-membered ring heterocycles;

  6. Monoarticular Hip Involvement in Pseudogout

    Directory of Open Access Journals (Sweden)

    Figen Kocyigit

    2015-01-01

    Full Text Available Pseudogout is the acutest form of arthritis in the elderly. Although clinical manifestations vary widely, polyarticular involvement is typical mimicking osteoarthritis or rheumatoid arthritis. Monoarticular involvement is relatively rare and is generally provoked by another medical condition. There are reported cases of hip involvement by pseudogout in monoarticular form. However, all of the cases were presented as septic arthritis. In this report, we present a case of monoarticular hip involvement mimicking soft tissue abscess. We confirmed the pseudogout diagnosis after ultrasonographic evaluation of the involved hip joint and pathological and biochemical analysis of synovial fluid analysis. Diagnosis is important to avoid unnecessary medical and surgical treatment in cases of the bizarre involvement of hip in pseudogout.

  7. Skin involvement in Dupuytren's disease.

    OpenAIRE

    Wade, R.; Igali, L; Figus, A

    2016-01-01

    Whether the palmar skin has a role in the development, propagation or recurrence of Dupuytren's disease remains unclear. Clinical assessment for skin involvement is difficult and its correlation with histology uncertain. We prospectively biopsied the palmar skin of consecutive patients undergoing single digit fasciectomy (for primary Dupuytren's disease without clinically involved skin) and dermofasciectomy (for clinically involved skin or recurrence) in order to investigate this relationship...

  8. cDNA cloning of glucose-6-phosphate isomerase from crucian carp (Carassius carassius) and expression of the active region as myofibril-bound serine proteinase inhibitor in Escherichia coli.

    Science.gov (United States)

    Han, Long; Cao, Min-Jie; Shi, Chao-lan; Wei, Xiao-Nan; Li, Huan; Du, Cui-Hong

    2014-02-01

    Glucose-6-phosphate isomerase (GPI) (EC 5.3.1.9) can act as a myofibril-bound serine proteinase (MBSP) inhibitor (MBSPI) in fish. In order to better understand the biological information of the GPI and its functional domain for inhibiting MBSP, the cDNA of GPI was cloned from crucian carp (Carassius carassius) with RT-PCR, nested-PCR and 3'-RACE. The result of sequencing showed that the GPI cDNA had an open reading frame of 1662bp encoding 553 amino acid residues. After constructing and comparing the three-dimensional structures of GPI and MBSP, the middle fragment of crucian carp GPI (GPI-M) was predicted as a functional domain for inhibiting MBSP. Then the crucian carp GPI-M gene was cloned and expressed in Escherichia coli. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the recombinant GPI-M (rGPI-M) with molecular mass of approximately 21kDa in the form of inclusion bodies. The rGPI-M was obtained at an electrophoresis level purity of approximately 95% after denaturation and dialysis renaturation.

  9. Down-regulation of triose phosphate isomerase in Vineristine-resistant gastric cancer SGC7901 cell line identified by immobilized pH gradient two-dimensional gel electrophoresis and mierosequencing

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective:To exkplore new multidrug-resistance-related proteins in gastric SC7901 cells and clarify their mechanisms.Methods:Two-dimensional(2-D) polyacrylamide gel electrophoresis with immobilized pH gradients(IPG) was applied to compare the differential expression of multidrug-resistance-related proteins in gastric cancer SGC7901 cells and Vineristine-resistant SGC7901 cells (SGC7901/VCR) induced by vincristine sulfate.The 2-D gels were silver-stained.Then,preparative 2-D PAGE was performed.The differential proteins of PVDF membranes were cxcised and identified by N-terminal microsequencing.The mRNA expressions of differential proteins were detected in SGC 7901 cells and SGC7901/VCR cells by RT-PCR.Results:Approximatedly 680 protein sports were resolved on each 2-D gel by silver staining.Most protein spots showed no difference in composition,shape or density.25 proteins differed in abundance (6 higher in SGC7901/VCR cells;19 higher in 7901 cells);5 proteins were unique to one kind of cell or the othe(3 in SGC7901/VRC cells,2 in 7901 cells).One drug-resistance-related protein,which was down-regulated in SGC7901/VCR cells,was identified as trisephosphate isomerase(TPI),a glycolytic pathway enzyme.Conclusions:the results suggest that these differential proteins including TPI may be related to the Vincristine-resistant mechanism in human gastric cancer SGC7901/VCR cell line.

  10. Direct Employee Involvement Quality (DEIQ)

    NARCIS (Netherlands)

    Torka, Nicole; Woerkom, van Marianne; Looise, Jan-Kees

    2008-01-01

    This paper focuses on one aspect of human resource management (HRM) that is important for innovative employee behaviour: direct employee involvement quality (DEIQ). However, research has also shown that employee involvement is often in serious need of improvement. This paper presents evidence from t

  11. Involvement of inositol in reproduction.

    NARCIS (Netherlands)

    Beemster, P.; Groenen, P.; Steegers-Theunissen, R.P.M.

    2002-01-01

    Inositol is involved in several aspects of reproduction. It affects overall embryogenesis, may prevent neural tube defects, and stimulates the production of lung surfactant. This article will review the involvement of inositol in reproduction. After describing the biologic function of inositol and i

  12. Preparing Teachers for Parent Involvement.

    Science.gov (United States)

    Safran, Daniel

    This paper examines the potential impact of parent involvement in the formal education of their children and suggests ways that teacher education can be restructured to prepare teachers to work with parents. This paper attempts to answer five questions: (1) Why should parents be involved in the formal education of their children? (2) Why should…

  13. Citizen Involvement in Public Television.

    Science.gov (United States)

    Wenner, Lawrence A.

    The purpose of this study was to evaluate the amount and quality of citizen involvement in public television. From the perspective of the "average citizen," the concept of involvement is considered with regard to the Carnegie Commission, the Corporation for Public Broadcasting (CPB) borad of directors, the National Citizens Committee for…

  14. Parental Involvement and Academic Achievement

    Science.gov (United States)

    Goodwin, Sarah Christine

    2015-01-01

    This research study examined the correlation between student achievement and parent's perceptions of their involvement in their child's schooling. Parent participants completed the Parent Involvement Project Parent Questionnaire. Results slightly indicated parents of students with higher level of achievement perceived less demand or invitations…

  15. Involvement of anti-oxidative enzymes, photosynthetic pigments and flavonoid metabolism in the adaptation of Reaumuria soongorica to salt stress

    Institute of Scientific and Technical Information of China (English)

    YuBing Liu; Bo Cao; MeiLing Liu

    2013-01-01

    Reaumuria soongorica is a short woody shrub widely found in semi-arid areas of China. It can survive severe environ-mental stress including high salinity in its natural habitat. Thus, we investigated the involvement of anti-oxidative enzymes, photosynthetic pigments and flavonoid metabolism in the adaptation of R. soongorica to saline environments. R. soon-gorica was treated with 0, 100, 200 and 400 mM NaCl solutions for 14 days. Soil salt content increased significantly by watering with high content of NaCl solution, and no variation between 8 and 14 days during treatment. The levels of pe-roxidation of lipid membranes (measured by malondialdehyde content) and the activities of three antioxidant enzymes (superoxide dismutase (SOD), peroxidase (POD) and ascorbate peroxidase (APX)) increased under salt stress. Chlorophyll and carotenoid content decreased with increasing salt content. The ratio of Chl a/Chl b and carotenoid/Chl exhibited sig-nificant increase under 400 mM NaCl. However, total flavonoid and anthocyanin contents and key enzyme activities in the flavonoid pathway including phenylalanine ammonialyase (PAL) and Chalcone isomerase (CHI) decreased under salt stress. These findings possibly suggest that R. soongorica has an adaptation protection mechanism against salt-induced oxidative damage by inducing the activity of antioxidant enzymes and maintaining a steady level of carotenoid/Chl.

  16. Ocular involvement in pemphigus vulgaris.

    Science.gov (United States)

    Akhyani, Maryam; Keshtkar-Jafari, Alireza; Chams-Davatchi, Cheyda; Lajevardi, Vahide; Beigi, Sara; Aghazadeh, Nessa; Rayati Damavandi, Maede; Arami, Shabnam

    2014-07-01

    Pemphigus vulgaris (PV) is an autoimmune disorder affecting the skin and mucous membranes. Ocular involvement in PV has been reported but its prevalence and clinical characteristics are not well defined. This prospective cross-sectional study of 103 PV patients was designed to determine the prevalence, clinical types and epidemiological trends of ocular involvement in a population of Iranian patients with PV. Ocular involvement was present in 17 (16.5%) patients. Conjunctivitis was the most prevalent type of ocular involvement (9/17, 52.9%), followed by erosion of the palpebral conjunctiva (7/17, 41.2%). Erosion of the bulbar conjunctiva was noted in only one patient (5.9%). The most commonly reported symptoms were eye irritation (76.5%) and redness (76.5%). No significant relation was found between ocular involvement and disease activity (partial remission or relapse). Mucoid discharge was significantly more common in patients with conjunctival erosions as compared to patients with conjunctivitis (P = 0.038). We conclude that ocular involvement is not rare in PV; 16.5% of PV patients develop ocular disease independent of the disease activity and extension. Conjunctivitis is the most common type of involvement, however, palpebral conjunctival erosion is more frequent than previously realized.

  17. GOVERNMENT INVOLVEMENT IN CONSUMPTION BEHAVIOUR

    Directory of Open Access Journals (Sweden)

    CRISTINA ZAMFIR

    2010-01-01

    Full Text Available In this article, we will follow the involvement that the government has,through its expenses, on the consumption behavior. The involvement that the government has inthe consumption behavior is made through fees and taxes that are applied on income. Fees andtaxes are applied to the different forms of income but in this article we will be focused only onthe influence of them on wages. In order to analyze the involvement of government expenses onconsumption behavior an utility model will be used.

  18. User Involvement And Entrepreneurial Action

    Directory of Open Access Journals (Sweden)

    Eva Heiskanen

    2007-01-01

    Full Text Available Involving users in the innovation process is a subject of much research, experimentation, and debate. Less attention has been given to the limits to user involvement that ensue from specific organizational characteristics. This article explores barriers to the utilization of users’ input in two small companies developing interactive digital applications. We contrast our findings to earlier research involving large companies to identify features of entrepreneurial sensemaking and action that influence the utilization of users’ input. We find that the small companies follow a distinct action rationality, leading to rapid implementation of some user inputs, and defensiveness toward others. Both sets of data also reveal common features that are often overlooked in the literature. We reconceptualize user involvement as a form of interaction between users and innovating companies that is facilitated and constrained by micro-sociological processes, on the one hand, and the nature of the competitive environment, on the other.

  19. Strategy Innovation with Employee Involvement

    DEFF Research Database (Denmark)

    Friis, Ole Uhrskov; Koch, Christian

    2015-01-01

    if the managers still dominate, some processes of direct involvement of employees occur, in particular when employees are asked to supplement overall strategic goals and when they directly shape several sub-strategies. Strategy practices found include strategy planning, an open space workshop and organised......The purpose of this article is to investigate how employees can be involved in strategy innovation processes and how new strategy practices (new tools and procedures) are used to change strategy praxis in order to sustain value creation. In the strategizing actions, we found that even...... strategy projects. Especially the latter two are important in facilitating the employee involvement. The case, however, also exhibits enterprise-situated praxises related to unplanned events, like the mitigation of taboos....

  20. Involving Employees in Strategy innovation

    DEFF Research Database (Denmark)

    Friis, Ole Uhrskov; Koch, Christian

    2011-01-01

    some of its employees. Even if managers still dominate, some processes of direct involvement of employees occur. The employees are in particular active in supplementing overall strategic goals and directly shaping one sub strategy, that of ‘process’. Strategy practices include planning, Porterian......Strategy as a practice and continuous innovation approaches are combined to conceptualise dilemmas of short versus long term and to analyse a case of employee participation as a particular example of strategy innovation. The case is a medium size textile company developing its strategy involving...... and Balanced Score Card consultancy, an ‘open space’ workshop and organized strategy projects. Especially the latter two are important in facilitating the employee involvement. The case however also exhibit enterprise situated praxis’s like mitigation of taboos....

  1. User involvement in care work

    DEFF Research Database (Denmark)

    Dybbroe, Betina; Kamp, Annette

    effectiveness and shared responsibility for care pathways. While NPM position users as consumers making their free choice, the user involvement paradigm underlines the users’ active participation in the mastering of their problems and disease. Research is scarce on this theme, and has until now primarily......In recent years user involvement has become a paradigm for transforming the health and social care sector. This development–also labelled empowerment, co-creation, partnership, patient-centeredness - is seen as a means to reform organizations in ways that enhance quality, economic cost...... addressed the way this paradigm affects the users, in specific sectors. However user involvement also affects working life. It may imply change and redistribution of tasks and identities between users and professionals, and may also transform the relations of care. In this paper we explore the possible...

  2. Gastrointestinal involvement in systemic sclerosis.

    Science.gov (United States)

    Savarino, Edoardo; Furnari, Manuele; de Bortoli, Nicola; Martinucci, Irene; Bodini, Giorgia; Ghio, Massimo; Savarino, Vincenzo

    2014-10-01

    Systemic sclerosis is an autoimmune chronic disease characterised by microvascular, muscular and immunologic abnormalities that lead to progressive and systemic deposition of connective tissue in the skin and internal organs. The gastrointestinal tract is often overlooked by physicians but it is the most affected organ after the skin, from the mouth to the anus. Indeed, 80% of SSc patients may present with gastrointestinal involvement. Gastrointestinal manifestations range from bloating and heartburn to dysphagia and anorectal dysfunction to severe weight loss and malabsorption. However, the gastrointestinal involvement is rarely the direct cause of death, but has great impact on quality of life and leads to several comorbidities that subsequently affect patients' survival. Treatments, including nutritional support and prokinetics provide limited benefits and do not arrest the progressive course of the disease, but earlier detection of gastrointestinal involvement may reduce the risk of complications such as malnutrition.

  3. Histochemical activity of 5-4-isomerase-3-B hydroxy steroid dehydrogenase in the ovary of the viviparous mexican lizardSceloporus mucronatus (Reptilia:Prhynosomatidae) and interelationship with progesterone levels during pregnancy

    Institute of Scientific and Technical Information of China (English)

    Martn Martnez-torres; E Martha Prez-armendariz; M Elena Hernndez Caballero; Juana luis; guadalupe ortz-Lpez

    2012-01-01

    Objective:To relate the histological characteristics and histochemicalΔ5-4-isomerase-3 beta hydroxy steroid dehydrogenase(Δ5-43β-HSD) activity of the corpora lutea(CL) and the atresic vitellogenic follicles(AVF) with progesterone(P4) plasma concentrations in three different times of gestation (early, medium and late) in the viviparous lizardSceloporus mucronatus (S. mucronatus).Methods:The histological characteristics as well as histochemical activity ofΔ5-43β-HSD of theCL andAVF and their relationship with plasmaP4 levels were studied during three different times of pregnancy of the viviparous lizardS. mucronatus.Results:Corpora lutea develops during the first third of gestation.In second third, the luteal tissue reaches maturity and starts the first regressive changes.The last third of gestation was characterized by a considerably advance in the luteolysis.Activity ofΔ5-43β-HSD was observed in he luteal cell mas.The activity of this enzyme were high during the first third and scantle activity was detected in the last third.Even though atresic vitellogenic follicles are found throughout the whole period of gestation,Δ5-43β-HSD activity is very low in relation with showed byCL and does not change significantly in the studied period of time.Another hand, we observed a direct relationship among the histological aspect of the corpus luteum,Δ5-43β-HSD activity and progesterone levels. Conclusions:These observations suggests that the corpus luteum is the most important source of ovarian progesterone(P4) during pregnancy and that the participation of theAVF in the production of this hormone is little or non-existent.

  4. The Activity and Localization of 3β-hydroxysteroid Dehydrogenase/Δ5-Δ4 Isomerase and Release of Androstenedione and Progesterone by Uterine Tissues During Early Pregnancy and the Estrous Cycle in Pigs

    Science.gov (United States)

    WOJCIECHOWICZ, Bartosz; KOTWICA, Genowefa; KOLAKOWSKA, Justyna; FRANCZAK, Anita

    2012-01-01

    Abstract Steroid hormones are produced by the porcine uterus. We hypothesized that the uterus in pigs possesses active 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD) responsible for progesterone and androstenedione production, that uterine steroids may supplement the amount of steroid hormones produced by embryos and corpus luteum and that these steroids are necessary for maintenance of pregnancy. In this study, we examined 1) endometrial and myometrial expression of 3β-HSD mRNA, 2) uterine 3β-HSD protein activity and 3) in vitro production of A4 and P4 by uterine slices harvested from pigs on days 10 to 11, 12 to 13 and 15 to 16 of pregnancy and the estrous cycle. The expression of 3β-HSD and the presence and activity of 3β-HSD protein were different in the endometrium and the myometrium during the examined periods of pregnancy and the estrous cycle. Production of A4 by the endometrium and myometrium was highest on days 12 to 13 of pregnancy and the estrous cycle. Endometrial secretion of P4 did not differ in the course of early pregnancy and on the respective days of the estrous cycle. The gravid myometrium was the highest source of P4 in pregnant pigs on days 12 to 13. The release of P4 by the cyclic myometrium rose during the examined days of the estrous cycle. The steroidogenic activity of the uterus, as described in this study, may support early pregnancy or the luteal phase of the estrous cycle in pigs. PMID:23095516

  5. The activity and localization of 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase and release of androstenedione and progesterone by uterine tissues during early pregnancy and the estrous cycle in pigs.

    Science.gov (United States)

    Wojciechowicz, Bartosz; Kotwica, Genowefa; Kolakowska, Justyna; Franczak, Anita

    2013-01-01

    Steroid hormones are produced by the porcine uterus. We hypothesized that the uterus in pigs possesses active 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3β-HSD) responsible for progesterone and androstenedione production, that uterine steroids may supplement the amount of steroid hormones produced by embryos and corpus luteum and that these steroids are necessary for maintenance of pregnancy. In this study, we examined 1) endometrial and myometrial expression of 3β-HSD mRNA, 2) uterine 3β-HSD protein activity and 3) in vitro production of A(4) and P(4) by uterine slices harvested from pigs on days 10 to 11, 12 to 13 and 15 to 16 of pregnancy and the estrous cycle. The expression of 3β-HSD and the presence and activity of 3β-HSD protein were different in the endometrium and the myometrium during the examined periods of pregnancy and the estrous cycle. Production of A(4) by the endometrium and myometrium was highest on days 12 to 13 of pregnancy and the estrous cycle. Endometrial secretion of P(4) did not differ in the course of early pregnancy and on the respective days of the estrous cycle. The gravid myometrium was the highest source of P(4) in pregnant pigs on days 12 to 13. The release of P(4) by the cyclic myometrium rose during the examined days of the estrous cycle. The steroidogenic activity of the uterus, as described in this study, may support early pregnancy or the luteal phase of the estrous cycle in pigs.

  6. Mutagenesis breeding of Lactobacillus bulgaricus and enzymatic properties of linoleate isomerase%产亚油酸异构酶菌株的诱变选育及其酶学性质研究

    Institute of Scientific and Technical Information of China (English)

    游庆红; 尹秀莲; 蒋中海

    2011-01-01

    Using Lactobacillus bulgaricus as original strain, two mutated high enzyme-producing strains A1 and A2 were obtained by ultraviolet mutation. The enzyme activity of the mutant strains A1 and A2 were 30. 1U/ml and 32. 1U/mi, which increased by 45% and 54% to the original strain, respectively. Study on enzymatic properties indicated that for stain A2, the optimum linoleate isomerase-producing temperature and pH was 40℃ and 6. 5 respectively, and the enzyme was stable when the temperature lower than 40℃ and pH in the range of 6. 0 ~ 7. 0. The maximum reaction rate Km and Vm were 0. 039mmol/L and 0. 24mmol/( h · mg), respectively when determined with enzyme dynamics experiment.%以保加利亚乳杆菌为出发菌株,经紫外诱变选育,获得2株高产酶突变株A1及A2,其酶活分别为30.1U/mL、32.1U/mL,酶活较出发菌株分别提高45%和54%.酶学性质研究表明,A2菌株产亚油酸异构酶的最适反应温度分别为40℃,最适pH 6.5,酶在低于40%时具有良好的热稳定性,pH 6.0~7.0时较稳定.以Lineweaver-Burk双倒数作图法求得亚油酸异构酶的Km为0.039 mmol/L,Vmax为0.24mmol/(h·mg).

  7. [Heel involvement in rheumatoid polyarthritis].

    Science.gov (United States)

    Bouysset, M; Tebib, J G; Vianey, J C; Berthier, M; Nemoz, J C; Chaumentin, G; Schnepp, J; Llorca, G; Bouvier, M

    1990-11-01

    Calcaneus involvement during the course of RA is poorly known. A clinical and radiological study of 408 consecutive rheumatoid feet are then reported. If talalgia was seldomly noted (3.7 p. cent), plantar calcaneitis was found in 29.7 p. cent as plantar spur. Similarly, posterior exostosis was displayed in 30.5 p. cent of patients. These radiological abnormalities are increased in RA but appeared more as a consequence of the statical modification of the foot secondary to RA process than as a direct involvement. Logical orthopedic therapeutics are then proposed.

  8. Multisystem involvement in neuromyelitis optica

    Directory of Open Access Journals (Sweden)

    Megan M Langille

    2015-01-01

    Full Text Available We describe a case of pediatric neuromyelitis optica (NMO with muscle and lung involvement in addition to central nervous system disease. Our patient initially presented with features of area postrema syndrome, then subsequently with optic neuritis. The patient also had recurrent hyperCKemia that responded to corticosteroids. Finally, axillary and hilar adenopathy with pulmonary consolidation were noted as well and responded to immunomodulation. Our case highlights multisystem involvement in NMO including non-infectious pulmonary findings which have not been described in the pediatric population previously.

  9. Vertebral osteomyelitis without disc involvement

    Energy Technology Data Exchange (ETDEWEB)

    Kamani, I.; Syed, I.; Saifuddin, A. E-mail: asaifuddin@aol.com; Green, R.; MacSweeney, F

    2004-10-01

    Vertebral osteomyelitis is most commonly due to pyogenic or granulomatous infection and typically results in the combined involvement of the intervertebral disc and adjacent vertebral bodies. Non-infective causes include the related conditions of chronic recurrent multifocal osteomyelitis (CRMO) and SAPHO (synovitis, acne, pustulosis, hyperostosis, and osteitis) syndrome. Occasionally, these conditions may present purely within the vertebral body, resulting in various combinations of vertebral marrow oedema and sclerosis, destructive lesions of the vertebral body and pathological vertebral collapse, thus mimicking neoplastic disease. This review illustrates the imaging features of vertebral osteomyelitis without disc involvement, with emphasis on magnetic resonance imaging (MRI) findings.

  10. Promoting Active Involvement in Classrooms

    Science.gov (United States)

    Conderman, Greg; Bresnahan, Val; Hedin, Laura

    2012-01-01

    This article presents a rationale for using active involvement techniques, describes large- and small-group methods based on their documented effectiveness and applicability to K-12 classrooms, and illustrates their use. These approaches include ways of engaging students in large groups (e.g., unison responses, response cards, dry-erase boards,…

  11. Parent Involvement as Ritualized Practice

    Science.gov (United States)

    Doucet, Fabienne

    2011-01-01

    This article examines parent involvement (PI) as a ritual system using Turner's concept of root paradigms. Through a twofold analysis, I argue that the highly ritualized nature of PI practices creates a group identity among mainstream parents and schools that marginalizes diverse families. First, I point out three root paradigms in the ritual…

  12. Putaminal involvement in Rasmussen encephalitis

    Energy Technology Data Exchange (ETDEWEB)

    Rajesh, Bhagavatheeswaran; Ashalatha, Radhakrishnan [Sree Chitra Tirunal Institute for Medical Sciences and Technology, Department of Neurology, Trivandrum, Kerala (India); Kesavadas, Chandrasekharan; Thomas, Bejoy [Sree Chitra Tirunal Institute for Medical Sciences and Technology, Department of Imaging Sciences and Interventional Radiology, Trivandrum, Kerala (India)

    2006-08-15

    Rasmussen encephalitis (RE) is a rare devastating disease of childhood causing progressive neurological deficits and intractable seizures, typically affecting one hemisphere. Characteristic MRI features include progressive unihemispheric focal cortical atrophy and grey- or white-matter high-signal changes and basal ganglion involvement, particularly of the caudate nucleus. To analyse the pattern of involvement of different brain structures in a series of patients with RE and to attempt clinical correlation. We reviewed the medical records and neuroimaging data of 12 patients diagnosed with RE satisfying the European Consensus Statement diagnostic criteria. The disease manifested as seizures in all patients and was refractory; epilepsia partialis continua was a notable feature (nine patients). Hemiparesis of varying grades was noted in all but one patient; none had extrapyramidal signs. Neuroimaging showed cortical involvement in the insular/periinsular regions in 11 patients. Caudate atrophy was noted in ten patients. Putaminal atrophy was seen in nine patients, six of whom had additional hyperintense signal changes. Our study highlights frequent putaminal atrophy and signal changes in RE, which suggests a more extensive basal ganglion involvement than emphasized previously. Recognition of putaminal changes may be a useful additional tool in the radiological diagnosis of RE. (orig.)

  13. Managing Parent Involvement during Crisis

    Science.gov (United States)

    Merriman, Lynette S.

    2008-01-01

    In the wake of 9/11, Hurricane Katrina, and the Virginia Tech shooting tragedy, it is no surprise that concern for students' safety is the primary reason attributed to parents' increased involvement. Parents and university administrators share in their commitment to student safety. However, college and university staff who assume responsibility…

  14. Cyclophilin A associates with enterovirus-71 virus capsid and plays an essential role in viral infection as an uncoating regulator.

    Directory of Open Access Journals (Sweden)

    Jie Qing

    2014-10-01

    Full Text Available Viruses utilize host factors for their efficient proliferation. By evaluating the inhibitory effects of compounds in our library, we identified inhibitors of cyclophilin A (CypA, a known immunosuppressor with peptidyl-prolyl cis-trans isomerase activity, can significantly attenuate EV71 proliferation. We demonstrated that CypA played an essential role in EV71 entry and that the RNA interference-mediated reduction of endogenous CypA expression led to decreased EV71 multiplication. We further revealed that CypA directly interacted with and modified the conformation of H-I loop of the VP1 protein in EV71 capsid, and thus regulated the uncoating process of EV71 entry step in a pH-dependent manner. Our results aid in the understanding of how host factors influence EV71 life cycle and provide new potential targets for developing antiviral agents against EV71 infection.

  15. PIN1 in breast development and cancer: a clinical perspective.

    Science.gov (United States)

    Rustighi, Alessandra; Zannini, Alessandro; Campaner, Elena; Ciani, Yari; Piazza, Silvano; Del Sal, Giannino

    2017-02-01

    Mammary gland development, various stages of mammary tumorigenesis and breast cancer progression have the peptidyl-prolyl cis/trans isomerase PIN1 at their centerpiece, in virtue of the ability of this unique enzyme to fine-tune the dynamic crosstalk between multiple molecular pathways. PIN1 exerts its action by inducing conformational and functional changes on key cellular proteins, following proline-directed phosphorylation. Through this post-phosphorylation signal transduction mechanism, PIN1 controls the extent and direction of the cellular response to a variety of inputs, in physiology and disease. This review discusses PIN1's roles in normal mammary development and cancerous progression, as well as the clinical impact of targeting this enzyme in breast cancer patients.

  16. Import of periplasmic bacteriocins targeting the murein.

    Science.gov (United States)

    Braun, Volkmar; Helbig, Stephanie; Patzer, Silke I

    2012-12-01

    Colicins are the only proteins imported by Escherichia coli and thus serve as tools to study the protein import mechanism. Most of the colicins studied degrade DNA, 16S RNA or tRNA in the cytoplasm, or form pores in the cytoplasmic membrane. Two bacteriocins, Cma (colicin M) and Pst (pesticin), affect the murein structure in the periplasm. These two bacteriocins must be imported only across the outer membrane and therefore represent the simplest system for studying protein import. Cma can be reversibly translocated across the outer membrane. Cma and Pst unfold during import. The crystal structure of Pst reveals a phage T4L (T4 lysozyme) fold of the activity domain. Both bacteriocins require energy for import which is translocated from the cytoplasmic membrane into the outer membrane by the Ton system. Cma kills cells only when the periplasmic FkpA PPIase (peptidylprolyl cis-trans isomerase)/chaperone is present.

  17. Genetic manipulation of carotenoid biosynthesis in the green sulfur bacterium Chlorobium tepidum

    DEFF Research Database (Denmark)

    Frigaard, Niels-Ulrik; Maresca, Julia A; Yunker, Colleen E

    2004-01-01

    The green sulfur bacterium Chlorobium tepidum is a strict anaerobe and an obligate photoautotroph. On the basis of sequence similarity with known enzymes or sequence motifs, nine open reading frames encoding putative enzymes of carotenoid biosynthesis were identified in the genome sequence of C....... tepidum, and all nine genes were inactivated. Analysis of the carotenoid composition in the resulting mutants allowed the genes encoding the following six enzymes to be identified: phytoene synthase (crtB/CT1386), phytoene desaturase (crtP/CT0807), zeta-carotene desaturase (crtQ/CT1414), gamma......-carotene desaturase (crtU/CT0323), carotenoid 1',2'-hydratase (crtC/CT0301), and carotenoid cis-trans isomerase (crtH/CT0649). Three mutants (CT0180, CT1357, and CT1416 mutants) did not exhibit a discernible phenotype. The carotenoid biosynthetic pathway in C. tepidum is similar to that in cyanobacteria and plants...

  18. Parasite cyclophilins and antiparasite activity of cyclosporin A.

    Science.gov (United States)

    Page, A P; Kumar, S; Carlow, C K

    1995-10-01

    Cyclosporin A (CsA) was initially developed as an immunosuppressive drug. In the past several years, it has been shown to possess antiparasite activity independent of the immune system. It is not known how the drug exerts these antiparasite effects, or why it is stage and/or species specific. The answers may lie in the enzymatic function of cyclophilins. The cyclophilins are a growing family of proteins that exhibit peptidyl-prolyl cis-trans isomerase (PPiase) activity and bid CsA to varying degrees. PPiases have been shown to play a role in the folding of many essential proteins. Antony Page, Sanjay Kumar and Clotilde Carlow here review parasite cyclophilins and their association with CsA. The possible biological function of parasite cyclophilins and their potential role in future drug discovery are also discussed.

  19. The Emerging Roles of Early Protein Folding Events in the Secretory Pathway in the Development of Neurodegenerative Maladies

    Science.gov (United States)

    Dubnikov, Tatyana; Cohen, Ehud

    2017-01-01

    Although, protein aggregation and deposition are unifying features of various neurodegenerative disorders, recent studies indicate that different mechanisms can lead to the development of the same malady. Among these, failure in early protein folding and maturation emerge as key mechanistic events that lead to the manifestation of a myriad of illnesses including Alzheimer's disease and prion disorders. Here we delineate the cascade of maturation steps that nascent polypeptides undergo in the secretory pathway to become functional proteins, and the chaperones that supervise and assist this process, focusing on the subgroup of proline cis/trans isomerases. We also describe the chaperones whose failure was found to be an underlying event that initiates the run-up toward neurodegeneration as well as chaperones whose activity impairs protein homeostasis (proteostasis) and thus, promotes the manifestation of these maladies. Finally, we discuss the roles of aggregate deposition sites in the cellular attempt to maintain proteostasis and point at potential targets for therapeutic interventions. PMID:28223916

  20. Enhancement of lipase r27RCL production in Pichia pastoris by regulating gene dosage and co-expression with chaperone protein disulfide isomerase.

    Science.gov (United States)

    Sha, Chong; Yu, Xiao-Wei; Lin, Nai-Xin; Zhang, Meng; Xu, Yan

    2013-12-10

    Pichia pastoris has been successfully used in the production of many secreted and intracellular recombinant proteins, but there is still a large room of improvement for this expression system. Two factors drastically influence the lipase r27RCL production from Rhizopus chinensis CCTCC M201021, which are gene dosage and protein folding in the endoplasmic reticulum (ER). Regarding the effect of gene dosage, the enzyme activity for recombinant strain with three copies lipase gene was 1.95-fold higher than that for recombinant strain with only one copy lipase gene. In addition, the lipase production was further improved by co-expression with chaperone PDI involved in the disulfide bond formation in the ER. Overall, the maximum enzyme activity reached 355U/mL by the recombinant strain with one copy chaperone gene PDI plus five copies lipase gene proRCL in shaking flasks, which was 2.74-fold higher than that for the control strain with only one copy lipase gene. Overall, co-expression with PDI vastly increased the capacity for processing proteins of ER in P. pastoris.

  1. Liver involvement in systemic infection

    Institute of Scientific and Technical Information of China (English)

    Masami; Minemura; Kazuto; Tajiri; Yukihiro; Shimizu

    2014-01-01

    The liver is often involved in systemic infections,resulting in various types of abnormal liver function test results.In particular,hyperbilirubinemia in the range of 2-10 mg/dL is often seen in patients with sepsis,and several mechanisms for this phenomenon have been proposed.In this review,we summarize how the liver is involved in various systemic infections that are not considered to be primarily hepatotropic.In most patients with systemic infections,treatment for the invading microbes is enough to normalize the liver function tests.However,some patients may show severe liver injury or fulminant hepatic failure,requiring intensive treatment of the liver.

  2. Inclusive Briefing and User Involvement

    DEFF Research Database (Denmark)

    Jensen, Per Anker

    2011-01-01

    Briefing is not just about specifying needs as requirements but also about evaluating how well design proposals fulfil needs and aspirations. Furthermore, briefing is not only about building design. Briefing starts at the preproject stage to create a basis for the project decision and can include...... a number of different processes with varying purposes before and during the design and construction activities. Thus, briefing can be regarded as a continuous process but it should also be an inclusive and interactive process with the involvement of all stakeholders, including end users. This article...... includes a literature study on briefing and user involvement in building projects, and presents a case study of a major building project of a new headquarters and media centre for the Danish Broadcasting Corporation in Copenhagen. The building project was actively used as part of a corporate change process...

  3. Lupus panniculitis involving the breast

    Energy Technology Data Exchange (ETDEWEB)

    Sabate, Josep M.; Gomez, Antonio; Torrubia, Sofia; Salinas, Teresa; Clotet, Montse [Universitat Autonoma de Barcelona, Unity of Breast Imaging, Department of Diagnostic Radiology, Hospital de Sant Pau, Barcelona (Spain); Lerma, Enrique [Universitat Autonoma de Barcelona, Department of Pathology, Hospital de Sant Pau, Barcelona (Spain)

    2006-01-01

    Lupus panniculitis is an unusual immunological disease that characteristically affects the subcutaneous fat and occurs in 2% of patients with systemic lupus erythematosus. We report a case of lupus panniculitis involving the breast, which represents a very uncommon location. Mammographically, it presented as a suspicious irregular mass involving the subcutaneous fat pad with skin thickening. High echogenicity constituted the most relevant sonographic finding. To the best of our knowledge, the magnetic resonance (MR) features have not been previously described. High signal intensity was found on both T1- and T2-weighted precontrast MR images. A dynamic contrast-enhanced study revealed a suspicious focal mass with irregular margins and rim enhancement, with a type 3 time-signal intensity curve. Differential diagnosis with carcinoma and fat necrosis and the value of core biopsy are discussed. (orig.)

  4. Cardiac involvement in rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    V. De Gennaro Colonna

    2011-06-01

    Full Text Available Rheumatoid arthritis (RA is a systemic disease of unknown etiology characterized by a chronic inflammatory process mainly leading to destruction of synovial membrane of small and major diarthrodial joints. The prevalence of RA within the general adult population is about 1% and female subjects in fertile age result mostly involved. It’s an invalidating disease, associated with changes in life quality and a reduced life expectancy. Moreover, we can observe an increased mortality rate in this population early after the onset of the disease. The mortality excess can be partially due to infective, gastrointestinal, renal or pulmonary complications and malignancy (mainly lung cancer and non- Hodgkin lymphoma. Among extra-articular complications, cardiovascular (CV involvement represents one of the leading causes of morbidity and mortality. Every cardiac structure can be affected by different pathogenic pathways: heart valves, conduction system, myocardium, endocardium, pericardium and coronary arteries. Consequently, different clinical manifestations can be detected, including: pericarditis, myocarditis, myocardial fibrosis, arrhythmias, alterations of conduction system, coronaropathies and ischemic cardiopathy, valvular disease, pulmonary hypertension and heart failure. Considering that early cardiac involvement negatively affects the prognosis, it is mandatory to identify high CV risk RA patients to better define long-term management of this population.

  5. Laryngeal Involvement of Multiple Myeloma

    Directory of Open Access Journals (Sweden)

    Ariel B. Grobman

    2012-01-01

    Full Text Available The objectives of this paper are to discuss a rare cause of laryngeal multiple myeloma, to review unique pathologic findings associated with plasma cell neoplasms, to discuss epidemiology, differential diagnosis, and treatment options for plasma cell neoplasms of the larynx. Laryngeal multiple myeloma, also noted in the literature as “metastatic” multiple myeloma, presenting as a de novo laryngeal mass is extremely rare with few reported cases. Laryngeal involvement of extramedullary tumors is reported to be between 6% and 18% with the epiglottis, glottis, false vocal folds, aryepiglottic folds, and subglottis involved in decreasing the order of frequency. We present the case of a 58-year-old male with a history of IgA smoldering myeloma who presented to a tertiary care laryngological practice with a two-month history of dysphonia, which was found to be laryngeal involvement of multiple myeloma. We review the classification of and differentiation between different plasma cell neoplasms, disease workups, pathologic findings, and treatment options.

  6. Neuronal involvement in cisplatin neuropathy

    DEFF Research Database (Denmark)

    Krarup-Hansen, A; Helweg-Larsen, Susanne Elisabeth; Schmalbruch, H

    2007-01-01

    Although it is well known that cisplatin causes a sensory neuropathy, the primary site of involvement is not established. The clinical symptoms localized in a stocking-glove distribution may be explained by a length dependent neuronopathy or by a distal axonopathy. To study whether the whole neuron...... of large dorsal root ganglion cells. Motor conduction studies, autonomic function and warm and cold temperature sensation remained unchanged at all doses of cisplatin treatment. The results of these studies are consistent with degeneration of large sensory neurons whereas there was no evidence of distal...

  7. Modeling interdisciplinary activities involving Mathematics

    DEFF Research Database (Denmark)

    Iversen, Steffen Møllegaard

    2006-01-01

    In this paper a didactical model is presented. The goal of the model is to work as a didactical tool, or conceptual frame, for developing, carrying through and evaluating interdisciplinary activities involving the subject of mathematics and philosophy in the high schools. Through the terms...... domains (Michelsen, 2001, 2005a, 2005b). Furthermore the theoretical description rest on a series of qualitative interviews with teachers from the Danish high school (grades 9-11) conducted recently. The special case of concrete interdisciplinary activities between mathematics and philosophy is also...

  8. Neuronal involvement in cisplatin neuropathy

    DEFF Research Database (Denmark)

    Krarup-Hansen, A; Helweg-Larsen, Susanne Elisabeth; Schmalbruch, H;

    2007-01-01

    of large dorsal root ganglion cells. Motor conduction studies, autonomic function and warm and cold temperature sensation remained unchanged at all doses of cisplatin treatment. The results of these studies are consistent with degeneration of large sensory neurons whereas there was no evidence of distal......Although it is well known that cisplatin causes a sensory neuropathy, the primary site of involvement is not established. The clinical symptoms localized in a stocking-glove distribution may be explained by a length dependent neuronopathy or by a distal axonopathy. To study whether the whole neuron...

  9. Cloning and Expression Analysis of Chalcone Isomerase Gene from Narcissus tazetta var.chinensis%中国水仙查尔酮异构酶基因的克隆与表达分析

    Institute of Scientific and Technical Information of China (English)

    蔡雪玲; 陈晓静; 叶一江; 何玮毅; 申艳红

    2011-01-01

    Chalcone isomerase(CHI)is one of the key enzymes in the biosynthesis pathway of flavonoids and plays an important role in the process of flower color development. Three genes, named NtCHIW, NtCHIJ and NtCHIY respectively, were cloned from Narcissus tazetta var. Chinensis petal by RT-PCR and RACE. The open reading frame encompassed 735 bp encoding a polypeptide of 244 amino acids. Sequencing analysis showed that the amino acid sequence of Baihua Narcissus was 93.03%, 93.44%, 64.75%, 55.42%, 50.58%, 59.43%, and 58.20% homologous with CHI genes from Jinzhanyintai Narcissus, Huanghua Narcissus, Allium cepa, Elaeis oleifera, Vitis labrusca, Oryza saliva Japonica, Arabidopsis thaliana, respectively. The result of real time RT-PCR showed that the transcription expression of CHI gene changed accordingly during flower blooming, indicating the possible role of CHI genes in the change of flower color.%查尔酮异构酶(CHI)是影响类黄酮合成的一个重要限速酶,在植物花色发育过程中起着重要作用.通过RT-PCR和RACE技术从中国水仙的花瓣中克隆得到3条CHI基因,分别命名为NtCHIW、NtCHIJ和NtCHIY.3个基因均含有一个735 bp的开放阅读框(ORF),编码244个氨基酸.氨基酸序列分析表明:白花水仙与金盏银台、黄花水仙、洋葱、粳稻、拟南芥、油棕榈、葡萄相应基因的氨基酸序列同源性分别为93.03%、93.44%、64.75%、55.42%、50.58%、59.43%、58.20%.荧光定量PCR分析表明:随着花开放的过程,中国水仙的CHI基因转录水平发生变化,说明CHI基因可能参与花色变化的过程.

  10. The dehydrogenase region of the NADPH oxidase component Nox2 acts as a protein disulfide isomerase (PDI) resembling PDIA3 with a role in the binding of the activator protein p67phox

    Science.gov (United States)

    Bechor, Edna; Dahan, Iris; Fradin, Tanya; Berdichevsky, Yevgeny; Zahavi, Anat; Rafalowski, Meirav; Federman-Gross, Aya; Pick, Edgar

    2015-02-01

    The superoxide (O2.-)-generating NADPH oxidase of phagocytes consists of a membrane component, cytochrome b558 (a heterodimer of Nox2 and p22phox), and four cytosolic components, p47phox, p67phox, p40phox, and Rac. The catalytic component, responsible for O2.- generation, is Nox2. It is activated by the interaction of the dehydrogenase region (DHR) of Nox2 with the cytosolic components, principally with p67phox. Using a peptide-protein binding assay, we found that Nox2 peptides containing a 369CysGlyCys371 triad (CGC) bound p67phox with high affinity, dependent upon the establishment of a disulfide bond between the two cysteines. Serially truncated recombinant Nox2 DHR proteins bound p67phox only when they comprised the CGC triad. CGC resembles the catalytic motif (CGHC) of protein disulfide isomerases (PDIs). This led to the hypothesis that Nox2 establishes disulfide bonds with p67phox via a thiol-dilsulfide exchange reaction and, thus, functions as a PDI. Evidence for this was provided by the following: 1. Recombinant Nox2 protein, which contained the CGC triad, exhibited PDI-like disulfide reductase activity; 2. Truncation of Nox2 C-terminal to the CGC triad or mutating C369 and C371 to R, resulted in loss of PDI activity; 3. Comparison of the sequence of the DHR of Nox2 with PDI family members revealed three small regions of homology with PDIA3; 4. Two monoclonal anti-Nox2 antibodies, with epitopes corresponding to regions of Nox2/PDIA3 homology, reacted with PDIA3 but not with PDIA1; 5. A polyclonal anti-PDIA3 (but not an anti-PDIA1) antibody reacted with Nox2; 6. p67phox, in which all cysteines were mutated to serines, lost its ability to bind to a Nox2 peptide containing the CGC triad and had an impaired capacity to support oxidase activity in vitro. We propose a model of oxidase assembly in which binding of p67phox to Nox2 via disulfide bonds, by virtue of the intrinsic PDI activity of Nox2, stabilizes the primary interaction between the two components.

  11. Subfascial involvement in glomuvenous malformation

    Energy Technology Data Exchange (ETDEWEB)

    Shaikh, Raja; Alomari, Ahmad I.; Chaudry, Gulraiz [Boston Children' s Hospital, Division of Interventional Radiology, Boston, MA (United States); Mulliken, John B. [Boston Children' s Hospital, Division of Plastic Surgery, Boston, MA (United States); Fishman, Steven J. [Boston Children' s Hospital, Department of Surgery, Boston, MA (United States); Kozakewich, Harry P.W. [Boston Children' s Hospital, Department of Pathology, Boston, MA (United States)

    2014-07-15

    Glomuvenous malformation (GVM) is an inherited autosomal dominant trait. The lesions, which appear as bluish nodules or plaque-like cutaneous elevations, are usually tender and more firm than sporadic venous malformations. Conventionally, the lesions are thought to be limited to the cutaneous and subcutaneous tissue planes. The objective was to characterize the depth of involvement of GVM lesions. Magnetic resonance imaging (MRI) findings in GVM were retrospectively evaluated by two radiologists. The signal characteristics, tissue distribution, pattern of contrast enhancement of the lesions in GVM were documented. Thirty patients (19 female) aged 1-35 years (mean 18 years) were diagnosed with GVM based on clinical features (n = 20) and/or histopathological findings (n = 10). The lesions were present in the lower extremity (n = 15), upper extremity (n = 6), cervico-facial region (n = 6), pelvis (n = 2), and chest wall (n = 1). All patients had skin and subcutaneous lesions. Fifty percent of the patients (n = 15) demonstrated subfascial intramuscular (n = 15), intra-osseous (n = 1), and intra-articular involvement (n = 1). Contrary to the conventional belief that GVMs are generally limited to the skin and subcutaneous tissue, deep subfascial extension of the lesions is common. (orig.)

  12. CHANGE AT CERN - BE INVOLVED

    CERN Multimedia

    2002-01-01

    The encouragement of individual members of staff to contribute to the work of the task forces by making suggestions via the form on the Users'page of the web has been successful. Therefore, as announced by the Director General in his talk to the Staff in April, the management would like staff to continue to be involved in the change process by making further suggestions. Suggestions received will be distributed to the Director(s)/Division Leader(s) concerned, for reply and action where appropriate. The Director General has set up a small Panel that will ensure the proper processing of the ideas and will present a regular overview to the Director General. Members of the Panel are: Jan van der Boon, Vincent Hatton, Karl-Heinz Kissler, Thomas Pettersson, Florian Sonnemann, and Horst Wenninger.

  13. Metronidazole neurotoxicity: Sequential neuroaxis involvement

    Directory of Open Access Journals (Sweden)

    Kyung-Il Park

    2011-01-01

    Full Text Available Neurological manifestation of metronidazole toxicity include neuropathy and encephalopathy. We report a 67-year-old man with progressive painful paresthesias involving all the four limbs of 3 weeks′ duration before admission. He had been treated with metronidazole and cephalosporin for 10 weeks for a hepatic abscess. Five weeks after the symptom onset, he complained of dysarthria and limb ataxia. Magnetic resonance imaging revealed signal abnormalities in the splenium of the corpus callosum and bilateral dentate nuclei. A few hours after brain imaging, the patient exhibited excessive diaphoresis and fluctuation in blood pressure, which resolved within several hours after discontinuation of metronidazole. Whereas his speech returned to near normal within approximately 1 week, a burning sensation was not completely relieved, even 6 months after discharge.

  14. Charmless B decays involving baryons

    Science.gov (United States)

    Gronau, Michael; Rosner, Jonathan L.

    1988-02-01

    Predictions are made for the fraction of B-meson decays involving specific final states of NN¯+nπ (n>=0), as functions of (a) decay dynamics, (b) models for multipion production, (c) the isospin of the final state, and (d) the ratio ||Vbu/Vbc|| of Kobayashi-Maskawa matrix elements. From recent observations of B+-->pp¯π+(+c.c.) and B0-->pp¯π+π- by the ARGUS Collaboration, it is concluded that ||Vbu/Vbc||>~0.08, similar to the ARGUS Collaboration's own estimate of 0.07. However, a more likely value for this ratio is near its present experimental upper limit. Predictions are made for further final states in NN¯+nπ and in other charmless B decays. We also comment briefly on prospects for observing CP violation in B-->NN¯+nπ.

  15. Measuring Purchase‐decision involvement ,

    Directory of Open Access Journals (Sweden)

    Naser Azad

    2014-08-01

    Full Text Available Nowadays, increasing competition is forcing businesses to pay more attention on customer satisfaction providing strong customer services. Increased competition has also increased marketing activities. This paper presents an empirical investigation to determine important factors influencing purchase decision involvement in food industry in city of Tehran, Iran. The study designs a questionnaire in Likert scale, distributes it among 270 experts in food industry and, using principle component (PCA analysis, extracts important group of factors. The questionnaire consists of 27 questions, which is reduced to 23 questions because of sensitivity of the PCA to Skewness of data. Cronbach alpha is calculated as 0.81, which is well above the minimum acceptable level. The results indicate that there were four factors including individual differences, product validation, triggers and dependent behavior influencing purchasing decisions.

  16. Kidney involvement in rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    P. Lazzarini

    2011-09-01

    Full Text Available Rheumatoid Arthritis (RA is a widespread disease and its renal involvement, relatively common, is clinically significant because worsens course and mortality of the primary disease. There is still no agreement on the prevalence of renal disorders in RA: data analysis originates from different sources, as death certificates, autopsies, clinical and laboratory findings and kidney biopsies, each with its limitations. Histoimmunological studies on bioptical specimens of patients with RA and kidney damage, led to clarify prevalent pathologies. In order of frequency: glomerulonephritis and amyloidosis (60-65% and 20-30% respectively, followed by acute or chronic interstitial nephritis. Kidney injury during RA includes secondary renal amyloidosis, nephrotoxic effects of antirheumatic drugs and nephropathies as extra-articular manifestations (rheumatoid nephropathy. Amyloidosis affects survival, increases morbidity and is the main cause of end stage renal disease in patients with RA and nephropathy. Strong association between RA activity and amyloidosis needs the use of immunosuppressive and combined therapies, to prevent this complication and reduce risk of dialysis. Long-lasting and combined RA pharmacotherapy involves various renal side effects. In this review we describe NSAIDs and DMARDs (Disease-Modifying Antirheumatic Drugs nephrotoxicity, particularly by gold compounds, D-penicillamine, cyclosporine A and methotrexate. Rare cases of IgA glomerulonephritis during immunomodulating therapy with leflunomide and TNF blocking receptor (etanercept are reported; real clinical significance of this drug-related nephropathy will be established by development of RA treatment. In RA nephropathies, mesangial glomerulonephritis is the most frequent histological lesion (35-60 % out of biopsies from patients with urinary abnormalities and/or kidney impairment, followed by minimal change glomerulopathy (3-14% and p-ANCA positive necrotizing crescentic

  17. Drug hypersensitivity reactions involving skin.

    Science.gov (United States)

    Hausmann, Oliver; Schnyder, Benno; Pichler, Werner J

    2010-01-01

    Immune reactions to drugs can cause a variety of diseases involving the skin, liver, kidney, lungs, and other organs. Beside immediate, IgE-mediated reactions of varying degrees (urticaria to anaphylactic shock), many drug hypersensitivity reactions appear delayed, namely hours to days after starting drug treatment, showing a variety of clinical manifestations from solely skin involvement to fulminant systemic diseases which may be fatal. Immunohistochemical and functional studies of drug-specific T cells in patients with delayed reactions confirmed a predominant role for T cells in the onset and maintenance of immune-mediated delayed drug hypersensitivity reactions (type IV reactions). In these reactions, drug-specific CD4+ and CD8+ T cells are stimulated by drugs through their T cell receptors (TCR). Drugs can stimulate T cells in two ways: they can act as haptens and bind covalently to larger protein structures (hapten-carrier model), inducing a specific immune response. In addition, they may accidentally bind in a labile, noncovalent way to a particular TCR of the whole TCR repertoire and possibly also major histocompatibility complex (MHC)-molecules - similar to their pharmacologic action. This seems to be sufficient to reactivate certain, probably in vivo preactivated T cells, if an additional interaction of the drug-stimulated TCR with MHC molecules occurs. The mechanism was named pharmacological interaction of a drug with (immune) receptor and thus termed the p-i concept. This new concept may explain the frequent skin symptoms in drug hypersensitivity to oral or parenteral drugs. Furthermore, the various clinical manifestations of T cell-mediated drug hypersensitivity may be explained by distinct T cell functions leading to different clinical phenotypes. These data allowed a subclassification of the delayed hypersensitivity reactions (type IV) into T cell reactions which, by releasing certain cytokines and chemokines, preferentially activate and recruit

  18. PULMONARY INVOLVEMENT IN RHEUMATOID ARTHRITIS

    Directory of Open Access Journals (Sweden)

    D. V. Bestaev

    2014-01-01

    Full Text Available Rheumatoid arthritis (RA is an autoimmune disease with erosive and destructive polyarthritis and systemic manifestations. Pulmonary involvement (PI is common in RA. With high-resolution computed tomography, the detection rate of PI in RA is as high as 50%. PI is a direct cause of death in 10–20% of patients with RA. Autoimmune mechanisms play a leading part in the development of PI in RA. Under the hypothesis advanced by M. Selman et al., that impaired alveolocyte regeneration processes after injury rather inflammation underlie the pathogenesis of pulmonary fibrosis. The pathological process is triggered by damaged alveolocytes and characterized by the migration and proliferation of fibroblasts and myofibroblasts, the suppressed apoptosis of the latter, and the enhanced activity of pneumofibrosis-stimulating cytokines. This gives rise to remodeling of the extracellular matrix, including destruction of the basement membrane, angiogenesis, and fibrosis. The paper considers the types of lung injury in RA and main methods for diagnosis and therapy.

  19. Evaluating Parent Involvement. Issue Paper No. 1.

    Science.gov (United States)

    Safran, Daniel

    This paper poses a series of questions to assist programs in deciding what it is about parent involvement that they wish to evaluate. The questions focus on the nature of parent involvement, why parent involvement is needed, and what evaluation of parent involvement should include. A conceptual framework for research on the impact of parent…

  20. Defining Parental Involvement: Perception of School Administrators

    Science.gov (United States)

    Young, Clara Y.; Austin, Sheila M.; Growe, Roslin

    2013-01-01

    There remains a plaguing question of how to get parents involved with their child's education. Many parents and educators have different perceptions of what parental involvement means. Miscommunication between the two groups often exists because of how parental involvement is conceptualized. While educators define parental involvement as…

  1. Cardiovascular involvement in psoriatic arthritis

    Directory of Open Access Journals (Sweden)

    V. De Gennaro Colonna

    2011-11-01

    Full Text Available Psoriasis is a chronic, genetically determined and immunomediated inflammatory skin disease that affects 2-3% of the Caucasian population. A considerable proportion of these patients develop a form of inflammatory arthritis known as psoriatic arthritis (PsA, although the prevalence of this has not been well defined. Patients with PsA have a higher mortality rate than the general population and the risk of mortality is related to disease severity at the time of presentation. Endothelial dysfunction and early atherosclerosis have been found in patients with PsA without any cardiovascular disease (CVD risk factors, and experts believe that CVD is one of the leading causes of death, as it is in patients with rheumatoid arthritis (RA. Various disease-related mechanisms may be involved in the development of premature vascular damage in both cases, including an increased synthesis of proinflammatory mediators (such as cytokines, chemokines and adhesion molecules, autoantibodies against endothelial cell components, perturbations in T-cell subsets, genetic polymorphisms, hyperhomocysteinemia, oxidative stress, abnormal vascular repair, and iatrogenic factors. In a recent study of 22 patients with PsA without any signs of CVD, we found that the plasma concentration of asymmetric dimethylarginine (ADMA levels were significantly high and coronary flow reserve (CFR was significantly reduced. Moreover, there was a significant correlation between CFR and plasma ADMA levels in the PsA group. The significant correlation between the reduced CRF and increased ADMA levels suggests that, like patients with early RA, PsA patients suffer from endothelial dysfunction and impaired coronary microcirculation. Active PsA is a risk factor for CVD, and so PsA patients should be screened for subclinical forms of the disease and its risk factors, and an early treatment approach should be adopted.

  2. Genetics Home Reference: glucose phosphate isomerase deficiency

    Science.gov (United States)

    ... have episodes of more severe hemolysis, called hemolytic crises, which can be triggered by bacterial or viral ... the enzyme plays a role in a critical energy-producing process known as glycolysis, also called the ...

  3. Cyclophilin A enhances cell proliferation and tumor growth of liver fluke-associated cholangiocarcinoma

    Directory of Open Access Journals (Sweden)

    Sawanyawisuth Kanlayanee

    2011-08-01

    Full Text Available Abstract Background Cyclophilin A (CypA expression is associated with malignant phenotypes in many cancers. However, the role and mechanisms of CypA in liver fluke-associated cholangiocarcinoma (CCA are not presently known. In this study, we investigated the expression of CypA in CCA tumor tissues and CCA cell lines as well as regulation mechanisms of CypA in tumor growth using CCA cell lines. Methods CypA expression was determined by real time RT-PCR, Western blot or immunohistochemistry. CypA silence or overexpression in CCA cells was achieved using gene delivery techniques. Cell proliferation was assessed using MTS assay or Ki-67 staining. The effect of silencing CypA on CCA tumor growth was determined in nude mice. The effect of CypA knockdown on ERK1/2 activation was assessed by Western blot. Results CypA was upregulated in 68% of CCA tumor tissues. Silencing CypA significantly suppressed cell proliferation in several CCA cell lines. Likewise, inhibition of CypA peptidyl-prolyl cis-trans isomerase (PPIase activity using cyclosporin A (CsA decreased cell proliferation. In contrast, overexpression of CypA resulted in 30% to 35% increases in proliferation of CCA cell lines. Interestingly, neither silence nor overexpression of CypA affected cell proliferation of a non-tumor human cholangiocyte cell line, MMNK1. Suppression of CypA expression attenuated ERK1/2 activity in CCA M139 cells by using both transient and stable knockdown methods. In the in vivo study, there was a 43% reduction in weight of tumors derived from CypA-silenced CCA cell lines compared with control vector CCA tumors in mice; these tumors with stable CypA silencing showed a reduced cell proliferation. Conclusions CypA is upregulated in majority of CCA patients' tissues and confers a significant growth advantage in CCA cells. Suppression of CypA expression decreases proliferation of CCA cell lines in vitro and reduces tumor growth in the nude mouse model. Inhibition of Cyp

  4. Surface-associated lipoprotein PpmA of Streptococcus pneumoniae is involved in colonization in a strain-specific manner.

    NARCIS (Netherlands)

    Cron, L.E.; Bootsma, H.J.; Noske, N.; Burghout, P.J.; Hammerschmidt, S.; Hermans, P.W.M.

    2009-01-01

    Streptococcus pneumoniae produces two surface-associated lipoproteins that share homology with two distinct families of peptidyl-prolyl isomerases (PPIases), the streptococcal lipoprotein rotamase A (SlrA) and the putative proteinase maturation protein A (PpmA). Previously, we have demonstrated that

  5. Bioconversion of D-fructose to D-allose by novel isomerases%以D-果糖为原料利用新型异构酶转化生产D-阿洛糖

    Institute of Scientific and Technical Information of China (English)

    柏玮; 朱玥明; 门燕; 李晓波; 何森健; 孙媛霞

    2012-01-01

    Rare sugar is a kind of important low-energy monosaccharide that is rarely found in nature and difficult to synthesize chemically. D-allose, a six-carbon aldose, is an important rare sugar with unique physiological functions. It is radical scavenging active and can inhibit cancer cell proliferation. To obtain D-allose, the microorganisms deriving D-psicose 3-epimerase (DPE) and L-rhamnose isomerase (L-Rhl) have drawn intense attention. In this paper, DPE from Clostridium cellulolyticum H10 was cloned and expressed in Bacillus subtilis, and L-Rhl from Bacillus subtilis 168 was cloned and expressed in Escherichia coli BL21 (DE3). The obtained crude DPE and L-Rhl were then purified through a HisTrap HP affinity chromatography column and an anion-exchange chromatography column. The purified DPE and L-Rhl were employed for the production of rare sugars at last, in which DPE catalyzed D-fructose into D-psicose while L-Rhl converted D-psicose into D-allose. The conversion of D-fructose into D-psicose by DPE was 27.34%, and the conversion of D-psicose into D-allose was 34.64%.%稀少糖是自然界中含量稀少、化学合成困难的一类低热量单糖.D-阿洛糖是一种重要的稀少己醛糖,其具有减少活性自由基、抑制癌细胞增殖等独特的生理学功能.因此,以微生物发酵生产D-阿洛酮糖-3-差向异构酶(DPE)和L-鼠李糖异构酶(L-RhI)转化生产D-阿洛糖,成为近几年来国际研究的热点之一.文中分别克隆了来源于解纤维梭菌Clostridium cellulolyticum H10的DPE基因以及来源于枯草芽胞杆菌Bacillus subtilis 168的L-RhI基因,并分别使其在宿主菌B.subtilis及大肠杆菌Escherichia coli BL21 (DE3)中得到了表达.进一步利用镍亲和层析和阴离子交换色谱等手段对这两种酶进行了纯化,并对这两种纯化后酶的转化能力进行了分析测定.结果表明,以D-果糖为原料利用两种异构酶依次转化获得D-阿洛酮糖及D-阿洛糖,其

  6. Patient involvement in Danish health care

    DEFF Research Database (Denmark)

    Vrangbaek, Karsten

    2015-01-01

    PURPOSE: The purpose of this paper is to investigate different types of patient involvement in Denmark, and to discuss the potential implications of pursuing several strategies for patient involvement simultaneously. DESIGN/METHODOLOGY/APPROACH: The paper presents a preliminary framework...... for analysis of patient involvement in health care. This framework is used to analyze key governance features of patient involvement in Denmark based on previous research papers and reports describing patient involvement in Danish health care. FINDINGS: Patient involvement is important in Denmark...... implications for the development of patient involvement in health care. ORIGINALITY/VALUE: This paper fulfills a need to study different types of patient involvement and to develop a theoretical framework for characterizing and analyzing such involvement strategies....

  7. Quality assurance - how to involve the employees

    DEFF Research Database (Denmark)

    Jørgensen, Michael Søgaard

    1996-01-01

    An overview of strategies for involvement of employees in quality assurance developement and implementation.......An overview of strategies for involvement of employees in quality assurance developement and implementation....

  8. Involving Nepali academics in health research

    DEFF Research Database (Denmark)

    Neupane, Dinesh; van Teijlingen, E; Khanal, V;

    2013-01-01

    Many academics from Nepal do not involve in research activities. There are several factors hindering the involvement such as inadequate human resources and lack of financial resources. Despite limited human and financial resources, we believe it is still possible to attract many Nepali academics...... in health research. This paper purposes some ideas to increase involvement of Nepali academics in health research....

  9. Community Involvement and Disadvantaged Students: A Review.

    Science.gov (United States)

    Nettles, Saundra Murray

    1991-01-01

    Community involvement is conceptualized as a typology of the following processes of social change: (1) conversion; (2) mobilization; (3) allocation of resources; and (4) instruction. The effects of these forms of involvement are considered in a review of 13 evaluations of programs for disadvantaged youth involving the community. (SLD)

  10. Parental Involvement in Traditional and Online Education

    Science.gov (United States)

    Chang, Cheng-Sian; Chen, Hsi-Mei

    2011-01-01

    While parental involvement benefits the learning performance of elementary students and the internet changes the learning environment, few studies have examined how parents are involved in the virtual world. This two-year project analysed the effects of parental involvement at home, in school and on internet use. The first stage of our research…

  11. Parental Involvement in Special Education Curriculum

    Science.gov (United States)

    Westwood-Robinette, Nicole M.

    2014-01-01

    Educators and researchers have long considered parental involvement an integral part in the success of students and researchers have concluded that there is a connection between parental involvement and the retention rates of students who are involved in regular education curriculum. However, much less information is available regarding the…

  12. 36 CFR 1010.12 - Public involvement.

    Science.gov (United States)

    2010-07-01

    ... 36 Parks, Forests, and Public Property 3 2010-07-01 2010-07-01 false Public involvement. 1010.12 Section 1010.12 Parks, Forests, and Public Property PRESIDIO TRUST ENVIRONMENTAL QUALITY § 1010.12 Public involvement. The Trust will make public involvement an essential part of its environmental review...

  13. Brainstem involvement in subacute sclerosing panencephalitis

    Directory of Open Access Journals (Sweden)

    Pawan Sharma

    2011-01-01

    Full Text Available The parieto-occipital region of the brain is most frequently and severely affected in subacute sclerosing panencephalitis (SSPE. The basal ganglia, cerebellum and corpus callosum are less commonly involved. Brainstem involvement is rarely described in SSPE, and usually there is involvement of other regions of the brain. We describe a patient with subacute sclerosing panencephalitis with brain magnetic resonance imaging showing extensive brainstem involvement without significant involvement of other cortical structures. Though rarely described in SSPE, one should be aware of such brainstem and cerebellum involvement, and SSPE should be kept in mind when brainstem signal changes are seen in brain MRI with or without involvement of other regions of brain to avoid erroneous reporting.

  14. Who and What Does Involvement Involve? A Multi-Sited Field Study of Involvement of Relatives in Danish Psychiatry.

    Science.gov (United States)

    Oute, Jeppe; Petersen, Anders; Huniche, Lotte

    2015-01-01

    This article gives an account of aspects of a multi-sited field study of involvement of relatives in Danish psychiatry. By following metaphors of involvement across three sites of the psychiatric system-a family site, a clinical site and a policy site-the first author (J.O.) investigated how, and on what grounds, involvement of relatives is perceived in Danish psychiatry. Paradoxically, the current understanding of involvement of relatives fails to take into consideration the perspectives of the relatives per se and families that were being studied. By analyzing involvement from a discourse theoretical perspective laid out by Ernesto Laclau and Chantal Mouffe, the aim of this study is to show how the dominant discourse about involvement at the political and clinical sites is constituted by understandings of mentally ill individuals and by political objectives of involvement. The analysis elucidates how a psycho-ideological discourse positions the mentally ill person as weak, incapable, and ineffective. By contrast, the supporting relative is positioned as a strong, capable, and effective co-therapist. Furthermore, the analysis considers how this dominant discourse of involvement is constituted by a broader discourse of neoliberalism and market orientation, which justifies involvement as a subtle institutionalization of social control. The article highlights that the role of the relative as a co-therapist may be contested by the families' discourse, which emphasizes issues concerning the responsibility toward the mental health of the ill individual as well as toward the psychological milieu of the family.

  15. Top Management Involvement in New Product Development

    DEFF Research Database (Denmark)

    Felekoglu, Burcu; Maier, Anja; Moultrie, James

    2010-01-01

    Involvement of top managers in new product development (NPD) is a critical factor affecting NPD performance and frequently considered to be the participation of top management to certain activities in NPD or their NPD related behaviours. However, “Top management involvement in NPD” occupies...... a broader conceptual space than this participation. This paper reviews the literature on top management involvement in new product development (NPD) and discusses relevance of different theoretical perspectives from other disciplines such as management, organisational behaviour and communication to analyse...... antecedents, realisation and consequences of top management involvement in NPD. It is argued that top management has different involvement at different NPD levels: organisation level and project level. Resulting from this literature review, a tentative framework for top management involvement in NPD...

  16. Dual Headquarters Involvement in Subsidiary Innovation

    DEFF Research Database (Denmark)

    Dellestrand, Henrik; Kappen, Philip; Nell, Phillip Christopher

    2014-01-01

    innovation importance, i.e., an innovation that is important for the division and the corporate level, drives dual headquarters involvement in innovation development. Contrary to expectations, no significant effect of dual embeddedness on dual headquarters involvement is found. The network size......The strategy and international business literature has identified the overall potential for headquarters to add value by allocating resources to subsidiary activities, but little is known about the extent to which multiple headquarters simultaneously involves itself in subsidiary operations...

  17. Liver involvement in Langerhans cell histiocytosis

    Energy Technology Data Exchange (ETDEWEB)

    Wong, Adelaine; Ortiz-Neira, Clara L.; Abou Reslan, Walid; Kaura, Deepak [Alberta Children' s Hospital, Department of Diagnostic Imaging, Calgary, Alberta (Canada); Sharon, Raphael; Anderson, Ronald [Alberta Children' s Hospital, Department of Oncology, Calgary, AB (Canada); Pinto-Rojas, Alfredo [Alberta Children' s Hospital, Department of Pathology, Calgary, AB (Canada)

    2006-10-15

    Liver involvement in Langerhans cell histiocytosis (LCH) typically presents with hepatomegaly and other signs of liver dysfunction. We present an 11-month-old child having only minimally elevated liver enzymes as an indication of liver involvement. Using sonography as the initial diagnostic tool followed by MRI, LCH of the liver was revealed. A review of sonographic, CT, MRI and MR cholangiopancreatography findings in liver LCH is presented. We recommend that physicians consider sonography and MRI screening for liver involvement in patients with newly diagnosed LCH, as periportal involvement may be present with little or no liver function abnormality present, as in this patient. (orig.)

  18. Perceptions of Parent Involvement in Academic Achievement

    Science.gov (United States)

    DePlanty, Jennifer; Coulter-Kern, Russell; Duchane, Kim A.

    2007-01-01

    The authors sought to understand the types of parent involvement that teachers, parents, and students believe affect the academic achievement of adolescent learners at the junior high school level. Research that included focus groups, interviews, and surveys indicated that teachers and students believed that parent involvement at school was…

  19. Involving Migrant Families in Education. ERIC Digest.

    Science.gov (United States)

    Martinez, Yolanda G.; Velazquez, Jose A.

    This digest describes parent involvement in their children's education from the perspective of migrant parents and educators and offers strategies to enhance the experience of schooling for migrant students and their families. Teachers often perceive parent involvement as preparing children for school, attending school events, and fulfilling…

  20. Citizen Involvement in Local Security Networks

    NARCIS (Netherlands)

    Terpstra, J.B.

    2009-01-01

    This paper deals with the involvement of citizens (and local businesspersons) in the prevention and control of crime and disorder. Four models of citizen involvement in local security networks are distinguished. In each of these models the role of citizens concentrates on different functions: (1) p

  1. Involving Program Constituencies in the Evaluation Process.

    Science.gov (United States)

    Deshler, David

    1984-01-01

    Two case studies provide examples of citizen and program participant involvement in program evaluation. The Reflective Appraisal of Programs at Cornell Cooperative Extension involves volunteer interviewers of participants; the Lancaster County (Pennsylvania) Office of Mental Health/Mental Retardation is reviewed by a volunteer citizens' committee.…

  2. Bullying Prevention and the Parent Involvement Model

    Science.gov (United States)

    Kolbert, Jered B.; Schultz, Danielle; Crothers, Laura M.

    2014-01-01

    A recent meta-analysis of bullying prevention programs provides support for social-ecological theory, in which parent involvement addressing child bullying behaviors is seen as important in preventing school-based bullying. The purpose of this manuscript is to suggest how Epstein and colleagues' parent involvement model can be used as a…

  3. Turkish Early Childhood Educators on Parental Involvement

    Science.gov (United States)

    Hakyemez, Sevcan

    2015-01-01

    Research conducted over recent decades show that parental involvement plays a significant role in children's academic achievement as well as their cognitive, social and emotional development. For effective parental involvement, understanding the conceptualization of early childhood educators should be significant. This research investigated the…

  4. Examining Teacher's Comfort Level of Parental Involvement

    Science.gov (United States)

    Jensen, Deborah Ann

    2011-01-01

    The connection between home and school is of utmost importance. Therefore, an important concern for those educating teachers is to help teachers recognize the need for and importance of establishing parental involvement and to help them create avenues in which communication can occur. Knowing that parental involvement is important and putting that…

  5. Defining stakeholder involvement in participatory design processes

    NARCIS (Netherlands)

    Vink, P.; Imada, A.S.; Zink, K.J.

    2008-01-01

    A participatory approach could be used to implement work place or organizational improvements. However, the question is which participants should be involved and how. In this paper the theoretical involvement in different steps of a linear stepwise approach is described and compared with the latest

  6. Secret-involved Information System Security Audit

    Institute of Scientific and Technical Information of China (English)

    ZHANG; Ya-lan

    2015-01-01

    Secret-involved information system security audit is a network security technology developing rapidly in recent years.It uses various technical to detect the problem of secret-involved information system,and uses certain audit methods to analyze all kinds of suspicious behavior and irregularities.

  7. Strategic Management: Managing Change by Employee Involvement

    Directory of Open Access Journals (Sweden)

    Dr. Fareeha Zafar

    2014-05-01

    Full Text Available Strategic management attempts to link strategic planning with decision making and these decisions implementation. The role of involvement -who should be involved and how that involvement should be managed is essential for effectual decision making. This paper scrutinizes the significance of involvement in decision making generally and demonstrate how involvement management principles apply to the strategic management process explicitly. Strategic management composed of three important steps: creation of a documented plan, making strategic planning an effectual part of the management systems, and appropriately manage involvement in the planning process. It requires that an organization's managers must be involved in a process of objectives identification, strategies development and implementation plans to accomplish those objectives, and sporadically evaluate the decisions implementation of decisions. Decision making, implementation, feedback, and evaluation; is a continuous process. Basically, effective decision making and implementation means congregating the right mix of people, convincing them that what they are doing is significant to the senior executive, and effectively managing their involvement in the decision making process. This paper provides some proven techniques that add value to many organizations.

  8. Peer Involvement in Adolescent Dating Violence

    Science.gov (United States)

    Stephenson, Pam S.; Martsolf, Donna; Draucker, Claire Burke

    2013-01-01

    This study investigated the ways in which peers are involved in adolescent dating violence. Eighty-eight young adults aged 18-21 were interviewed and asked to reflect on aggressive dating relationships they experienced as teens. The researchers used grounded theory to analyze the data. Findings showed that male and female peers were involved in…

  9. Macrodystrophia lipomatosa of foot involving great toe.

    Science.gov (United States)

    Gaur, A K; Mhambre, A S; Popalwar, H; Sharma, R

    2014-06-01

    Macrodystrophia lipomatosa is a rare form of congenital disorder in which there is localized gigantism characterized by progressive overgrowth of all mesenchymal elements with a disproportionate increase in the fibroadipose tissues. The adipose tissue infiltration involves subcutaneous tissue, periosteum, nerves and bone marrow. Most of the cases reported have hand or foot involvement. Patient seeks medical help for improving cosmesis or to get the size of the involved part reduced in order to reduce mechanical problems. We report a case of macrodystrophia lipomatosa involving medial side of foot with significant enlargement of great toe causing concern for cosmesis and inconvenience due to mechanical problems. The X-rays showed increased soft tissue with more of adipose tissue and increased size of involved digits with widening of ends. Since the patient's mother did not want any surgical intervention he was educated about foot care and proper footwear design was suggested.

  10. Secretomic analysis of large cell lung cancer cell lines using two-dimensional gel electrophoresis coupled to mass spectrometry

    Directory of Open Access Journals (Sweden)

    Zahra Yousefi

    2012-10-01

    Full Text Available

    The secretome of cancer cells is a valuable source of biomarkers that can ultimately reach the serum or other body fluids. Cancer biomarkers can facilitate early diagnosis and monitoring of the disease, contribute to our understanding of tumor biology, and support the development of more efficient therapies. Recently, high-throughput proteomic analysis of the conditioned media of cancer cell lines has shown potential to identify novel biomarkers in cancer. We used two-dimensional gel electrophoresis coupled to liquid chromatography tandem mass spectrometry to identify the secretome of the large cell lung cancer cell lines QU-DB and Mehr-80, which they were established from a Canadian and a Persian patient, respectively. A total of 130 distinct protein species were identified. Of these, 124 were previously found in serum or other body fluids, the membrane compartment or conditioned media of other cancer cell lines. Some of the proteins that we identified, e.g. IL-6, triosephosphate isomerase, PGP9.5, α-enolase, Dickkopf-1, and peroxiredoxin-1 are known putative serum markers for lung cancer, whereas others might be candidate markers for further validation in lung cancer body fluids such as IL-25, stathmin, vimentin, peptidyl-prolyl cis-trans isomerase A, transgelin-2, and chloride intracellular channel protein 4.

  11. 胶质瘤中PIN1-Nanog相关通路表达的研究%Relation of peptidyl-prolyi isomerase 1 and Nanog expressions in human gliomas

    Institute of Scientific and Technical Information of China (English)

    倪升远; 牛朝诗; 杨洋; 张翔宇

    2015-01-01

    目的 观察肽基脯氨酰异构酶1(PIN1)、Nanog在胶质瘤组织中的表达以及PIN1抑制剂PiB对U87细胞增殖能力及PIN1、Nanog表达的影响. 方法 选取安徽医科大学附属省立医院神经外科自2013年3月至2014年8月间手术切除的84例胶质瘤和10例正常脑组织标本,免疫组化染色检测标本PIN1、Nanog蛋白的表达.体外常规培养U87胶质瘤细胞,用0.2、0.5、1.0、2.0 μg/mL PiB分别作用24、48 h,MTT检测PiB对细胞增殖的抑制作用并计算抑制率;以1.0μmol/L PiB处理U87细胞24 h为1.0 μmol/L PiB组,未经任何处理的U87细胞作为对照组,免疫荧光双染检测细胞PIN1、Nanog的表达,RT-PCR和Westem blotting分别检测细胞PIN1、Nanog mRNA和蛋白的表达. 结果 WHOⅡ级、Ⅲ级、Ⅳ级胶质瘤中PIN1、Nanog高表达率不同,差异有统计学意义(P<0.05),且胶质瘤级别越高,PIN1、Nanog高表达率越高.MTT检测显示PiB对U87细胞的增殖有抑制作用,且呈时间和剂量依赖性.与对照组比较,1.0 μmol/L PiB组PIN1、Nanog及PIN1/Nanog阳性细胞百分比降低,差异有统计学意义(P<0.05);对照组和1.0 μmol/L PiB组细胞PIN1 mRNA的表达量分别为1.82±0.03和0.94±0.20,Nanog mRNA的表达量分别为1.47±0.04和0.82±0.19,差异均有统计学意义(P<0.05);对照组和1.0 μmol/L PiB组细胞PIN1蛋白表达量分别为2.96±0.05和1.15±0.26,Nanog蛋白表达量分别为1.52±0.16和0.75±0.05,差异均有统计学意义(P<0.05). 结论 PIN1、Nanog在胶质瘤的发生发展中起重要作用,且与肿瘤的恶性程度相关.PIN1可参与调控Nanog,两者之间存在相关通路,下调PIN1-Nanog通路后,胶质瘤细胞增殖能力下降.%Objective To obsevre the peptidyl-prolyi isomerase 1 (PIN1) and Nanog expreesions in glioma tissues and the effect of PIN1 inhibitor polyiso-butylene (PiB) on their expressions and U87 cell proliferation.Methods Eighty-four glioma samples (15 with WHO graded Ⅱ,27 with WHO graded

  12. KIN241: a gene involved in cell morphogenesis in Paramecium tetraurelia reveals a novel protein family of cyclophilin-RNA interacting proteins (CRIPs) conserved from fission yeast to man.

    Science.gov (United States)

    Krzywicka, A; Beisson, J; Keller, A M; Cohen, J; Jerka-Dziadosz, M; Klotz, C

    2001-10-01

    In this study, we report cloning, by functional complementation of the KIN241 gene involved in Paramecium cell morphogenesis, cortical organization and nuclear reorganization. This gene is predicted to encode a protein of a novel type, comprising a cyclophilin-type, peptidyl-prolyl isomerase domain, an RNA recognition motif, followed by a region rich in glutamate and lysine (EK domain) and a C-terminal string of serines. As homologues of this protein are present in the genomes of Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana and Homo sapiens, the Kin241p predicted sequence defines a new family of proteins that we propose to call 'CRIP', for cyclophilin-RNA interacting protein. We demonstrate that, in Paramecium, Kin241p is localized in the nucleus and that deletion of some nuclear localization signals (NLSs) decreases transport of the protein into the nucleus. No Kin241-1 protein is present in mutant cells, suggesting that the C-terminal serine-rich region is responsible for protein stability.

  13. The psychology of male (non) involvement.

    Science.gov (United States)

    1977-01-01

    The primary reason for male involvement in family planning is that the current approach of focusing all family planning attention on women is not working. Unwanted pregnancy continues to increase despite the advent of the oral contraceptive and the IUD and despite the great amount of money and effort that have been invested in making women better contraceptors. Whether or not a contraceptive is effectively used depends a lot on the attitude of the man. The great majority of the studies conducted on male attitudes toward ocntraception indicates that boys and men are interested in the subject. Despite the discrepancy between what people say and what they actually do, there is a vast population of men who are receptive to education and guidance. If the approach is right, the majority of men are willing to become involved. The men who are resistant to involvement in family planning are a minority, and probably nothing can be done about this group. Although the majority of men are not resistant, they see no reason for involvement. In the last 20 years men have been taught that contraception is none of their business. This teaching has been done by the media, family planning agencies, some feminist writers and by women themselves. Most of the resistance to male involvement comes from family planning agencies rather than from the men. How to get men involved in actually a small issues. If there is a change in thinking in the agencies and a willingness to involve men, many effective ways will be found.

  14. Liver Involvement with Acute Myeloid Leukemia

    Directory of Open Access Journals (Sweden)

    Emily Mathews

    2008-03-01

    Full Text Available Liver involvement with acute myeloid leukemia (AML is rarely reported. The majority of published cases suggest a cholestatic picture and obstructive jaundice at presentation. On the contrary, our patient presented with transaminitis without cholestasis. Elevated liver function tests persisted in our patient despite cholecystectomy; however, they normalized with chemotherapy administration, suggesting that AML was the causative effect of the hepatitis-like picture. Our review of the literature revealed that most reported cases of AML with liver involvement had short-lived remissions and an overall ominous prognosis. In our opinion, patients who have liver involvement with AML should be offered alternative investigational therapies with a low hepatic toxicity profile.

  15. Comparative transcriptome analysis of genes involved in anthocyanin biosynthesis in the red and yellow fruits of sweet cherry (Prunus avium L..

    Directory of Open Access Journals (Sweden)

    Hairong Wei

    Full Text Available Fruit color is one of the most important economic traits of the sweet cherry (Prunus avium L.. The red coloration of sweet cherry fruit is mainly attributed to anthocyanins. However, limited information is available regarding the molecular mechanisms underlying anthocyanin biosynthesis and its regulation in sweet cherry.In this study, a reference transcriptome of P. avium L. was sequenced and annotated to identify the transcriptional determinants of fruit color. Normalized cDNA libraries from red and yellow fruits were sequenced using the next-generation Illumina/Solexa sequencing platform and de novo assembly. Over 66 million high-quality reads were assembled into 43,128 unigenes using a combined assembly strategy. Then a total of 22,452 unigenes were compared to public databases using homology searches, and 20,095 of these unigenes were annotated in the Nr protein database. Furthermore, transcriptome differences between the four stages of fruit ripening were analyzed using Illumina digital gene expression (DGE profiling. Biological pathway analysis revealed that 72 unigenes were involved in anthocyanin biosynthesis. The expression patterns of unigenes encoding phenylalanine ammonia-lyase (PAL, 4-coumarate-CoA ligase (4CL, chalcone synthase (CHS, chalcone isomerase (CHI, flavanone 3-hydroxylase (F3H, flavanone 3'-hydroxylase (F3'H, dihydroflavonol 4-reductase (DFR, anthocyanidin synthase (ANS and UDP glucose: flavonol 3-O-glucosyltransferase (UFGT during fruit ripening differed between red and yellow fruit. In addition, we identified some transcription factor families (such as MYB, bHLH and WD40 that may control anthocyanin biosynthesis. We confirmed the altered expression levels of eighteen unigenes that encode anthocyanin biosynthetic enzymes and transcription factors using quantitative real-time PCR (qRT-PCR.The obtained sweet cherry transcriptome and DGE profiling data provide comprehensive gene expression information that lends insights

  16. Adipocyte Accumulation of Long-Chain Fatty Acids in Obesity is Multifactorial, Resulting from Increased Fatty Acid Uptake and Decreased Activity of Genes Involved in Fat Utilization

    Science.gov (United States)

    Walewski, José L.; Ge, Fengxia; Gagner, Michel; Inabnet, William B.; Pomp, Alfons; Branch, Andrea D.

    2010-01-01

    Background The obesity epidemic causes significant morbidity and mortality. Knowledge of cellular function and gene expression in obese adipose tissue will yield insights into obesity pathogenesis and suggest therapeutic targets. The aim of this work is to study the processes determining fat accumulation in adipose tissue from obese patients. Methods Omental fat was collected from two cohorts of obese bariatric surgery patients and sex-matched normal-weight donors. Isolated adipocytes were compared for cell size, volume, and long-chain fatty acid (LCFA) uptake. Omental fat RNAs were screened by 10K microarray (cohort 1: three obese, three normal) or Whole Genome microarray (cohort 2: seven obese, four normal). Statistical differences in gene and pathway expression were identified in cohort 1 using the GeneSifter Software (Geospiza) with key results confirmed in cohort 2 samples by microarray, quantitative real-time polymerase chain reaction, and pathway analysis. Results Obese omental adipocytes had increased surface area, volume, and Vmax for saturable LCFA uptake. Dodecenoyl-coenzyme A delta isomerase, central to LCFA metabolism, was approximately 1.6-fold underexpressed in obese fat in cohorts 1 and 2. Additionally, the Kyoto Encyclopedia of Genes and Genomics pathway analysis identified oxidative phosphorylation and fatty acid metabolism pathways as having coordinate, nonrandom down-regulation of gene expression in both cohorts. Conclusions In obese omental fat, saturable adipocyte LCFA uptake was greater than in controls, and expression of key genes involved in lipolysis, β-oxidation, and metabolism of fatty acids was reduced. Thus, both increased uptake and reduced metabolism of LCFAs contribute to the accumulation of LCFAs in obese adipocytes. PMID:19866242

  17. 地芽孢杆菌Y565-5分离鉴定及其木糖异构酶基因xylA的克隆表达和酶学性质%Cloning, expression and characterization of xylose isomerase, XylA from Geobacillus sp.Y565-5

    Institute of Scientific and Technical Information of China (English)

    张洁; 黄志勇; 王钦宏; 王永莉; 王硕

    2011-01-01

    从甘肃玉门油田地表土中分离到一株嗜热木糖利用菌,地芽孢杆菌Y565-5.利用PCR方法从该菌株中克隆得到一个木糖异构酶基因,xylA.该基因开放阅读框长1 182 bp,编码394个氨基酸,XylA氨基酸序列与Geobacillus sp.Y412MC52相似性达到99%.将xylA基因克隆到原核表达载体pET-28a(+)上,得到重组质粒pET-28a(+)-xylA,然后将此重组质粒转化至BL21(DE3)中,经IPTG诱导后,通过SDS-PAGE电泳检测出明显的45 kD(相对分子质量)特异性蛋白质条带,并且通过半胱氨酸咔唑法检测出表达产物具有木糖异构酶的活性.对其酶学性质的研究发现,XylA最适温度为90℃,最适pH值为8.0.%Xylose-utilizing and thermophilic Geobacillus sp. Y565-5 was isolated from surface soil of an oilfield in Yumen Town, Gansu Province, China. A xylose isomerase (XylA) gene was cloned from the strain by PCR. The open reading frame of xylA (1 182 bp) encoded a protein of 394 amino acids,which showed high sequence homology (99% identity) with that of Geobacillus sp. Y412MC52. The intact coding region was subcloned into pET28a(+) vector and expressed in Escherichia coli BL21(DE3).The molecular weight of the recombinant protein was 45 kD based on SDS-PAGE and its xylose isomerase activity was detected through cysteine welts thiazole method after the induction of isopropyl β-D-1-thiogalactopyranoside (IPTG). The optimum temperature and pH for the partially purified recombinant XylA activity were 90 ℃ and pH 8.0, respectively.

  18. Peer Effects and Academics’ Industry Involvement

    DEFF Research Database (Denmark)

    Aschhoff, Birgit; Grimpe, Christoph

    This study explores the interaction between professional imprinting and age in the context of industry-science collaboration. Specifically, we examine the impact of localized and personal peer effects on academics’ involvement with industry and how these effects are moderated by the career age...... of the scientist. We suggest that both localized and personal peer effects drive industry involvement but that the effects from such imprinting are more pronounced for younger researchers, suggesting that professional imprinting takes place in the early stages of a scientist’s academic career. Based on a sample...... of 330 German academics in the field of biotechnology and publication data from the Science Citation Index Expanded (SCIE), we find that scientists with industry-oriented co-authors are more likely to be involved with industry (personal peer effect). Moreover, we find that the scientist’s involvement...

  19. Fires and Burns Involving Home Medical Oxygen

    Science.gov (United States)

    ... nfpa.org Fires and Burns Involving Home Medical Oxygen The air is normally 21% oxygen. Oxygen is not flammable, but fire needs it to burn. ¾ When more oxygen is present, any fire that starts will burn ...

  20. Nurses' political involvement: responsibility versus privilege.

    Science.gov (United States)

    Boswell, Carol; Cannon, Sharon; Miller, Joyce

    2005-01-01

    Nursing apathy toward participation in the political process is pandemic. Never more so than today has the profession needed a strong united stand within the political arena. Political involvement encompasses being knowledgeable about issues, laws, and health policy. Barriers to political activism are thought to encompass several spectra including heavy workloads, feelings of powerlessness, time constraints, sex issues, and lack of understanding of a complex political process. The implementation of a political role for a nurse is based on three levels of commitment including survival, success, and significance. Survival includes individual involvement within communities. Success accepts challenges in addressing injustices especially within the health-care arena. Significant involvement uses visionary nurses toward the betterment of the nurse profession. Strategies for involvement include political awareness, incorporation of course/program expectations on both undergraduate and graduate levels and teamwork. As patient advocates, nurses cannot continue to be spectators in the political arena.