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Sample records for dihydrofolate reductase knockdown

  1. Expression and site-directed mutagenesis of human dihydrofolate reductase

    Energy Technology Data Exchange (ETDEWEB)

    Prendergast, N.J.; Delcamp, T.J.; Smith, P.L.; Freisheim, J.H.

    1988-05-17

    A procaryotic high-level expression vector for human dihydrofolate reductase has been constructed and the protein characterized as a first step toward structure-function studies of this enzyme. A vector bearing the tac promoter, four synthetic oligodeoxynucleotides, and a restriction fragment from the dihydrofolate reductase cDNA were ligated in a manner which optimized the transcriptional and translational frequency of the enzyme mRNA. The reductase, comprising ca. 17% of the total soluble protein in the host bacteria, was purified to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and characterized by amino acid composition, partial amino acid sequence, and steady-sate kinetic analysis. This expression vector has been used as a template for double-stranded plasmid DNA site-specific mutagenesis. Functional studies on a Cys-6 ..-->.. Ser-6 mutant enzyme support the contention that Cys-6 is obligatory for organomercurial activation of human dihydrofolate reductase. The Ser-6 mutant enzyme was not activated to any extent following a 24-h incubation with p-(hydroxymercuri)benzoate and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH), whereas the k/sub cat/ for Cys-6 reductase increased 2-fold under identical conditions. The specific activities of the Cys-6 and Ser-6 enzymes were virtually identical as determined by methotrexate titration as were the K/sub m/ values for both dihydrofolate and NADPH. The Ser-6 mutant showed a decreased temperature stability and was more sensitive to inactivation by ..cap alpha..-chymotrypsin when compared to the wild-type enzyme. These results suggest that the Ser-6 mutant reductase is conformationally altered relative to the Cys-6 native enzyme.

  2. Expression and site-directed mutagenesis of human dihydrofolate reductase

    International Nuclear Information System (INIS)

    Prendergast, N.J.; Delcamp, T.J.; Smith, P.L.; Freisheim, J.H.

    1988-01-01

    A procaryotic high-level expression vector for human dihydrofolate reductase has been constructed and the protein characterized as a first step toward structure-function studies of this enzyme. A vector bearing the tac promoter, four synthetic oligodeoxynucleotides, and a restriction fragment from the dihydrofolate reductase cDNA were ligated in a manner which optimized the transcriptional and translational frequency of the enzyme mRNA. The reductase, comprising ca. 17% of the total soluble protein in the host bacteria, was purified to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and characterized by amino acid composition, partial amino acid sequence, and steady-sate kinetic analysis. This expression vector has been used as a template for double-stranded plasmid DNA site-specific mutagenesis. Functional studies on a Cys-6 → Ser-6 mutant enzyme support the contention that Cys-6 is obligatory for organomercurial activation of human dihydrofolate reductase. The Ser-6 mutant enzyme was not activated to any extent following a 24-h incubation with p-(hydroxymercuri)benzoate and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH), whereas the k/sub cat/ for Cys-6 reductase increased 2-fold under identical conditions. The specific activities of the Cys-6 and Ser-6 enzymes were virtually identical as determined by methotrexate titration as were the K/sub m/ values for both dihydrofolate and NADPH. The Ser-6 mutant showed a decreased temperature stability and was more sensitive to inactivation by α-chymotrypsin when compared to the wild-type enzyme. These results suggest that the Ser-6 mutant reductase is conformationally altered relative to the Cys-6 native enzyme

  3. [Comparison of Physico-chemical Aspects between E. coli and Human Dihydrofolate Reductase: an Equilibrium Unfolding Study].

    Science.gov (United States)

    Thapliyal, Charu; Jain, Neha; Chaudhuri, Pratima

    2015-01-01

    A protein, differing in origin, may exhibit variable physicochemical behaviour, difference in sequence homology, fold and function. Thus studying structure-function relationship of proteins from altered sources is meaningful in the sense that it may give rise to comparative aspects of their sequence-structure-function relationship. Dihydrofolate reductase is an enzyme involved in cell cycle regulation. It is a significant enzyme as.a target for developing anticancer drugs. Hence, detailed understanding of structure-function relationships of wide variants of the enzyme dihydrofolate reductase would be important for developing an inhibitor or an antagonist against the enzyme involved in the cellular developmental processes. In this communication, we have reported the comparative structure-function relationship between E. coli and human dihydrofolate reductase. The differences in the unfolding behaviour of these two proteins have been investigated to understand various properties of these two proteins like relative' stability differences and variation in conformational changes under identical denaturing conditions. The equilibrium unfolding mechanism of dihydrofolate reductase proteins using guanidine hydrochloride as a denaturant in the presence of various types of osmolytes has been monitored using loss in enzymatic activity, intrinsic tryptophan fluorescence and an extrinsic fluorophore 8-anilino-1-naphthalene-sulfonic acid as probes. It has been observed that osmolytes, such as 1M sucrose, and 30% glycerol, provided enhanced stability to both variants of dihydrofolate reductase. Their level of stabilisation has been observed to be dependent on intrinsic protein stability. It was observed that 100 mM proline does not show any 'significant stabilisation to either of dihydrofolate reductases. In the present study, it has been observed that the human protein is relatively less stable than the E.coli counterpart.

  4. Isolation and expression of the Pneumocystis carinii dihydrofolate reductase gene

    DEFF Research Database (Denmark)

    Edman, J C; Edman, U; Cao, Mi-Mi

    1989-01-01

    Pneumocystis carinii dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) cDNA sequences have been isolated by their ability to confer trimethoprim resistance to Escherichia coli. Consistent with the recent conclusion that P. carinii is a member of the Fungi...

  5. para-Aminosalicylic acid is a prodrug targeting dihydrofolate reductase in Mycobacterium tuberculosis.

    Science.gov (United States)

    Zheng, Jun; Rubin, Eric J; Bifani, Pablo; Mathys, Vanessa; Lim, Vivian; Au, Melvin; Jang, Jichan; Nam, Jiyoun; Dick, Thomas; Walker, John R; Pethe, Kevin; Camacho, Luis R

    2013-08-09

    para-Aminosalicylic acid (PAS) is one of the antimycobacterial drugs currently used for multidrug-resistant tuberculosis. Although it has been in clinical use for over 60 years, its mechanism(s) of action remains elusive. Here we report that PAS is a prodrug targeting dihydrofolate reductase (DHFR) through an unusual and novel mechanism of action. We provide evidences that PAS is incorporated into the folate pathway by dihydropteroate synthase (DHPS) and dihydrofolate synthase (DHFS) to generate a hydroxyl dihydrofolate antimetabolite, which in turn inhibits DHFR enzymatic activity. Interestingly, PAS is recognized by DHPS as efficiently as its natural substrate para-amino benzoic acid. Chemical inhibition of DHPS or mutation in DHFS prevents the formation of the antimetabolite, thereby conferring resistance to PAS. In addition, we identified a bifunctional enzyme (riboflavin biosynthesis protein (RibD)), a putative functional analog of DHFR in a knock-out strain. This finding is further supported by the identification of PAS-resistant clinical isolates encoding a RibD overexpression mutation displaying cross-resistance to genuine DHFR inhibitors. Our findings reveal that a metabolite of PAS inhibits DHFR in the folate pathway. RibD was shown to act as a functional analog of DHFR, and as for DHFS, both were shown to be associated in PAS resistance in laboratory strains and clinical isolates.

  6. Role of Lysine-54 in determining cofactor specificity and binding in human dihydrofolate reductase

    International Nuclear Information System (INIS)

    Huang, Shaoming; Tan, Xuehai; Thompson, P.D.; Freisheim, J.H.; Appleman, J.R.; Blakley, R.L.; Sheridan, R.P.; Venkataraghavan, R.

    1990-01-01

    Lysine-54 of human dihydrofolate reductase (hDHFR) appears to be involved in the interaction with the 2'-phosphate of NADPH and is conserved as a basic residue in other species. Studies have suggested that in Lactobacillus casei dihydrofolate reductase Arg-43, the homologous residue at this position, plays an important role in the binding of NADPH and in the differentiation of K m values for NADPH and NADH. A Lys-54 to Gln-54 mutant (K54Q) of hDHFR has been constructed by oligodeoxynucleotide-directed mutagenesis in order to study the role of Lys-54 in differentiating K m and k cat values for NADPH and NADH as well as in other functions of hDHFR. The purpose of this paper is to delineate in quantitative terms the magnitude of the effect of the Lys-54 to Gln-54 replacement on the various kinetic parameters of hDHFR. Such quantitative effects cannot be predicted solely on the basis of X-ray structures. The ratio of K m (NADH)/K m (NADPH) decreases from 69 in the wild-type enzyme to 4.7 in the K54Q enzyme, suggesting that Lys-54, among other interactions between protein side-chain residues and the 2'-phosphate, makes a major contribution in terms of binding energy and differentiation of K m values for NADPH and NADH. Agents at concentrations that show activating effects on the wild-type enzyme such as potassium chloride and urea all inactivate the K54Q enzyme. There appear to be no gross conformational differences between wild-type and K54Q enzyme molecules as judged by competitive ELISA using peptide-specific antibodies against human dihydrofolate reductase and from protease susceptibility studies on both wild-type and K54Q mutant enzymes. The pH-rate profiles using NADPH for K54Q and wild-type enzymes show divergences at certain pH values, suggesting the possibility of alteration(s) in the steps of the catalytic pathway for the K54Q enzyme

  7. Isolation and characterization of dihydrofolate reductase from trimethoprim-susceptible and trimethoprim-resistant Pseudomonas cepacia.

    OpenAIRE

    Burns, J L; Lien, D M; Hedin, L A

    1989-01-01

    Trimethoprim resistance was investigated in cystic fibrosis isolates of Pseudomonas cepacia. Determination of the MIC of trimethoprim for 111 strains revealed at least two populations of resistant organisms, suggesting the presence of more than one mechanism of resistance. Investigation of the antibiotic target, dihydrofolate reductase, was undertaken in both a susceptible strain and a strain with high-level resistance (MIC, greater than 1,000 micrograms/ml). The enzyme was purified by using ...

  8. Dihydrofolate reductase and dihydropteroate synthase genotypes associated with in vitro resistance of Plasmodium falciparum to pyrimethamine, trimethoprim, sulfadoxine, and sulfamethoxazole

    DEFF Research Database (Denmark)

    Khalil, Insaf; Rønn, Anita M; Alifrangis, Michael

    2003-01-01

    A total of 70 Plasmodium falciparum isolates were tested in vitro against pyrimethamine (PYR), trimethoprim (TRM), sulfadoxine (SDX), and sulfamethoxazole (SMX), and their dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genotypes were determined. dhfr genotypes correlated...

  9. Fragment Discovery for the Design of Nitrogen Heterocycles as Mycobacterium tuberculosis Dihydrofolate Reductase Inhibitors.

    Science.gov (United States)

    Shelke, Rupesh U; Degani, Mariam S; Raju, Archana; Ray, Mukti Kanta; Rajan, Mysore G R

    2016-08-01

    Fragment-based drug design was used to identify Mycobacterium tuberculosis (Mtb) dihydrofolate reductase (DHFR) inhibitors. Screening of ligands against the Mtb DHFR enzyme resulted in the identification of multiple fragment hits with IC50 values in the range of 38-90 μM versus Mtb DHFR and minimum inhibitory concentration (MIC) values in the range of 31.5-125 μg/mL. These fragment scaffolds would be useful for anti-tubercular drug design. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Selection of drug resistant mutants from random library of Plasmodium falciparum dihydrofolate reductase in Plasmodium berghei model

    OpenAIRE

    Tipsuwan, Wachiraporn; Srichairatanakool, Somdet; Kamchonwongpaisan, Sumalee; Yuthavong, Yongyuth; Uthaipibull, Chairat

    2011-01-01

    Abstract Background The prevalence of drug resistance amongst the human malaria Plasmodium species has most commonly been associated with genomic mutation within the parasites. This phenomenon necessitates evolutionary predictive studies of possible resistance mutations, which may occur when a new drug is introduced. Therefore, identification of possible new Plasmodium falciparum dihydrofolate reductase (PfDHFR) mutants that confer resistance to antifolate drugs is essential in the process of...

  11. Crystallographic investigation of the cooperative interaction between trimethoprim, reduced cofactor and dihydrofolate reductase

    International Nuclear Information System (INIS)

    Champness, J.N.; Stammers, D.K.; Beddell, C.R.

    1986-01-01

    The structure of the complex between E. coli form I dihydrofolate reductase, the antibacterial trimethoprim and NADPH has been determined by X-ray crystallography. The inhibitor and cofactor are in mutual contact. A flexible chain segment which includes Met 20 is in contact with the inhibitor in the presence of NADPH, but more distant in its absence. By contrast, the inhibitor conformation is little changed with NADPH present. The authors discuss these observations with regard to the mutually cooperative binding of these ligands to the protein, and to the associated enhancement of inhibitory selectivity shown by trimethoprim for bacterial as opposed to vertebrate enzyme. (Auth.)

  12. Multiple ligand-binding modes in bacterial R67 dihydrofolate reductase

    Science.gov (United States)

    Alonso, Hernán; Gillies, Malcolm B.; Cummins, Peter L.; Bliznyuk, Andrey A.; Gready, Jill E.

    2005-03-01

    R67 dihydrofolate reductase (DHFR), a bacterial plasmid-encoded enzyme associated with resistance to the drug trimethoprim, shows neither sequence nor structural homology with the chromosomal DHFR. It presents a highly symmetrical toroidal structure, where four identical monomers contribute to the unique central active-site pore. Two reactants (dihydrofolate, DHF), two cofactors (NADPH) or one of each (R67•DHF•NADPH) can be found simultaneously within the active site, the last one being the reactive ternary complex. As the positioning of the ligands has proven elusive to empirical determination, we addressed the problem from a theoretical perspective. Several potential structures of the ternary complex were generated using the docking programs AutoDock and FlexX. The variability among the final poses, many of which conformed to experimental data, prompted us to perform a comparative scoring analysis and molecular dynamics simulations to assess the stability of the complexes. Analysis of ligand-ligand and ligand-protein interactions along the 4 ns trajectories of eight different structures allowed us to identify important inter-ligand contacts and key protein residues. Our results, combined with published empirical data, clearly suggest that multipe binding modes of the ligands are possible within R67 DHFR. While the pterin ring of DHF and the nicotinamide ring of NADPH assume a stacked endo-conformation at the centre of the pore, probably assisted by V66, Q67 and I68, the tails of the molecules extend towards opposite ends of the cavity, adopting multiple configurations in a solvent rich-environment where hydrogen-bond interactions with K32 and Y69 may play important roles.

  13. Mechanism of the reaction catalyzed by dihydrofolate reductase from Escherichia coli: pH and deuterium isotope effects with NADPH as the variable substrate

    International Nuclear Information System (INIS)

    Morrison, J.F.; Stone, S.R.

    1988-01-01

    The variations with pH of the kinetic parameters and primary deuterium isotope effects for the reaction of NADPH with dihydrofolate reductase from Escherichia coli have been determined. The aims of the investigations were to elucidate the chemical mechanism of the reaction and to obtain information about the location of the rate-limiting steps. The V and V/K/sub NADPH/ profiles indicate that a single ionizing group at the active center of the enzyme must be protonated for catalysis, whereas the K/sub i/ profiles show that the binding of NADPH to the free enzyme and of ATP-ribose to the enzyme-dihydrofolate complex is pH independent. From the results of deuterium isotope effects on V/K/sub NADPH/, it is concluded that NADPH behaves as a sticky substrate. It is this stickiness that raises artificially the intrinsic pK value of 6.4 for the Asp-27 residue of the enzyme-dihydrofolate complex to an observed value of 8.9. Thus, the binary enzyme complex is largely protonated at neutral pH. The elevation of the intrinsic pK value of 6.4 for the ternary enzyme-NADPH-dihydrofolate complex to 8.5 is not due to the kinetic effects of substrates. Rather, it is the consequence of the lower, pH-independent rate of product release and the faster pH-dependent catalytic step. The data for deuterium isotope and deuterium solvent isotope effects are consistent with the postulate that, for the reduction of dihydrofolate to tetrahydrofolate, protonation precedes hydride transfer. A scheme is proposed for the indirect transfer of a proton from the enzyme to dihydrofolate

  14. Photoaffinity analogues of methotrexate as folate antagonist binding probes. 1. Photoaffinity labeling of murine L1210 dihydrofolate reductase and amino acid sequence of the binding region

    International Nuclear Information System (INIS)

    Price, E.M.; Smith, P.L.; Klein, T.E.; Freisheim, J.H.

    1987-01-01

    N/sup α/-(4-Amino-4-deoxy-10-methylpteroyl)-N/sup epsilon/-(4-azido-5-[ 125 I]iodosalicylyl)-L-lysine, a photoaffinity analogue of methotrexate, is only 2-fold less potent than methotrexate in the inhibition of murine L1210 dihydrofolate reductase. Irradiation of the enzyme in the presence of an equimolar concentration of the 125 I-labeled analogue ultimately leads to an 8% incorporation of the photoprobe. A 100-fold molar excess of methotrexate essentially blocks this incorporation. Cyanogen bromide digestion of the labeled enzyme, followed by high-pressure liquid chromatography purification of the generated peptides, indicates that greater than 85% of the total radioactivity is incorporated into a single cyanogen bromide peptide. Sequence analysis revealed this peptide to be residues 53-111, with a majority of the radioactivity centered around residues 63-65 (Lys-Asn-Arg). These data demonstrate that the photoaffinity analogue specifically binds to dihydrofolate reductase and covalently modifies the enzyme following irradiation and is therefore a photolabeling agent useful for probing the inhibitor binding domain of the enzyme

  15. Protein dynamics and stability: The distribution of atomic fluctuations in thermophilic and mesophilic dihydrofolate reductase derived using elastic incoherent neutron scattering

    International Nuclear Information System (INIS)

    Meinhold, Lars; Clement, David; Tehei, M.; Daniel, R.M.; Finney, J.L.; Smith, Jeremy C.

    2008-01-01

    The temperature dependence of the dynamics of mesophilic and thermophilic dihydrofolate reductase is examined using elastic incoherent neutron scattering. It is demonstrated that the distribution of atomic displacement amplitudes can be derived from the elastic scattering data by assuming a (Weibull) functional form that resembles distributions seen in molecular dynamics simulations. The thermophilic enzyme has a significantly broader distribution than its mesophilic counterpart. Furthermore, although the rate of increase with temperature of the atomic mean-square displacements extracted from the dynamic structure factor is found to be comparable for both enzymes, the amplitudes are found to be slightly larger for the thermophilic enzyme. Therefore, these results imply that the thermophilic enzyme is the more flexible of the two

  16. Rational Design of Novel Allosteric Dihydrofolate Reductase Inhibitors Showing Antibacterial Effects on Drug-Resistant Escherichia coli Escape Variants.

    Science.gov (United States)

    Srinivasan, Bharath; Rodrigues, João V; Tonddast-Navaei, Sam; Shakhnovich, Eugene; Skolnick, Jeffrey

    2017-07-21

    In drug discovery, systematic variations of substituents on a common scaffold and bioisosteric replacements are often used to generate diversity and obtain molecules with better biological effects. However, this could saturate the small-molecule diversity pool resulting in drug resistance. On the other hand, conventional drug discovery relies on targeting known pockets on protein surfaces leading to drug resistance by mutations of critical pocket residues. Here, we present a two-pronged strategy of designing novel drugs that target unique pockets on a protein's surface to overcome the above problems. Dihydrofolate reductase, DHFR, is a critical enzyme involved in thymidine and purine nucleotide biosynthesis. Several classes of compounds that are structural analogues of the substrate dihydrofolate have been explored for their antifolate activity. Here, we describe 10 novel small-molecule inhibitors of Escherichia coli DHFR, EcDHFR, belonging to the stilbenoid, deoxybenzoin, and chalcone family of compounds discovered by a combination of pocket-based virtual ligand screening and systematic scaffold hopping. These inhibitors show a unique uncompetitive or noncompetitive inhibition mechanism, distinct from those reported for all known inhibitors of DHFR, indicative of binding to a unique pocket distinct from either substrate or cofactor-binding pockets. Furthermore, we demonstrate that rescue mutants of EcDHFR, with reduced affinity to all known classes of DHFR inhibitors, are inhibited at the same concentration as the wild-type. These compounds also exhibit antibacterial activity against E. coli harboring the drug-resistant variant of DHFR. This discovery is the first report on a novel class of inhibitors targeting a unique pocket on EcDHFR.

  17. Fitness trade-offs in the evolution of dihydrofolate reductase and drug resistance in Plasmodium falciparum.

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    Marna S Costanzo

    Full Text Available Patterns of emerging drug resistance reflect the underlying adaptive landscapes for specific drugs. In Plasmodium falciparum, the parasite that causes the most serious form of malaria, antifolate drugs inhibit the function of essential enzymes in the folate pathway. However, a handful of mutations in the gene coding for one such enzyme, dihydrofolate reductase, confer drug resistance. Understanding how evolution proceeds from drug susceptibility to drug resistance is critical if new antifolate treatments are to have sustained usefulness.We use a transgenic yeast expression system to build on previous studies that described the adaptive landscape for the antifolate drug pyrimethamine, and we describe the most likely evolutionary trajectories for the evolution of drug resistance to the antifolate chlorcycloguanil. We find that the adaptive landscape for chlorcycloguanil is multi-peaked, not all highly resistant alleles are equally accessible by evolution, and there are both commonalities and differences in adaptive landscapes for chlorcycloguanil and pyrimethamine.Our findings suggest that cross-resistance between drugs targeting the same enzyme reflect the fitness landscapes associated with each particular drug and the position of the genotype on both landscapes. The possible public health implications of these findings are discussed.

  18. Chemical Ligation and Isotope Labeling to Locate Dynamic Effects during Catalysis by Dihydrofolate Reductase.

    Science.gov (United States)

    Luk, Louis Y P; Ruiz-Pernía, J Javier; Adesina, Aduragbemi S; Loveridge, E Joel; Tuñón, Iñaki; Moliner, Vincent; Allemann, Rudolf K

    2015-07-27

    Chemical ligation has been used to alter motions in specific regions of dihydrofolate reductase from E. coli and to investigate the effects of localized motional changes on enzyme catalysis. Two isotopic hybrids were prepared; one with the mobile N-terminal segment containing heavy isotopes ((2) H, (13) C, (15) N) and the remainder of the protein with natural isotopic abundance, and the other one with only the C-terminal segment isotopically labeled. Kinetic investigations indicated that isotopic substitution of the N-terminal segment affected only a physical step of catalysis, whereas the enzyme chemistry was affected by protein motions from the C-terminal segment. QM/MM studies support the idea that dynamic effects on catalysis mostly originate from the C-terminal segment. The use of isotope hybrids provides insights into the microscopic mechanism of dynamic coupling, which is difficult to obtain with other studies, and helps define the dynamic networks of intramolecular interactions central to enzyme catalysis. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

  19. Biosynthetic incorporation of telluromethionine into dihydrofolate reductase and crystallographic analysis of the distribution of tellurium atoms in the protein molecule

    Energy Technology Data Exchange (ETDEWEB)

    Kunkle, M.G.; Lewinski, K.; Boles, J.O.; Dunlap, R.B.; Odom, J.D.; Lebioda, L. [Univ. of South Carolina, Columbia, SC (United States)

    1994-12-01

    Recent successes in crystallographic studies of proteins with methionine (Met) residues replaced with SeMet, pioneered by Hendrickson and coworkers, inspired us to replace Met with TeMet in Escherichia coli dihydrofolate reductase (DHFR). E. coli DHFR, which catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate, consists of 159 residues, 5 of which are Met. TeMet was incorporated into DHFR using the Met auxotroph, E. coli DL41, carrying the expression vector pWT8 with an IPTG inducible promoter and ampicillin resistance gene. The enzyme was purified by successive chromatography on Q-Sepharose and PHenyl Sepharose resins, yielding milligram quantities of homogeneous enzyme with a specific activity of 40 units/mg. TeMet DHFR exhibits kinetic properties similar to those of wt DHFR. Amino acid analysis indicated 3 authentic Met residues in TeMet DHFR, whereas atomic absorption spectroscopy detected 2 Te per protein molecule. Amino acid sequence analysis results suggested that only authentic Met was present in the first three Met positions (1,16,and 20). Crystals of Te-DHFR were grown in the presence of methotrexate from PEG 4000 and were isomorphous with wt-DHFR crystals grown from ethanol. Difference Fourier maps and restrained least-squares refinement show very little, if any, Te in the first three Met positions: Met{sup 1}, Met{sup 16}, and Met{sup 20}, whereas the occupancy of Te in positions 42 and 92 is 0.64. Apparently, the process of folding, subsequent purification, and crystallization select DHFR molecules with Te in Met{sup 42} and Met{sup 92}. Replacing Met with TeMet provides an internal probe that should facilitate structural and mechanistic studies of proteins.

  20. Effect of gamma rays at the dihydrofolate reductase locus: deletions and inversions

    International Nuclear Information System (INIS)

    Urlaub, G.; Mitchell, P.J.; Kas, E.; Chasin, L.A.; Funanage, V.L.; Myoda, T.T.; Hamlin, J.

    1986-01-01

    A series 11 gamma-ray-induced mutants at the dihydrofolate reductase (dhfr) locus in Chinese hamster ovary cells has been examined for the types of DNA sequence change brought about by this form of ionizing radiation. All 11 mutants were found to have suffered major structural changes affecting the dhfr gene. In eight of the mutants, all or part of the dhfr gene has been deleted. The extent of these deletions was examined in seven of these mutants and, for comparison, in two deletion mutants that were induced by UV irradiation. For this purpose, probes from an overlapping set of cosmids that span 210 kb of DNA in this region were used. Three of seven gamma-ray-induced mutants and one UV-induced mutant were shown to have deleted the entire 210-kb region. In the remaining mutants, endpoints ranging from within the dhfr gene to 100 kb downstream were observed. No upstream endpoints were detected, so that an upper limit on the size of these large deletions could not be assigned. Three of the 11 gamma-ray-induced mutants contained an interruption in the dhfr gene without any detectable loss of sequence. Restriction analysis of these interrupted mutants showed that at least 8-14 kb of foreign DNA sequence became joined to the gene at the point of disruption. Cytogenetic analysis of these mutants showed that in two cases an inversion of the banding pattern on chromosome Z-2 had taken place. The inverted dhfr mutants contain very low amounts of dhfr RNA sequences, and the 5' end of an inversion mutant gene exhibits the same pattern of DNA methylation and DNase I-hypersensitivity as the wild-type gene. Our results suggest that ionizing radiation causes primarily, if not exclusively, large deletions and inversions in mammalian cells

  1. A simple, high-throughput method to detect Plasmodium falciparum single nucleotide polymorphisms in the dihydrofolate reductase, dihydropteroate synthase, and P. falciparum chloroquine resistance transporter genes using polymerase chain reaction- and enzyme-linked immunosorbent

    DEFF Research Database (Denmark)

    Alifrangis, Michael; Enosse, Sonia; Pearce, Richard

    2005-01-01

    Single nucleotide polymorphisms (SNPs) in the Plasmodium falciparum dihydrofolate reductase (dhfr), and dihydropteroate synthetase (dhps), and chloroquine resistance transporter (Pfcrt) genes are used as molecular markers of P. falciparum resistance to sulfadoxine/pyrimethamine and chloroquine....... However, to be a practical tool in the surveillance of drug resistance, simpler methods for high-throughput haplotyping are warranted. Here we describe a quick and simple technique that detects dhfr, dhps, and Pfcrt SNPs using polymerase chain reaction (PCR)- and enzyme-linked immunosorbent assay (ELISA...

  2. Structures of dihydrofolate reductase-thymidylate synthase of Trypanosoma cruzi in the folate-free state and in complex with two antifolate drugs, trimetrexate and methotrexate

    Energy Technology Data Exchange (ETDEWEB)

    Senkovich, Olga; Schormann, Norbert; Chattopadhyay, Debasish; (UAB)

    2010-11-22

    The flagellate protozoan parasite Trypanosoma cruzi is the pathogenic agent of Chagas disease (also called American trypanosomiasis), which causes approximately 50 000 deaths annually. The disease is endemic in South and Central America. The parasite is usually transmitted by a blood-feeding insect vector, but can also be transmitted via blood transfusion. In the chronic form, Chagas disease causes severe damage to the heart and other organs. There is no satisfactory treatment for chronic Chagas disease and no vaccine is available. There is an urgent need for the development of chemotherapeutic agents for the treatment of T. cruzi infection and therefore for the identification of potential drug targets. The dihydrofolate reductase activity of T. cruzi, which is expressed as part of a bifunctional enzyme, dihydrofolate reductase-thymidylate synthase (DHFR-TS), is a potential target for drug development. In order to gain a detailed understanding of the structure-function relationship of T. cruzi DHFR, the three-dimensional structure of this protein in complex with various ligands is being studied. Here, the crystal structures of T. cruzi DHFR-TS with three different compositions of the DHFR domain are reported: the folate-free state, the complex with the lipophilic antifolate trimetrexate (TMQ) and the complex with the classical antifolate methotrexate (MTX). These structures reveal that the enzyme is a homodimer with substantial interactions between the two TS domains of neighboring subunits. In contrast to the enzymes from Cryptosporidium hominis and Plasmodium falciparum, the DHFR and TS active sites of T. cruzi lie on the same side of the monomer. As in other parasitic DHFR-TS proteins, the N-terminal extension of the T. cruzi enzyme is involved in extensive interactions between the two domains. The DHFR active site of the T. cruzi enzyme shows subtle differences compared with its human counterpart. These differences may be exploited for the development of

  3. Direct 19F NMR observation of the conformational selection of optically active rotamers of the antifolate compound fluoronitropyrimethamine bound to enzyme dihydrofolate reductase

    International Nuclear Information System (INIS)

    Tendler, S.J.B.; Birdsall, B.; Feeney, J.; Griffin, R.J.; Stevens, M.F.G.; Roberts, G.C.K.

    1988-01-01

    The molucular basis of the binding of the lipophilic antifolate compound fluoronitropyrimethamine to its target enzyme dihydrofolate reductase has been investigated using a combination of 19 F NMR spectroscopy and molecular mechanical calculations 19 F NMR reveals the presence of two different conformational states for the fluoronitropyrimethamine-Lactobacillus casei enzyme complex. MM2 molecular mechanical calculations predict restricted rotation about the C5-C1 bond of the ligand and this give rise to two slowly interconverting rotamers which are an enantiomeric pair. The results of 19 F NMR spectroscopy reveal that both these isomers bind to the enzyme, with different affinities. There is no detectable interconversion of the bound rotamers themselves on the NMR timescale. The effect of the addition of co-enzyme to the sample is to reverse the preference the enzyme has for each rotamer. (author). 11 refs.; 3 figs

  4. Thioredoxin reductase 1 knockdown enhances selenazolidine cytotoxicity in human lung cancer cells via mitochondrial dysfunction

    Science.gov (United States)

    Poerschke, Robyn L.; Moos, Philip J.

    2010-01-01

    Thioredoxin reductase (TR1) is a selenoprotein that is involved in cellular redox status control and deoxyribonucleotide biosynthesis. Many cancers, including lung, overexpress TR1, making it a potential cancer therapy target. Previous work has shown that TR1 knockdown enhances the sensitivity of cancer cells to anticancer treatments, as well as certain selenocompounds. However, it is unknown if TR1 knockdown produces similar effect on the sensitivity of human lung cancer cells. To further elucidate the role of TR1 in the mechanism of selenocompounds in lung cancer, a lentiviral microRNA delivery system to knockdown TR1 expression in A549 human lung adenocarcinoma cells was utilized. Cell viability was assessed after 48 hr treatment with the selenocysteine prodrug selenazolidines 2-butylselenazolidine-4(R)-carboxylic acid (BSCA) and 2-cyclohexylselenazolidine-4-(R)-carboxylic acid (ChSCA), selenocystine (SECY), methylseleninic acid (MSA), 1,4-phenylenebis(methylene)selenocyanate (p-XSC), and selenomethionine (SEM). TR1 knockdown increased the cytotoxicity of BSCA, ChSCA, and SECY but did not sensitize cells to MSA, SEM, or p-XSC. GSH and TR1 depletion together decreased cell viability, while no change was observed with GSH depletion alone. Reactive oxygen species generation was induced only in TR1 knockdown cells treated with the selenazolidines or SECY. These three compounds also decreased total intracellular glutathione levels and oxidized thioredoxin, but in a TR1 independent manner. TR1 knockdown increased selenazolidine and SECY-induced mitochondrial membrane depolarization, as well as DNA strand breaks and AIF translocation from the mitochondria. These results indicate the ability of TR1 to modulate the cytotoxic effects of BSCA, ChSCA and SECY in human lung cancer cells through mitochondrial dysfunction. PMID:20920480

  5. Defining the Structural Basis for Allosteric Product Release from E. coli Dihydrofolate Reductase Using NMR Relaxation Dispersion.

    Science.gov (United States)

    Oyen, David; Fenwick, R Bryn; Aoto, Phillip C; Stanfield, Robyn L; Wilson, Ian A; Dyson, H Jane; Wright, Peter E

    2017-08-16

    The rate-determining step in the catalytic cycle of E. coli dihydrofolate reductase is tetrahydrofolate (THF) product release, which can occur via an allosteric or an intrinsic pathway. The allosteric pathway, which becomes accessible when the reduced cofactor NADPH is bound, involves transient sampling of a higher energy conformational state, greatly increasing the product dissociation rate as compared to the intrinsic pathway that obtains when NADPH is absent. Although the kinetics of this process are known, the enzyme structure and the THF product conformation in the transiently formed excited state remain elusive. Here, we use side-chain proton NMR relaxation dispersion measurements, X-ray crystallography, and structure-based chemical shift predictions to explore the structural basis of allosteric product release. In the excited state of the E:THF:NADPH product release complex, the reduced nicotinamide ring of the cofactor transiently enters the active site where it displaces the pterin ring of the THF product. The p-aminobenzoyl-l-glutamate tail of THF remains weakly bound in a widened binding cleft. Thus, through transient entry of the nicotinamide ring into the active site, the NADPH cofactor remodels the enzyme structure and the conformation of the THF to form a weakly populated excited state that is poised for rapid product release.

  6. Nuclear magnetic resonance study of interaction of ligands with Streptococcus faecium dihydrofolate reductase labeled with [#betta#-13C]tryptophan

    International Nuclear Information System (INIS)

    London, R.E.; Groff, J.P.; Cocco, L.; Blakley, R.L.

    1982-01-01

    Dihydrofolate reductase from Streptococcus faecium has been labeled with [#betta#- 13 C]tryptophan. We have determined changes occurring in the chemical shifts and line widths of the four resonances of the 13 C NMR spectrum of the labeled enzyme, due to its interaction with various ligands. These include the coenzyme, NPDPH and related nucleotides, folate and its polyglutamate derivatives, and many inhibitors including methotrexate and trimethoprim. In addition, paramagnetic relaxation effects produced by a bound spin-labeled analogue of 2'-phosphoadenosine-5'-diphosphoribose on the tryptophan C/sup #betta#/ carbons have been measured. Distances calculated from the relaxation data have been compared with corresponding distances in the crystallographic model of the NADPH-methotrexate ternary complex of Lactobacillus casei reductase. The paramagnetic relaxation data indicate that the two downfield resonances (1 and 2) correspond to tryptophans (W/sub A/ and W/sub B/) that are more remote from the catalytic site, and from the crystallographic model these are seen to be Trp-115 and Trp-160. The upfield resonances (3 and 4) that show broadening due to chemical exchange correspond to closer residues (W/sub C/ and W/sub D/), and these are identified with Trp-6 and Trp-22. However, the relaxation data do not permit specific assignments within the nearer and farther pairs. Although resonance 3, which is split due to chemical exchange, was formerly assigned to Trp-6, data obtained for the enzyme in the presence of various ligands are better interpreted if resonance 3 is assigned to Trp-22, which is located on a loop that joins elements of secondary structure and forms one side of the ligand-binding cavity

  7. Multiple origins of resistance-conferring mutations in Plasmodium vivax dihydrofolate reductase

    Directory of Open Access Journals (Sweden)

    O'Neil Michael T

    2008-04-01

    Full Text Available Abstract Background In order to maximize the useful therapeutic life of antimalarial drugs, it is crucial to understand the mechanisms by which parasites resistant to antimalarial drugs are selected and spread in natural populations. Recent work has demonstrated that pyrimethamine-resistance conferring mutations in Plasmodium falciparum dihydrofolate reductase (dhfr have arisen rarely de novo, but spread widely in Asia and Africa. The origin and spread of mutations in Plasmodium vivax dhfr were assessed by constructing haplotypes based on sequencing dhfr and its flanking regions. Methods The P. vivax dhfr coding region, 792 bp upstream and 683 bp downstream were amplified and sequenced from 137 contemporary patient isolates from Colombia, India, Indonesia, Papua New Guinea, Sri Lanka, Thailand, and Vanuatu. A repeat motif located 2.6 kb upstream of dhfr was also sequenced from 75 of 137 patient isolates, and mutational relationships among the haplotypes were visualized using the programme Network. Results Synonymous and non-synonymous single nucleotide polymorphisms (SNPs within the dhfr coding region were identified, as was the well-documented in-frame insertion/deletion (indel. SNPs were also identified upstream and downstream of dhfr, with an indel and a highly polymorphic repeat region identified upstream of dhfr. The regions flanking dhfr were highly variable. The double mutant (58R/117N dhfr allele has evolved from several origins, because the 58R is encoded by at least 3 different codons. The triple (58R/61M/117T and quadruple (57L/61M/117T/173F, 57I/58R/61M/117T and 57L/58R/61M/117T mutant alleles had at least three independent origins in Thailand, Indonesia, and Papua New Guinea/Vanuatu. Conclusion It was found that the P. vivax dhfr coding region and its flanking intergenic regions are highly polymorphic and that mutations in P. vivax dhfr that confer antifolate resistance have arisen several times in the Asian region. This contrasts

  8. Structural analysis of dihydrofolate reductases enables rationalization of antifolate binding affinities and suggests repurposing possibilities.

    Science.gov (United States)

    Bhosle, Amrisha; Chandra, Nagasuma

    2016-03-01

    Antifolates are competitive inhibitors of dihydrofolate reductase (DHFR), a conserved enzyme that is central to metabolism and widely targeted in pathogenic diseases, cancer and autoimmune disorders. Although most clinically used antifolates are known to be target specific, some display a fair degree of cross-reactivity with DHFRs from other species. A method that enables identification of determinants of affinity and specificity in target DHFRs from different species and provides guidelines for the design of antifolates is currently lacking. To address this, we first captured the potential druggable space of a DHFR in a substructure called the 'supersite' and classified supersites of DHFRs from 56 species into 16 'site-types' based on pairwise structural similarity. Analysis of supersites across these site-types revealed that DHFRs exhibit varying extents of dissimilarity at structurally equivalent positions in and around the binding site. We were able to explain the pattern of affinities towards chemically diverse antifolates exhibited by DHFRs of different site-types based on these structural differences. We then generated an antifolate-DHFR network by mapping known high-affinity antifolates to their respective supersites and used this to identify antifolates that can be repurposed based on similarity between supersites or antifolates. Thus, we identified 177 human-specific and 458 pathogen-specific antifolates, a large number of which are supported by available experimental data. Thus, in the light of the clinical importance of DHFR, we present a novel approach to identifying differences in the druggable space of DHFRs that can be utilized for rational design of antifolates. © 2016 Federation of European Biochemical Societies.

  9. Two crystal structures of dihydrofolate reductase-thymidylate synthase from Cryptosporidium hominis reveal protein–ligand interactions including a structural basis for observed antifolate resistance

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Amy C., E-mail: aca@dartmouth.edu [Dartmouth College, Department of Chemistry, Burke Laboratories, Hanover, NH 03755 (United States)

    2005-03-01

    An analysis of the protein–ligand interactions in two crystal structures of DHFR-TS from C. hominis reveals a possible structural basis for observed antifolate resistance in C. hominis DHFR. A comparison with the structure of human DHFR reveals residue substitutions that may be exploited for the design of species-selective inhibitors. Cryptosporidium hominis is a protozoan parasite that causes acute gastrointestinal illness. There are no effective therapies for cryptosporidiosis, highlighting the need for new drug-lead discovery. An analysis of the protein–ligand interactions in two crystal structures of dihydrofolate reductase-thymidylate synthase (DHFR-TS) from C. hominis, determined at 2.8 and 2.87 Å resolution, reveals that the interactions of residues Ile29, Thr58 and Cys113 in the active site of C. hominis DHFR provide a possible structural basis for the observed antifolate resistance. A comparison with the structure of human DHFR reveals active-site differences that may be exploited for the design of species-selective inhibitors.

  10. Fragment-based design of symmetrical bis-benzimidazoles as selective inhibitors of the trimethoprim-resistant, type II R67 dihydrofolate reductase.

    Science.gov (United States)

    Bastien, Dominic; Ebert, Maximilian C C J C; Forge, Delphine; Toulouse, Jacynthe; Kadnikova, Natalia; Perron, Florent; Mayence, Annie; Huang, Tien L; Vanden Eynde, Jean Jacques; Pelletier, Joelle N

    2012-04-12

    The continuously increasing use of trimethoprim as a common antibiotic for medical use and for prophylactic application in terrestrial and aquatic animal farming has increased its prevalence in the environment. This has been accompanied by increased drug resistance, generally in the form of alterations in the drug target, dihydrofolate reductase (DHFR). The most highly resistant variants of DHFR are known as type II DHFR, among which R67 DHFR is the most broadly studied variant. We report the first attempt at designing specific inhibitors to this emerging drug target by fragment-based design. The detection of inhibition in R67 DHFR was accompanied by parallel monitoring of the human DHFR, as an assessment of compound selectivity. By those means, small aromatic molecules of 150-250 g/mol (fragments) inhibiting R67 DHFR selectively in the low millimolar range were identified. More complex, symmetrical bis-benzimidazoles and a bis-carboxyphenyl were then assayed as fragment-based leads, which procured selective inhibition of the target in the low micromolar range (K(i) = 2-4 μM). The putative mode of inhibition is discussed according to molecular modeling supported by in vitro tests. © 2012 American Chemical Society

  11. Trade-offs with stability modulate innate and mutationally acquired drug-resistance in bacterial dihydrofolate reductase enzymes.

    Science.gov (United States)

    Matange, Nishad; Bodkhe, Swapnil; Patel, Maitri; Shah, Pooja

    2018-06-05

    Structural stability is a major constraint on the evolution of protein sequences. However, under strong directional selection, mutations that confer novel phenotypes but compromise structural stability of proteins may be permissible. During the evolution of antibiotic resistance, mutations that confer drug resistance often have pleiotropic effects on the structure and function of antibiotic-target proteins, usually essential metabolic enzymes. In this study, we show that trimethoprim-resistant alleles of dihydrofolate reductase from Escherichia coli (EcDHFR) harbouring the Trp30Gly, Trp30Arg or Trp30Cys mutations are significantly less stable than the wild type making them prone to aggregation and proteolysis. This destabilization is associated with lower expression level resulting in a fitness cost and negative epistasis with other TMP-resistant mutations in EcDHFR. Using structure-based mutational analysis we show that perturbation of critical stabilizing hydrophobic interactions in wild type EcDHFR enzyme explains the phenotypes of Trp30 mutants. Surprisingly, though crucial for the stability of EcDHFR, significant sequence variation is found at this site among bacterial DHFRs. Mutational and computational analyses in EcDHFR as well as in DHFR enzymes from Staphylococcus aureus and Mycobacterium tuberculosis demonstrate that natural variation at this site and its interacting hydrophobic residues, modulates TMP-resistance in other bacterial DHFRs as well, and may explain the different susceptibilities of bacterial pathogens to trimethoprim. Our study demonstrates that trade-offs between structural stability and function can influence innate drug resistance as well as the potential for mutationally acquired drug resistance of an enzyme. ©2018 The Author(s).

  12. Selection of drug resistant mutants from random library of Plasmodium falciparum dihydrofolate reductase in Plasmodium berghei model

    Directory of Open Access Journals (Sweden)

    Yuthavong Yongyuth

    2011-05-01

    Full Text Available Abstract Background The prevalence of drug resistance amongst the human malaria Plasmodium species has most commonly been associated with genomic mutation within the parasites. This phenomenon necessitates evolutionary predictive studies of possible resistance mutations, which may occur when a new drug is introduced. Therefore, identification of possible new Plasmodium falciparum dihydrofolate reductase (PfDHFR mutants that confer resistance to antifolate drugs is essential in the process of antifolate anti-malarial drug development. Methods A system to identify mutations in Pfdhfr gene that confer antifolate drug resistance using an animal Plasmodium parasite model was developed. By using error-prone PCR and Plasmodium transfection technologies, libraries of Pfdhfr mutant were generated and then episomally transfected to Plasmodium berghei parasites, from which pyrimethamine-resistant PfDHFR mutants were selected. Results The principal mutation found from this experiment was S108N, coincident with the first pyrimethamine-resistance mutation isolated from the field. A transgenic P. berghei, in which endogenous Pbdhfr allele was replaced with the mutant PfdhfrS108N, was generated and confirmed to have normal growth rate comparing to parental non-transgenic parasite and also confer resistance to pyrimethamine. Conclusion This study demonstrated the power of the transgenic P. berghei system to predict drug-resistant Pfdhfr mutations in an in vivo parasite/host setting. The system could be utilized for identification of possible novel drug-resistant mutants that could arise against new antifolate compounds and for prediction the evolution of resistance mutations.

  13. Assessment of Folic Acid Supplementation in Pregnant Women by Estimation of Serum Levels of Tetrahydrofolic Acid, Dihydrofolate Reductase, and Homocysteine

    Directory of Open Access Journals (Sweden)

    Manisha Naithani

    2016-01-01

    Full Text Available Background. Status of folic acid use in pregnant women of the hilly regions in North India was little known. This study was carried out to assess the folic acid use and estimate folate metabolites in pregnant women of this region. Materials and Methods. This cross-sectional study is comprised of 76 pregnant women, whose folic acid supplementation was assessed by a questionnaire and serum levels of homocysteine, tetrahydrofolic acid (THFA, and dihydrofolate reductase (DHFR were estimated using Enzyme Linked Immunoassays. Results. The study data revealed awareness of folic acid use during pregnancy was present in 46.1% and 23.7% were taking folic acid supplements. The study depicted that there was no statistically significant difference between serum levels of THFA and DHFR in pregnant women with and without folic acid supplements (p=0.790. Hyperhomocysteinemia was present in 15.78% of the participants. Conclusion. Less awareness about folic acid supplementation and low use of folic acid by pregnant women were observed in this region. Sufficient dietary ingestion may suffice for the escalated requirements in pregnancy, but since this cannot be ensured, hence folic acid supplementation should be made as an integral part of education and reproductive health programs for its better metabolic use, growth, and development of fetus.

  14. An approximate but efficient method to calculate free energy trends by computer simulation: Application to dihydrofolate reductase-inhibitor complexes

    Science.gov (United States)

    Gerber, Paul R.; Mark, Alan E.; van Gunsteren, Wilfred F.

    1993-06-01

    Derivatives of free energy differences have been calculated by molecular dynamics techniques. The systems under study were ternary complexes of Trimethoprim (TMP) with dihydrofolate reductases of E. coli and chicken liver, containing the cofactor NADPH. Derivatives are taken with respect to modification of TMP, with emphasis on altering the 3-, 4- and 5-substituents of the phenyl ring. A linear approximation allows the encompassing of a whole set of modifications in a single simulation, as opposed to a full perturbation calculation, which requires a separate simulation for each modification. In the case considered here, the proposed technique requires a factor of 1000 less computing effort than a full free energy perturbation calculation. For the linear approximation to yield a significant result, one has to find ways of choosing the perturbation evolution, such that the initial trend mirrors the full calculation. The generation of new atoms requires a careful treatment of the singular terms in the non-bonded interaction. The result can be represented by maps of the changed molecule, which indicate whether complex formation is favoured under movement of partial charges and change in atom polarizabilities. Comparison with experimental measurements of inhibition constants reveals fair agreement in the range of values covered. However, detailed comparison fails to show a significant correlation. Possible reasons for the most pronounced deviations are given.

  15. NMR studies of differences in the conformations and dynamics of ligand complexes formed with mutant dihydrofolate reductases

    International Nuclear Information System (INIS)

    Birdsall, B.; Andrews, J.; Ostler, G.; Tendler, S.J.B.; Feeney, J.; Roberts, G.C.K.; Davies, R.W.; Cheung, H.T.A.

    1989-01-01

    Two mutants of Lactobacillus casei dihydrofolate reductase, Trp 21 → Leu and Asp 26 → Glu, have been prepared by using site-directed mutagenesis methods, and their ligand binding and structural properties have been compared with those of the wild-type enzyme. 1 H, 13 C, and 31 P NMR studies have been carried out to characterize the structural changes in the complexes of the mutant and wild-type enzymes. Replacement of the conserved Trp 21 by a Leu residue causes a decrease in activity of the enzyme and reduces the NADPH binding constant by a factor of 400. The binding of substrates and substrate analogues is only slightly affected. 1 H NMR studies of the Trp 21 → Leu enzyme complexes have confirmed the original resonance assignments for Trp 21. In complexes formed with methotrexate and the mutant enzyme, the results indicate some small changes in conformation occurring as much as 14 angstrom away from the site of substitution. For the enzyme-NADPH complexes, the chemical shifts of nuclei in the bound coenzyme indicate that the nicotinamide ring binds differently in complexes with the mutant and the wild-type enzyme. There are complexes where the wild-type enzyme has been shown to exist in solution as a mixture of conformations, and studies on the corresponding complexes with the Trp 21 → Leu mutant indicate that the delicately poised equilibria can be perturbed. Some conformational adjustments are required to allow the carboxylate of Glu 26 to bind effectively to the N1 proton of inhibitors such as methotrexate and trimethoprim

  16. In Silico Screening, Synthesis and In Vitro Evaluation of Some Quinazolinone and Pyridine Derivatives as Dihydrofolate Reductase Inhibitors for Anticancer Activity

    Directory of Open Access Journals (Sweden)

    A. G. Nerkar

    2009-01-01

    Full Text Available Dihydrofolate reductase (DHFR is the important target for anticancer drugs belonging to the class of antimetabolites as the enzyme plays important role in the de novo purine synthesis. We here report the in silico screening to obtain best fit molecules as DHFR inhibitors, synthesis of some ʻbest fitʼ quinazolinone from 2-phenyl-3-(substituted-benzilidine-amino quinazolinones (Quinazolinone Shiff's bases QSB1-5 and pyridine-4-carbohydrazide Shiff's bases (ISB1-5 derivatives and their in vitro anticancer assay. Synthesis of the molecules was performed using microwave assisted synthesis. The structures of these molecules were elucidated by IR and 1H-NMR. These compounds were then subjected for in vitro anticancer evaluation against five human cancer cell-lines for anticancer cyto-toxicity assay. Methotrexate (MTX was used as standard for this evaluation to give a comparable inhibition of the cell proliferation by DHFR inhibition. Placlitaxel, adriamycin and 5-fluoro-uracil were also used as standard to give a comparable activity of these compounds with other mechanism of anticancer activity. ISB3 (4-(N, N-dimethyl-amino-phenyl Schiff''s base derivative of pyridine carbohydrazide showed equipotent activity with the standards used in in vitro anticancer assay as per the NCI (National Cancer Institute guidelines.

  17. Probing the electrostatics of active site microenvironments along the catalytic cycle for Escherichia coli dihydrofolate reductase.

    Science.gov (United States)

    Liu, C Tony; Layfield, Joshua P; Stewart, Robert J; French, Jarrod B; Hanoian, Philip; Asbury, John B; Hammes-Schiffer, Sharon; Benkovic, Stephen J

    2014-07-23

    Electrostatic interactions play an important role in enzyme catalysis by guiding ligand binding and facilitating chemical reactions. These electrostatic interactions are modulated by conformational changes occurring over the catalytic cycle. Herein, the changes in active site electrostatic microenvironments are examined for all enzyme complexes along the catalytic cycle of Escherichia coli dihydrofolate reductase (ecDHFR) by incorporation of thiocyanate probes at two site-specific locations in the active site. The electrostatics and degree of hydration of the microenvironments surrounding the probes are investigated with spectroscopic techniques and mixed quantum mechanical/molecular mechanical (QM/MM) calculations. Changes in the electrostatic microenvironments along the catalytic environment lead to different nitrile (CN) vibrational stretching frequencies and (13)C NMR chemical shifts. These environmental changes arise from protein conformational rearrangements during catalysis. The QM/MM calculations reproduce the experimentally measured vibrational frequency shifts of the thiocyanate probes across the catalyzed hydride transfer step, which spans the closed and occluded conformations of the enzyme. Analysis of the molecular dynamics trajectories provides insight into the conformational changes occurring between these two states and the resulting changes in classical electrostatics and specific hydrogen-bonding interactions. The electric fields along the CN axes of the probes are decomposed into contributions from specific residues, ligands, and solvent molecules that make up the microenvironments around the probes. Moreover, calculation of the electric field along the hydride donor-acceptor axis, along with decomposition of this field into specific contributions, indicates that the cofactor and substrate, as well as the enzyme, impose a substantial electric field that facilitates hydride transfer. Overall, experimental and theoretical data provide evidence for

  18. Halophilic mechanism of the enzymatic function of a moderately halophilic dihydrofolate reductase from Haloarcula japonica strain TR-1.

    Science.gov (United States)

    Miyashita, Yurina; Ohmae, Eiji; Ikura, Teikichi; Nakasone, Kaoru; Katayanagi, Katsuo

    2017-05-01

    Dihydrofolate (DHF) reductase coded by a plasmid of the extremely halophilic archaeon Haloarcula japonica strain TR-1 (HjDHFR P1) shows moderate halophilicity on enzymatic activity at pH 6.0, although there is no significant effect of NaCl on its secondary structure. To elucidate the salt-activation and -inactivation mechanisms of this enzyme, we investigated the effects of pH and salt concentration, deuterium isotope effect, steady-state kinetics, and rapid-phase ligand-binding kinetics. Enzyme activity was increased eightfold by the addition of 500 mM NaCl at pH 6.0, fourfold by 250 mM at pH 8.0, and became independent of salt concentration at pH 10.0. Full isotope effects observed at pH 10.0 under 0-1000 mM NaCl indicated that the rate of hydride transfer, which was the rate-determining step at the basic pH region, was independent of salt concentration. Conversely, rapid-phase ligand-binding experiments showed that the amplitude of the DHF-binding reaction increased and the tetrahydrofolate (THF)-releasing rate decreased with increasing NaCl concentration. These results suggested that the salt-activation mechanism of HjDHFR P1 is via the population change of the anion-unbound and anion-bound conformers, which are binding-incompetent and -competent conformations for DHF, respectively, while that of salt inactivation is via deceleration of the THF-releasing rate, which is the rate-determining step at the neutral pH region.

  19. Site-specific bioconjugation of a murine dihydrofolate reductase enzyme by copper(I-catalyzed azide-alkyne cycloaddition with retained activity.

    Directory of Open Access Journals (Sweden)

    Sung In Lim

    Full Text Available Cu(I-catalyzed azide-alkyne cycloaddition (CuAAC is an efficient reaction linking an azido and an alkynyl group in the presence of copper catalyst. Incorporation of a non-natural amino acid (NAA containing either an azido or an alkynyl group into a protein allows site-specific bioconjugation in mild conditions via CuAAC. Despite its great potential, bioconjugation of an enzyme has been hampered by several issues including low yield, poor solubility of a ligand, and protein structural/functional perturbation by CuAAC components. In the present study, we incorporated an alkyne-bearing NAA into an enzyme, murine dihydrofolate reductase (mDHFR, in high cell density cultivation of Escherichia coli, and performed CuAAC conjugation with fluorescent azide dyes to evaluate enzyme compatibility of various CuAAC conditions comprising combination of commercially available Cu(I-chelating ligands and reductants. The condensed culture improves the protein yield 19-fold based on the same amount of non-natural amino acid, and the enzyme incubation under the optimized reaction condition did not lead to any activity loss but allowed a fast and high-yield bioconjugation. Using the established conditions, a biotin-azide spacer was efficiently conjugated to mDHFR with retained activity leading to the site-specific immobilization of the biotin-conjugated mDHFR on a streptavidin-coated plate. These results demonstrate that the combination of reactive non-natural amino acid incorporation and the optimized CuAAC can be used to bioconjugate enzymes with retained enzymatic activity.

  20. ORF Alignment: NC_002945 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available hain A, Dihydrofolate ... Reductase Of Mycobacterium Tuberculosis Complexed With ... Nadph And... Methotrexate pdb|1DG8|A Chain A, Dihydrofolate ... Reductase Of Mycobacterium Tuberculosis... Complexed With ... Nadph pdb|1DG7|A Chain A, Dihydrofolate Reductase Of ... Mycobacterium Tuberculosi...late ... Reductase Of Mycobacterium Tuberculosis Complexed With ... Nadph And Trimethoprim emb

  1. ORF Alignment: NC_000962 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available hain A, Dihydrofolate ... Reductase Of Mycobacterium Tuberculosis Complexed With ... Nadph And... Methotrexate pdb|1DG8|A Chain A, Dihydrofolate ... Reductase Of Mycobacterium Tuberculosis... Complexed With ... Nadph pdb|1DG7|A Chain A, Dihydrofolate Reductase Of ... Mycobacterium Tuberculosi...late ... Reductase Of Mycobacterium Tuberculosis Complexed With ... Nadph And Trimethoprim emb

  2. ORF Alignment: NC_002755 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available hain A, Dihydrofolate ... Reductase Of Mycobacterium Tuberculosis Complexed With ... Nadph And... Methotrexate pdb|1DG8|A Chain A, Dihydrofolate ... Reductase Of Mycobacterium Tuberculosis... Complexed With ... Nadph pdb|1DG7|A Chain A, Dihydrofolate Reductase Of ... Mycobacterium Tuberculosi...late ... Reductase Of Mycobacterium Tuberculosis Complexed With ... Nadph And Trimethoprim emb

  3. Design, Synthesis, and X-ray Crystal Structures of 2,4-Diaminofuro[2,3-d]pyrimidines as Multireceptor Tyrosine Kinase and Dihydrofolate Reductase Inhibitors

    Science.gov (United States)

    Gangjee, Aleem; Li, Wei; Lin, Lu; Zeng, Yibin; Ihnat, Michael; Warnke, Linda A.; Green, Dixy W.; Cody, Vivian; Pace, Jim; Queener, Sherry F.

    2009-01-01

    To optimize dual receptor tyrosine kinase (RTK) and dihydrofolate reductase (DHFR) inhibition, the E- and Z-isomers of 5-[2-(2-methoxyphenyl)prop-1-en-1-yl]furo[2,3-d]pyrimidine-2,4-diamines (1a and 1b) were separated by HPLC and the X-ray crystal structures (2.0 Å and 1.4 Å respectively) with mouse DHFR and NADPH as well as 1b with human DHFR (1.5 Å) were determined. The E- and Z-isomers adopt different binding modes when bound to mouse DHFR. A series of 2,4-diaminofuro[2,3-d]pyrimidines 2–13 were designed and synthesized using the X-ray crystal structures of 1a and 1b with DHFR to increase their DHFR inhibitory activity. Wittig reactions of appropriate 2-methoxyphenyl ketones with 2,4-diamino-6-chloromethyl furo[2,3-d]pyrimidine afforded the C8–C9 unsaturated compounds 2–7 and catalytic reduction gave the saturated 8–13. Homologation of the C9-methyl analog maintains DHFR inhibitory activity. In addition, inhibition of EGFR and PDGFR-β were discovered for saturated C9-homologated analogs 9 and 10 that were absent in the saturated C9-methyl analogs. PMID:19748785

  4. The crystal structure of the bifunctional deaminase/reductase RibD of the riboflavin biosynthetic pathway in Escherichia coli: implications for the reductive mechanism.

    Science.gov (United States)

    Stenmark, Pål; Moche, Martin; Gurmu, Daniel; Nordlund, Pär

    2007-10-12

    We have determined the crystal structure of the bi-functional deaminase/reductase enzyme from Escherichia coli (EcRibD) that catalyzes two consecutive reactions during riboflavin biosynthesis. The polypeptide chain of EcRibD is folded into two domains where the 3D structure of the N-terminal domain (1-145) is similar to cytosine deaminase and the C-terminal domain (146-367) is similar to dihydrofolate reductase. We showed that EcRibD is dimeric and compared our structure to tetrameric RibG, an ortholog from Bacillus subtilis (BsRibG). We have also determined the structure of EcRibD in two binary complexes with the oxidized cofactor (NADP(+)) and with the substrate analogue ribose-5-phosphate (RP5) and superposed these two in order to mimic the ternary complex. Based on this superposition we propose that the invariant Asp200 initiates the reductive reaction by abstracting a proton from the bound substrate and that the pro-R proton from C4 of the cofactor is transferred to C1 of the substrate. A highly flexible loop is found in the reductase active site (159-173) that appears to control cofactor and substrate binding to the reductase active site and was therefore compared to the corresponding Met20 loop of E. coli dihydrofolate reductase (EcDHFR). Lys152, identified by comparing substrate analogue (RP5) coordination in the reductase active site of EcRibD with the homologous reductase from Methanocaldococcus jannaschii (MjaRED), is invariant among bacterial RibD enzymes and could contribute to the various pathways taken during riboflavin biosynthesis in bacteria and yeast.

  5. Molecular epidemiology of malaria in Cameroon. XXX. sequence analysis of Plasmodium falciparum ATPase 6, dihydrofolate reductase, and dihydropteroate synthase resistance markers in clinical isolates from children treated with an artesunate-sulfadoxine-pyrimethamine combination.

    Science.gov (United States)

    Menemedengue, Virginie; Sahnouni, Khalifa; Basco, Leonardo; Tahar, Rachida

    2011-07-01

    Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes are reliable molecular markers for antifolate resistance. The P. falciparum ATPase 6 (pfatp6) gene has been proposed to be a potential marker for artemisinin resistance. In our previous clinical study, we showed that artesunate-sulfadoxine-pyrimethamine is highly effective against uncomplicated malaria in Yaoundé, Cameroon. In the present study, dhfr, dhps, and pfatp6 mutations in P. falciparum isolates obtained from children treated with artesunate-sulfadoxine-pyrimethamine were determined. All 61 isolates had wild-type Pfatp6 263, 623, and 769 alleles, and 11 (18%) had a single E431K substitution. Three additional mutations, E643Q, E432K, and E641Q, were detected. The results did not indicate any warning signal of serious concern (i.e., no parasites were seen with quintuple dhfr-dhps, DHFR Ile164Leu, or pfatp6 mutations), as confirmed by the high clinical efficacy of artesunate-sulfadoxine-pyrimethamine. Further studies are required to identify a molecular marker that reliably predicts artemisinin resistance.

  6. Imaging of dihydrofolate reductase fusion gene expression in xenografts of human liver metastases of colorectal cancer in living rats

    Energy Technology Data Exchange (ETDEWEB)

    Mayer-Kuckuk, Philipp; Bertino, Joseph R.; Banerjee, Debabrata [Molecular Pharmacology and Therapeutics Program, Memorial Sloan-Kettering Cancer Center, New York, NY (United States); The Cancer Institute of New Jersey, Robert Wood Johnson Medical School/UMDNJ, 195 Little Albany Street, NJ 08903, New Brunswick (United States); Doubrovin, Mikhail; Blasberg, Ronald; Tjuvajev, Juri Gelovani [Department of Neurooncology, Memorial Sloan-Kettering Cancer Center, New York, NY (United States); Gusani, Niraj J.; Fong, Yuman [Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, NY (United States); Gade, Terence; Koutcher, Jason A. [Department of Medical Physics, Memorial Sloan-Kettering Cancer Center, New York, NY (United States); Balatoni, Julius; Finn, Ronald [Radiochemistry/Cyclotron Core Facility, Memorial Sloan-Kettering Cancer Center, New York, NY (United States); Akhurst, Tim; Larson, Steven [Nuclear Medicine Service, Memorial Sloan-Kettering Cancer Center, New York, NY (United States)

    2003-09-01

    Radionuclide imaging has been demonstrated to be feasible to monitor transgene expression in vivo. We hypothesized that a potential application of this technique is to non-invasively detect in deep tissue, such as cancer cells metastatic to the liver, a specific molecular response following systemic drug treatment. Utilizing human colon adenocarcinoma cells derived from a patient's liver lesion we first developed a nude rat xenograft model for colorectal cancer metastatic to the liver. Expression of a dihydrofolate reductase-herpes simplex virus 1 thymidine kinase fusion (DHFR-HSV1 TK) transgene in the hepatic tumors was monitored in individual animals using the tracer [{sup 124}I]2'-fluoro-2'-deoxy-5-iodouracil-{beta}-d-arabinofuranoside (FIAU) and a small animal micro positron emission tomograph (microPET), while groups of rats were imaged using the tracer [{sup 131}I]FIAU and a clinical gamma camera. Growth of the human metastatic colorectal cancer cells in the rat liver was detected using magnetic resonance imaging and confirmed by surgical inspection. Single as well as multiple lesions of different sizes and sites were observed in the liver of the animals. Next, using a subset of rats bearing hepatic tumors, which were retrovirally bulk transduced to express the DHFR-HSV1 TK transgene, we imaged the fusion protein expression in the hepatic tumor of living rats using the tracer [{sup 124}I]FIAU and a microPET. The observed deep tissue signals were highly specific for the tumors expressing the DHFR-HSV1 TK fusion protein compared with parental untransduced tumors and other tissues as determined by gamma counting of tissue samples. A subsequent study used the tracer [{sup 131}I]FIAU and a gamma camera to monitor two groups of transduced hepatic tumor-bearing rats. Prior to imaging, one group was treated with trimetrexate to exploit DHFR-mediated upregulation of the fusion gene product. Imaging in the living animal as well as subsequent gamma

  7. Structural comparison of complexes of methotrexate analogues with Lactobacillus casei dihydrofolate reductase by two-dimensional 1H NMR at 500 MHz

    International Nuclear Information System (INIS)

    Hammond, S.J.; Birdsall, B.; Feeney, J.; Searle, M.S.; Roberts, G.C.K.; Cheung, H.T.A.

    1987-01-01

    The authors have used two-dimensional (2D) NMR methods to examine complexes of Lactobacillus casei dihydrofolate reductase and methotrexate (MTX) analogues having structural modifications of the benzoyl ring and also the glutamic acid moiety. Assignments of the 1 H signals in the spectra of the various complexes were made by comparison of their 2D spectra with those complexes containing methotrexate where we have previously assigned resonances from 32 of the 162 amino acid residues. In the complexes formed with the dihalomethotrexate analogues, the glutamic acid and pteridine ring moieties were shown to bind to the enzyme in a manner similar to that found in the methotrexate-enzyme complex. Perturbations in 1 H chemical shifts of protons in Phe-49, Leu-54, and Leu-27 and the methotrexate H7 and NMe protons were observed in the different complexes and were accounted for by changes in orientation of the benzoyl ring in the various complexes. Binding of oxidized or reduced coenzyme to the binary complexes did not result in different shifts for Leu-27, Leu-54, or Leu-19 protons, and thus, the orientation of the benzoyl ring of the methotrexate analogues is not perturbed greatly by the presence of either oxidized or reduced coenzyme. In the complex with the γ-monoamide analog, the 1 H signals of assigned residues in the protein had almost identical shifts with the corresponding protons in the methotrexate-enzyme complex for all residues except His-28 and, to a lesser extent, Leu-27. This indicates that while the His-28 interaction with the MTX γ-CO 2 - is no longer present in this complex with the γ-amide, there has not been a major change in the overall structure of the two complexes. This behavior contrasts to that of the α-amide complex where 1 H signals from protons in several amino acid residues are different compared with their values in the complex formed with methotrexate

  8. Purification and properties of the dihydrofolate synthetase from Serratia indica

    International Nuclear Information System (INIS)

    Ikeda, Masamichi; Iwai, Kazuo

    1976-01-01

    The dihydrofolate synthetase (EC6.3.2.12) responsible for catalyzing the synthesis of dihydrofolic acid from dihydropteroic acid and L-glutamic acid was purified about 130-fold from extracts of Serratia indica IFO 3759 by ammonium sulfate fractionation, DEAE-Sephadex column chromatography, Sephadex G-200 gel filtration, and DEAE-cellulose column chromatography. The enzyme preparation obtained was shown to be homogeneous by DEAE-cellulose column chromatography and ultracentrifugal analysis. The sedimentation coefficient of this enzyme was 3.9 S, and the molecular weight was determined to be about 47,000 by Sephadex G-100. The optimum pH for the reaction was 9.0. The enzymatic reaction required dihydropteroate, L-glutamate and ATP as substrates, and Mg 2+ and K + as cofactors. γ-L-Glutamyl-L-glutamic acid cannot replace L-glutamic acid as the substrate. Neither pteroic acid nor tetrahydropteroic acid can be used as the substrate. ATP was partially replaced by ITP or GTP. The enzyme reaction was inhibited by the addition of ADP, but not by AMP. One mole of dihydrofolate, 1 mole of ADP and 1 mole of orthophosphate were produced from each 1 mole of dihydropteroic acid, L-glutamic acid, and ATP. These results suggest that the systematic name for the dihydrofolate synthetase is 7,8-dihydropteroate: L-glutamate ligase (ADP). (auth.)

  9. JS-K, a Nitric Oxide Prodrug, Has Enhanced Cytotoxicity in Colon Cancer Cells with Knockdown of Thioredoxin Reductase 1

    Science.gov (United States)

    Edes, Kornelia; Cassidy, Pamela; Shami, Paul J.; Moos, Philip J.

    2010-01-01

    Background The selenoenzyme thioredoxin reductase 1 has a complex role relating to cell growth. It is induced as a component of the cellular response to potentially mutagenic oxidants, but also appears to provide growth advantages to transformed cells by inhibiting apoptosis. In addition, selenocysteine-deficient or alkylated forms of thioredoxin reductase 1 have also demonstrated oxidative, pro-apoptotic activity. Therefore, a greater understanding of the role of thioredoxin reductase in redox initiated apoptotic processes is warranted. Methodology The role of thioredoxin reductase 1 in RKO cells was evaluated by attenuating endogenous thioredoxin reductase 1 expression with siRNA and then either inducing a selenium-deficient thioredoxin reductase or treatment with distinct redox challenges including, hydrogen peroxide, an oxidized lipid, 4-hydroxy-2-nonenol, and a nitric oxide donating prodrug. Thioredoxin redox status, cellular viability, and effector caspase activity were measured. Conclusions/Significance In cells with attenuated endogenous thioredoxin reductase 1, a stably integrated selenocysteine-deficient form of the enzyme was induced but did not alter either the thioredoxin redox status or the cellular growth kinetics. The oxidized lipid and the nitric oxide donor demonstrated enhanced cytotoxicity when thioredoxin reductase 1 was knocked-down; however, the effect was more pronounced with the nitric oxide prodrug. These results are consistent with the hypothesis that attenuation of the thioredoxin-system can promote apoptosis in a nitric oxide-dependent manner. PMID:20098717

  10. The role of biliverdin reductase in colorectal cancer

    International Nuclear Information System (INIS)

    Bauer, M.

    2010-01-01

    In recent years, the effects of biliverdin and bilirubin have been studied extensively, and an inhibitory effect of bile pigments in cancer progression has been proposed. In this study we focused on the effects of biliverdin reductase, the enzyme that converts biliverdin to bilirubin, in colorectal cancer. For in vitro experiments we used a human colorectal carcinoma cell line and transfected it with an expression construct of shRNA specific for biliverdin reductase, to create cells with stable knock-down of enzyme expression. Cell proliferation was analyzed using the CASY model TT cell counting device. Western blot protein analysis was performed to study intracellular signaling cascades. Samples of human colorectal cancer were analyzed using immunohistochemistry. We were able to confirm the antiproliferative effects of bile pigments on cancer cells in vitro. However, this effect was attenuated in biliverdin reductase knock down cells. ERK and Akt activation seen under biliverdin and bilirubin treatment was also reduced in biliverdin reductase deficient cells. Immunohistochemical analysis of tumor samples from patients with colorectal cancer showed elevated biliverdin reductase levels. High enzyme expression was associated with lower overall and disease free patient survival. We conclude that BVR is required for bile pigment mediated effects regarding cancer cell proliferation and modulation of intracellular signaling cascades. The role of BVR overexpression in vivo and its exact influence on cancer progression and patient survival need to be further investigated. (author) [de

  11. Frequencies distribution of dihydrofolate reductase and dihydropteroate synthetase mutant alleles associated with sulfadoxine-pyrimethamine resistance in Plasmodium falciparum population from Hadhramout Governorate, Yemen.

    Science.gov (United States)

    Bamaga, Omar A A; Mahdy, Mohammed A K; Lim, Yvonne A L

    2015-12-22

    Malaria in Yemen is mainly caused by Plasmodium falciparum and 25% of the population is at high risk. Sulfadoxine-pyrimethamine (SP) had been used as monotherapy against P. falciparum. Emergence of chloroquine resistance led to the shift in anti-malarial treatment policy in Yemen to artemisinin-based combination therapy, that is artesunate (AS) plus SP as first-line therapy for uncomplicated malaria and artemether-lumefantrine as second-line treatment. This study aimed to screen mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes associated with SP resistance among P. falciparum population in Hadhramout governorate, Yemen. Genomic DNA was extracted from dried blood spots of 137 P. falciparum isolates collected from a community-based study. DNA was amplified using nested polymerase chain reaction (PCR) and subsequently sequenced for Pfdhfr and Pfdhps genes. Sequences were analysed for mutations in Pfdhfr gene codons 51, 59, 108, and 164 and in Pfdhps gene codons 436, 437, and 540. A total of 128 and 114 P. falciparum isolates were successfully sequenced for Pfdhfr and Pfdhps genes, respectively. Each Pfdhfr mutant allele (I51 and N108) in P. falciparum population had a frequency of 84%. Pfdhfr R59 mutant allele was detected in one isolate. Mutation at codon 437 (G437) in the Pfdhps gene was detected in 44.7% of falciparum malaria isolates. Frequencies of Pfdhfr double mutant genotype (I51C59N108I164) and Pfdhfr/Pfdhps triple mutant genotype (I51C59N108I164-S436G437K540) were 82.8 and 39.3%, respectively. One isolate harboured Pfdhfr triple mutant genotype (I51, R59, N108, I164) and Pfdhfr/Pfdhps quadruple mutant genotype (I51R59N108I164-S436G437K540). High frequencies of Pfdhfr and Pfdhps mutant alleles and genotypes in P. falciparum population in Hadhramout, Yemen, highlight the risk of developing resistance for SP, the partner drug of AS, which subsequently will expose the parasite to AS monotherapy increasing then the

  12. Knock-Down of the IFR1 Protein Perturbs the Homeostasis of Reactive Electrophile Species and Boosts Photosynthetic Hydrogen Production in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Venkanna, Deepak; Südfeld, Christian; Baier, Thomas; Homburg, Sarah V; Patel, Anant V; Wobbe, Lutz; Kruse, Olaf

    2017-01-01

    The protein superfamily of short-chain dehydrogenases/reductases (SDR), including members of the atypical type (aSDR), covers a huge range of catalyzed reactions and in vivo substrates. This superfamily also comprises isoflavone reductase-like (IRL) proteins, which are aSDRs highly homologous to isoflavone reductases from leguminous plants. The molecular function of IRLs in non-leguminous plants and green microalgae has not been identified as yet, but several lines of evidence point at their implication in reactive oxygen species homeostasis. The Chlamydomonas reinhardtii IRL protein IFR1 was identified in a previous study, analyzing the transcriptomic changes occurring during the acclimation to sulfur deprivation and anaerobiosis, a condition that triggers photobiological hydrogen production in this microalgae. Accumulation of the cytosolic IFR1 protein is induced by sulfur limitation as well as by the exposure of C. reinhardtii cells to reactive electrophile species (RES) such as reactive carbonyls. The latter has not been described for IRL proteins before. Over-accumulation of IFR1 in the singlet oxygen response 1 ( sor1 ) mutant together with the presence of an electrophile response element, known to be required for SOR1-dependent gene activation as a response to RES, in the promoter of IFR1 , indicate that IFR1 expression is controlled by the SOR1-dependent pathway. An implication of IFR1 into RES homeostasis, is further implied by a knock-down of IFR1 , which results in a diminished tolerance toward RES. Intriguingly, IFR1 knock-down has a positive effect on photosystem II (PSII) stability under sulfur-deprived conditions used to trigger photobiological hydrogen production, by reducing PSII-dependent oxygen evolution, in C. reinhardtii . Reduced PSII photoinhibition in IFR1 knock-down strains prolongs the hydrogen production phase resulting in an almost doubled final hydrogen yield compared to the parental strain. Finally, IFR1 knock-down could be

  13. High prevalence of dhfr triple mutant and correlation with high rates of sulphadoxine-pyrimethamine treatment failures in vivo in Gabonese children

    NARCIS (Netherlands)

    Mombo-Ngoma, G.; Oyakhirome, S.; Ord, R.; Gabor, J.J.; Greutélaers, K.C.; Profanter, K.; Greutélaers, B.; Kurth, F.; Lell, B.; Kun, J.F.J.; Issifou, S.; Roper, C.; Kremsner, P.G.; Grobusch, M.P.

    2011-01-01

    Background: Drug resistance contributes to the global malaria burden. Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) polymorphisms confer resistance to sulphadoxine-pyrimethamine (SP). Methods: The study assessed the frequency of SP resistance-conferring

  14. High prevalence of dhfr triple mutant and correlation with high rates of sulphadoxine-pyrimethamine treatment failures in vivo in Gabonese children

    NARCIS (Netherlands)

    Mombo-Ngoma, Ghyslain; Oyakhirome, Sunny; Ord, Rosalynn; Gabor, Julian J.; Greutélaers, Katja C.; Profanter, Katharina; Greutélaers, Benedikt; Kurth, Florian; Lell, Bertrand; Kun, Jürgen F. J.; Issifou, Saadou; Roper, Cally; Kremsner, Peter G.; Grobusch, Martin P.

    2011-01-01

    Drug resistance contributes to the global malaria burden. Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) polymorphisms confer resistance to sulphadoxine-pyrimethamine (SP). The study assessed the frequency of SP resistance-conferring polymorphisms in

  15. Ketopantoyl lactone reductase is a conjugated polyketone reductase.

    Science.gov (United States)

    Hata, H; Shimizu, S; Hattori, S; Yamada, H

    1989-03-01

    Ketopantoyl lactone reductase (EC 1.1.1.168) of Saccharomyces cerevisiae was found to catalyze the reduction of a variety of natural and unnatural conjugated polyketone compounds and quinones, such as isatin, ninhydrin, camphorquinone and beta-naphthoquinone in the presence of NADPH. 5-Bromoisatin is the best substrate for the enzyme (Km = 3.1 mM; Vmax = 650 mumol/min/mg). The enzyme is inhibited by quercetin, and several polyketones. These results suggest that ketopantoyl lactone reductase is a carbonyl reductase which specifically catalyzes the reduction of conjugated polyketones.

  16. Drug: D03771 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available D03771 Drug Diaveridine (USAN/INN) ... C13H16N4O2 D03771.gif ... Antiparasitic ... DG01932 ... Antifolate, antiprotoz...oal ... Veterinary medicine dihydrofolate reductase [KO:K13998] ... CAS: 5355-16-8 Pub

  17. The colon mucosa is a potential site for the initial triggering of Rheumatoid Arthritis

    DEFF Research Database (Denmark)

    Bennike, Tue Bjerg; Ellingsen, Torkell Juulsgaad; Glerup, Henning

    2017-01-01

    extracted by colonoscopy and analyzed by label-free quantitative proteomics, enabling the quantitation of 5366 proteins. The abundance of dihydrofolate reductase (DHFR) was statistically significantly increased in RA-patient biopsies compared with controls and correlated with the administered dosage...

  18. The binding sites on human heme oxygenase-1 for cytochrome p450 reductase and biliverdin reductase.

    Science.gov (United States)

    Wang, Jinling; de Montellano, Paul R Ortiz

    2003-05-30

    Human heme oxygenase-1 (hHO-1) catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, CO, and free iron. The biliverdin is subsequently reduced to bilirubin by biliverdin reductase. Earlier kinetic studies suggested that biliverdin reductase facilitates the release of biliverdin from hHO-1 (Liu, Y., and Ortiz de Montellano, P. R. (2000) J. Biol. Chem. 275, 5297-5307). We have investigated the binding of P450 reductase and biliverdin reductase to truncated, soluble hHO-1 by fluorescence resonance energy transfer and site-specific mutagenesis. P450 reductase and biliverdin reductase bind to truncated hHO-1 with Kd = 0.4 +/- 0.1 and 0.2 +/- 0.1 microm, respectively. FRET experiments indicate that biliverdin reductase and P450 reductase compete for binding to truncated hHO-1. Mutation of surface ionic residues shows that hHO-1 residues Lys18, Lys22, Lys179, Arg183, Arg198, Glu19, Glu127, and Glu190 contribute to the binding of cytochrome P450 reductase. The mutagenesis results and a computational analysis of the protein surfaces partially define the binding site for P450 reductase. An overlapping binding site including Lys18, Lys22, Lys179, Arg183, and Arg185 is similarly defined for biliverdin reductase. These results confirm the binding of biliverdin reductase to hHO-1 and define binding sites of the two reductases.

  19. A PHASE-II AND PHARMACOKINETIC STUDY WITH ORAL PIRITREXIM FOR METASTATIC BREAST-CANCER

    NARCIS (Netherlands)

    DEVRIES, EGE; GIETEMA, JA; WORKMAN, P; SCOTT, JE; CRAWSHAW, A; DOBBS, HJ; DENNIS, [No Value; MULDER, NH; SLEIJFER, DT; WILLEMSE, PHB

    Piritrexim is a lipid-soluble antifolate which, like methotrexate, has a potent capacity to inhibit dihydrofolate reductase. We performed a multicentre phase 11 study with piritrexim in patients with locally advanced or metastatic breast cancer. Twenty-four patients of which sixteen had received

  20. Dynamics of antifolate transport via the reduced folate carrier and the membrane folate receptor in murine leukaemia cells in vitro and in vivo

    NARCIS (Netherlands)

    Mauritz, Robert; Peters, Godefridus; Kathmann, Ietje; Teshale, Habte; Noordhuis, Paul; Comijn, Elizabeth; Pinedo, Herbert; Jansen, Gerrit

    Murine L1210 leukaemia cells expressing either the reduced folate carrier (RFC) or the membrane folate receptor (MFR) were studied in vitro and in vivo to assess the dynamics of membrane transport of two categories antifolates; folate-based inhibitors of dihydrofolate reductase (methotrexate,

  1. The A581G Mutation in the Gene Encoding Plasmodium falciparum Dihydropteroate Synthetase Reduces the Effectiveness of Sulfadoxine-Pyrimethamine Preventive Therapy in Malawian Pregnant Women

    NARCIS (Netherlands)

    Gutman, Julie; Kalilani, Linda; Taylor, Steve; Zhou, Zhiyong; Wiegand, Ryan E.; Thwai, Kyaw L.; Mwandama, Dyson; Khairallah, Carole; Madanitsa, Mwayi; Chaluluka, Ebbie; Dzinjalamala, Fraction; Ali, Doreen; Mathanga, Don P.; Skarbinski, Jacek; Shi, Ya Ping; Meshnick, Steve; ter Kuile, Feiko O.

    2015-01-01

    Background. The A581G mutation in the gene encoding Plasmodium falciparum dihydropteroate synthase (dhps), in combination with the quintuple mutant involving mutations in both dhps and the gene encoding dihydrofolate reductase (dhfr), the so-called sextuple mutant, has been associated with increased

  2. Drug: D05273 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available ... Trimethoprim antibacterial ... DG01931 ... Dihydrofolate reductase inhibitor, antibacterial ... Component of Rofenaid (TN) veterinary medi...cine Trimethoprim [DR:D00145] derivative ... CAS: 6981-18-6 PubChem: 47206982 ChEMBL: CHEMBL494760 LigandBox: D05273 NIKKAJI: J9.643A ...

  3. The evolutionary landscape of antifolate resistance in Plasmodium ...

    Indian Academy of Sciences (India)

    Permanent link: https://www.ias.ac.in/article/fulltext/jgen/090/02/0187-0190. Keywords. malaria; drug resistance; dihydrofolate reductase; pyrimethamine; chlorcycloguanil; antifolates. Author Affiliations. Marna S. Costanzo1 Daniel L. Hartl1. Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, ...

  4. Dhfr and dhps mutations in Plasmodium falciparum isolates in ...

    African Journals Online (AJOL)

    Sulfadoxine-pyrimethamine (SP), the current first line antimalarial drug in Tanzania, is compromised by evolution and spread of mutations in the parasite's dhfr and dhps genes. In the present study we established the baseline frequencies of Plasmodium falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate ...

  5. In silico design, construction and cloning of Trastuzumab humanized monoclonal antibody: A possible biosimilar for Herceptin

    Directory of Open Access Journals (Sweden)

    Soudabeh Akbarzadeh-Sharbaf

    2012-01-01

    Conclusions: This study was based on a recently developed technology for mAb expression in mammalian cells. The obtained constructs could be successfully used for biosimilar recombinant mAb production in CHO DG44 dihydrofolate reductase (DHFR gene deficient cell line in the suspension culture medium.

  6. Aldolase B knockdown prevents high glucose-induced methylglyoxal overproduction and cellular dysfunction in endothelial cells.

    Directory of Open Access Journals (Sweden)

    Jianghai Liu

    Full Text Available We used cultured endothelial cells as a model to examine whether up-regulation of aldolase B and enhanced methylglyoxal (MG formation play an important role in high glucose-induced overproduction of advanced glycosylation endproducts (AGEs, oxidative stress and cellular dysfunction. High glucose (25 mM incubation up-regulated mRNA levels of aldose reductase (an enzyme converting glucose to fructose and aldolase B (a key enzyme that catalyzes MG formation from fructose and enhanced MG formation in human umbilical vein endothelial cells (HUVECs and HUVEC-derived EA. hy926 cells. High glucose-increased MG production in EA. hy926 cells was completely prevented by siRNA knockdown of aldolase B, but unaffected by siRNA knockdown of aldolase A, an enzyme responsible for MG formation during glycolysis. In addition, inhibition of cytochrome P450 2E1 or semicarbazide-sensitive amine oxidase which produces MG during the metabolism of lipid and proteins, respectively, did not alter MG production. Both high glucose (25 mM and MG (30, 100 µM increased the formation of N(ε-carboxyethyl-lysine (CEL, a MG-induced AGE, oxidative stress (determined by the generation of oxidized DCF, H(2O(2, protein carbonyls and 8-oxo-dG, O-GlcNAc modification (product of the hexosamine pathway, membrane protein kinase C activity and nuclear translocation of NF-κB in EA. hy926 cells. However, the above metabolic and signaling alterations induced by high glucose were completely prevented by knockdown of aldolase B and partially by application of aminoguanidine (a MG scavenger or alagebrium (an AGEs breaker. In conclusion, efficient inhibition of aldolase B can prevent high glucose-induced overproduction of MG and related cellular dysfunction in endothelial cells.

  7. Direct C-H sulfenylation of purines and deazapurines

    Czech Academy of Sciences Publication Activity Database

    Klečka, Martin; Pohl, Radek; Čejka, Jan; Hocek, Michal

    2013-01-01

    Roč. 11, č. 31 (2013), s. 5189-5193 ISSN 1477-0520 Grant - others:GA ČR(CZ) GAP207/12/0205 Institutional support: RVO:61388963 Keywords : dihydrofolate-reductase * 6-(het)aryl-7-deazapurine ribonucleosides * thymidylate synthase Subject RIV: CC - Organic Chemistry Impact factor: 3.487, year: 2013

  8. Bioinformatic analysis of dihydrofolate reductase predicted in the ...

    African Journals Online (AJOL)

    olayemitoyin

    TASSER was used to predict the 3-D model structure of the protein and potential active binding site residues. ..... co-ordinate file model in PDB format was visualized with Jmol molecular .... evolutionary preservation of the protein dynamics to.

  9. Bioinformatic analysis of dihydrofolate reductase predicted in the ...

    African Journals Online (AJOL)

    Physiologic studies of Lactobacillus species show that some species cannot synthesize folate de novo, which is required for growth. Folate plays a critical role in regulating the amount of tetrahydrofolate in the cell that is utilized for DNA replication, and proliferation of the erythropoietic system. We recently sequenced the ...

  10. Immunocytochemical localization of APS reductase and bisulfite reductase in three Desulfovibrio species

    NARCIS (Netherlands)

    Kremer, D.R.; Veenhuis, M.; Fauque, G.; Peck Jr., H.D.; LeGall, J.; Lampreia, J.; Moura, J.J.G.; Hansen, T.A.

    1988-01-01

    The localization of APS reductase and bisulfite reductase in Desulfovibrio gigas, D. vulgaris Hildenborough and D. thermophilus was studied by immunoelectron microscopy. Polyclonal antibodies were raised against the purified enzymes from each strain. Cells fixed with formaldehyde/glutaraldehyde were

  11. Knockdown of NADPH-cytochrome P450 reductase results in reduced resistance to buprofezin in the small brown planthopper, Laodelphax striatellus (fallén).

    Science.gov (United States)

    Zhang, Yueliang; Wang, Yaming; Wang, Lihua; Yao, Jing; Guo, Huifang; Fang, Jichao

    2016-02-01

    NADPH-cytochrome P450 reductase (CPR) plays an important role in cytochrome P450 function, and CPR knockdown in several insects leads to increased susceptibility to insecticides. However, a putative CPR gene has not yet been fully characterized in the small brown planthopper Laodelphax striatellus, a notorious agricultural pest in rice that causes serious damage by transmitting rice stripe and rice black-streaked dwarf viruses. The objective of this study was to clone the cDNA and to knock down the expression of the gene that encodes L. striatellus CPR (LsCPR) to further determine whether P450s are involved in the resistance of L. striatellus to buprofezin. First, the full-length cDNA of LsCPR was cloned and found to contain an open reading frame (ORF) encoding a polypeptide of 679 amino acids with a calculated molecular mass and isoelectric point of 76.92kDa and 5.37, respectively. The deduced amino acid sequence shares high identity with the CPRs of other insects (98%, 97%, 75% and 68% for Sogatella furcifera, Nilaparvata lugens, Cimex lectularius and Anopheles gambiae, respectively) and possesses the characteristic features of classical CPRs, such as an N-terminal membrane anchor and conserved domains for flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) binding. Phylogenetic analysis revealed that LsCPR is located in a branch along with the CPRs of other hemipteran insects. LsCPR mRNA was detectable in all examined body parts and developmental stages of L. striatellus, as determined by real-time quantitative PCR (qPCR), and transcripts were most abundant in the adult abdomen and in first-instar nymphs and adults. Ingestion of 200μg/mL of LsCPR double-stranded RNA (dsLsCPR) by the planthopper for 5days significantly reduced the transcription level of LsCPR. Moreover, silencing of LsCPR caused increased susceptibility to buprofezin in a buprofezin-resistant (YN-BPF) strain but not in a

  12. RNCR3 knockdown inhibits diabetes mellitus-induced retinal reactive gliosis

    International Nuclear Information System (INIS)

    Liu, Chang; Li, Chao-peng; Wang, Jia-Jian; Shan, Kun; Liu, Xin; Yan, Biao

    2016-01-01

    Retinal reactive gliosis is an important pathological feature of diabetic retinopathy. Identifying the underlying mechanisms causing reactive gliosis will be important for developing new therapeutic strategies for treating diabetic retinopathy. Herein, we show that long noncoding RNA-RNCR3 knockdown significantly inhibits retinal reactive gliosis. RNCR3 knockdown leads to a marked reduction in the release of several cytokines. RNCR3 knockdown alleviates diabetes mellitus-induced retinal neurodegeneration, as shown by less apoptotic retinal cells and ameliorative visual function. RNCR3 knockdown could also decrease Müller glial cell viability and proliferation, and reduce the expression of glial reactivity-related genes including GFAP and vimentin in vitro. Collectively, this study shows that RNCR3 knockdown may be a promising strategy for the prevention of diabetes mellitus-induced retinal neurodegeneration. - Highlights: • RNCR3 knockdown inhibits retinal reactive gliosis. • RNCR3 knockdown causes a significant change in cytokine profile. • RNCR3 knockdown alleviates diabetes mellitus-induced retinal neurodegeneration. • RNCR3 knockdown affects Müller glial cell function in vitro.

  13. A cellular stress response (CSR) that interacts with NADPH-P450 reductase (NPR) is a new regulator of hypoxic response.

    Science.gov (United States)

    Oguro, Ami; Koyama, Chika; Xu, Jing; Imaoka, Susumu

    2014-02-28

    NADPH-P450 reductase (NPR) was previously found to contribute to the hypoxic response of cells, but the mechanism was not clarified. In this study, we identified a cellular stress response (CSR) as a new factor interacting with NPR by a yeast two-hybrid system. Overexpression of CSR enhanced the induction of erythropoietin and hypoxia response element (HRE) activity under hypoxia in human hepatocarcinoma cell lines (Hep3B), while knockdown of CSR suppressed them. This new finding regarding the interaction of NPR with CSR provides insight into the function of NPR in hypoxic response. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Replacement of the folC gene, encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli, with genes mutagenized in vitro.

    Science.gov (United States)

    Pyne, C; Bognar, A L

    1992-03-01

    The folylpolyglutamate synthetase-dihydrofolate synthetase gene (folC) in Escherichia coli was deleted from the bacterial chromosome and replaced by a selectable Kmr marker. The deletion strain required a complementing gene expressing folylpolyglutamate synthetase encoded on a plasmid for viability, indicating that folC is an essential gene in E. coli. The complementing folC gene was cloned into the vector pPM103 (pSC101, temperature sensitive for replication), which segregated spontaneously at 42 degrees C in the absence of selection. This complementing plasmid was replaced in the folC deletion strain by compatible pUC plasmids containing folC genes with mutations generated in vitro, producing strains which express only mutant folylpolyglutamate synthetase. Mutant folC genes expressing insufficient enzyme activity could not complement the chromosomal deletion, resulting in retention of the pPM103 plasmid. Some mutant genes expressing low levels of enzyme activity replaced the complementing plasmid, but the strains produced were auxotrophic for products of folate-dependent pathways. The folylpolyglutamate synthetase gene from Lactobacillus casei, which may lack dihydrofolate synthetase activity, replaced the complementing plasmid, but the strain was auxotrophic for all folate end products.

  15. Crystallographic Analysis Reveals a Novel Second Binding Site for Trimethoprim in Active Site Double Mutants of Human Dihydrofolate Reductase†,‡

    Science.gov (United States)

    Cody, Vivian; Pace, Jim; Piraino, Jennifer; Queener, Sherry F.

    2011-01-01

    In order to produce a more potent replacement for trimethoprim (TMP) used as a therapy for Pneumocystis pneumonia and targets dihydrofolate reductase from Pneumocystis jirovecii (pjDHFR), it is necessary to understand the determinants of potency and selectivity against DHFR from the mammalian host and fungal pathogen cells. To this end, active site residues in human (h)DHFR were replaced with those from pjDHFR. Structural data are reported for two complexes of TMP with the double mutants Gln35Ser/Asn64Phe (Q35S/N64F) and Gln35Lys/Asn64Phe (Q35K/N64F) of hDHFR that unexpectedly show evidence for the binding of two molecules of TMP: one molecule that binds in the normal folate binding site and the second molecule that binds in a novel subpocket site such that the mutated residue Phe64 is involved in van der Waals contacts to the trimethoxyphenyl ring of the second TMP molecule. Kinetic data for the binding of TMP to hDHFR and pjDHFR reveal an 84-fold selectivity of TMP against pjDHFR (Ki 49 nM) compared to hDHFR (Ki 4093 nM). Two mutants that contain one substitution from pj- and one from the closely related Pneumocystis carinii DHFR (pcDHFR) (Q35K/N64F and Q35S/N64F) show Ki values of 593 and 617 nM, respectively; these Ki values are well above both the Ki for pjDHFR and are similar to pcDHFR (Q35K/N64F) and Q35S/N64F) (305 nM). These results suggest that active site residues 35 and 64 play key roles in determining selectivity for pneumocystis DHFR, but that other residues contribute to the unique binding of inhibitors to these enzymes. PMID:21684339

  16. Shell Buckling Knockdown Factors

    Data.gov (United States)

    National Aeronautics and Space Administration — The Shell Buckling Knockdown Factor (SBKF) Project, NASA Engineering and Safety Center (NESC) Assessment #: 07-010-E, was established in March of 2007 by the NESC in...

  17. Ketopantoyl-lactone reductase from Candida parapsilosis: purification and characterization as a conjugated polyketone reductase.

    Science.gov (United States)

    Hata, H; Shimizu, S; Hattori, S; Yamada, H

    1989-02-24

    Ketopantoyl-lactone reductase (2-dehydropantoyl-lactone reductase, EC 1.1.1.168) was purified and crystallized from cells of Candida parapsilosis IFO 0708. The enzyme was found to be homogeneous on ultracentrifugation, high-performance gel-permeation liquid chromatography and SDS-polyacrylamide gel electrophoresis. The relative molecular mass of the native and SDS-treated enzyme is approximately 40,000. The isoelectric point of the enzyme is 6.3. The enzyme was found to catalyze specifically the reduction of a variety of natural and unnatural polyketones and quinones other than ketopantoyl lactone in the presence of NADPH. Isatin and 5-methylisatin are rapidly reduced by the enzyme, the Km and Vmax values for isatin being 14 microM and 306 mumol/min per mg protein, respectively. Ketopantoyl lactone is also a good substrate (Km = 333 microM and Vmax = 481 mumol/min per mg protein). Reverse reaction was not detected with pantoyl lactone and NADP+. The enzyme is inhibited by quercetin, several polyketones and SH-reagents. 3,4-Dihydroxy-3-cyclobutene-1,2-dione, cyclohexenediol-1,2,3,4-tetraone and parabanic acid are uncompetitive inhibitors for the enzyme, the Ki values being 1.4, 0.2 and 3140 microM, respectively, with isatin as substrate. Comparison of the enzyme with the conjugated polyketone reductase of Mucor ambiguus (S. Shimizu, H. Hattori, H. Hata and H. Yamada (1988) Eur. J. Biochem. 174, 37-44) and ketopantoyl-lactone reductase of Saccharomyces cerevisiae suggested that ketopantoyl-lactone reductase is a kind of conjugated polyketone reductase.

  18. The structure of apo and holo forms of xylose reductase, a dimeric aldo-keto reductase from Candida tenuis.

    Science.gov (United States)

    Kavanagh, Kathryn L; Klimacek, Mario; Nidetzky, Bernd; Wilson, David K

    2002-07-16

    Xylose reductase is a homodimeric oxidoreductase dependent on NADPH or NADH and belongs to the largely monomeric aldo-keto reductase superfamily of proteins. It catalyzes the first step in the assimilation of xylose, an aldose found to be a major constituent monosaccharide of renewable plant hemicellulosic material, into yeast metabolic pathways. It does this by reducing open chain xylose to xylitol, which is reoxidized to xylulose by xylitol dehydrogenase and metabolically integrated via the pentose phosphate pathway. No structure has yet been determined for a xylose reductase, a dimeric aldo-keto reductase or a family 2 aldo-keto reductase. The structures of the Candida tenuis xylose reductase apo- and holoenzyme, which crystallize in spacegroup C2 with different unit cells, have been determined to 2.2 A resolution and an R-factor of 17.9 and 20.8%, respectively. Residues responsible for mediating the novel dimeric interface include Asp-178, Arg-181, Lys-202, Phe-206, Trp-313, and Pro-319. Alignments with other superfamily members indicate that these interactions are conserved in other dimeric xylose reductases but not throughout the remainder of the oligomeric aldo-keto reductases, predicting alternate modes of oligomerization for other families. An arrangement of side chains in a catalytic triad shows that Tyr-52 has a conserved function as a general acid. The loop that folds over the NAD(P)H cosubstrate is disordered in the apo form but becomes ordered upon cosubstrate binding. A slow conformational isomerization of this loop probably accounts for the observed rate-limiting step involving release of cosubstrate. Xylose binding (K(m) = 87 mM) is mediated by interactions with a binding pocket that is more polar than a typical aldo-keto reductase. Modeling of xylose into the active site of the holoenzyme using ordered waters as a guide for sugar hydroxyls suggests a convincing mode of substrate binding.

  19. New antibacterials for the treatment of toxoplasmosis; a patent review.

    Science.gov (United States)

    Rodriguez, Juan Bautista; Szajnman, Sergio Hernán

    2012-03-01

    Toxoplasma gondii is an opportunistic protozoan parasite responsible for toxoplasmosis. T. gondii is able to infect a wide range of hosts, particularly humans and warm-blooded animals. Toxoplasmosis can be considered as one of the most prevalent parasitic diseases affecting close to one billion people worldwide, but its current chemotherapy is still deficient and is only effective in the acute phase of the disease. This review covers different approaches to toxoplasmosis chemotherapy focused on the metabolic differences between the host and the parasite. Selective action on different targets such as the isoprenoid pathway, dihydrofolate reductase, T. gondii adenosine kinase, different antibacterials, T. gondii histone deacetylase and calcium-dependent protein kinases is discussed. A new and safe chemotherapy is needed, as T. gondii causes serious morbidity and mortality in pregnant women and immunodeficient patients undergoing chemotherapy. A particular drawback of the available treatments is the lack of efficacy against the tissue cyst of the parasite. During this review a broad scope of several attractive targets for drug design have been presented. In this context, the isoprenoid pathway, dihydrofolate reductase, T. gondii histone deacetylase are promising molecular targets.

  20. The aldo-keto reductase superfamily homepage.

    Science.gov (United States)

    Hyndman, David; Bauman, David R; Heredia, Vladi V; Penning, Trevor M

    2003-02-01

    The aldo-keto reductases (AKRs) are one of the three enzyme superfamilies that perform oxidoreduction on a wide variety of natural and foreign substrates. A systematic nomenclature for the AKR superfamily was adopted in 1996 and was updated in September 2000 (visit www.med.upenn.edu/akr). Investigators have been diligent in submitting sequences of functional proteins to the Web site. With the new additions, the superfamily contains 114 proteins expressed in prokaryotes and eukaryotes that are distributed over 14 families (AKR1-AKR14). The AKR1 family contains the aldose reductases, the aldehyde reductases, the hydroxysteroid dehydrogenases and steroid 5beta-reductases, and is the largest. Other families of interest include AKR6, which includes potassium channel beta-subunits, and AKR7 the aflatoxin aldehyde reductases. Two new families include AKR13 (yeast aldose reductase) and AKR14 (Escherichia coli aldehyde reductase). Crystal structures of many AKRs and their complexes with ligands are available in the PDB and accessible through the Web site. Each structure has the characteristic (alpha/beta)(8)-barrel motif of the superfamily, a conserved cofactor binding site and a catalytic tetrad, and variable loop structures that define substrate specificity. Although the majority of AKRs are monomeric proteins of about 320 amino acids in length, the AKR2, AKR6 and AKR7 family may form multimers. To expand the nomenclature to accommodate multimers, we recommend that the composition and stoichiometry be listed. For example, AKR7A1:AKR7A4 (1:3) would designate a tetramer of the composition indicated. The current nomenclature is recognized by the Human Genome Project (HUGO) and the Web site provides a link to genomic information including chromosomal localization, gene boundaries, human ESTs and SNPs and much more.

  1. Tetrathionate reductase of Salmonella thyphimurium: a molybdenum containing enzyme

    International Nuclear Information System (INIS)

    Hinojosa-Leon, M.; Dubourdieu, M.; Sanchez-Crispin, J.A.; Chippaux, M.

    1986-01-01

    Use of radioactive molybdenum demonstrates that the tetrathionate reductase of Salmonella typhimurium is a molydenum containing enzyme. It is proposed that this enzyme shares with other molybdo-proteins, such as nitrate reductase, a common molybdenum containing cofactor the defect of which leads to the loss of the tetrathionate reductase and nitrate reductase activities

  2. Oxygen and xenobiotic reductase activities of cytochrome P450.

    NARCIS (Netherlands)

    Goeptar, A.R.; Scheerens, H.; Vermeulen, N.P.E.

    1995-01-01

    The oxygen reductase and xenobiotic reductase activities of cytochrome P450 (P450) are reviewed. During the oxygen reductase activity of P450, molecular oxygen is reduced to superoxide anion radicals (O

  3. Structure and mechanism of dimethylsulfoxide reductase, a molybdopterin-containing enzyme of DMSO reductase family

    International Nuclear Information System (INIS)

    McEwan, A.G.; Ridge, J.P.; McDevitt, C.A.; Hanson, G.R.

    2001-01-01

    Full text: Apart from nitrogenase, enzymes containing molybdenum are members of a superfamily, the molybdopterin-containing enzymes. Most of these enzymes catalyse an oxygen atom transfer and two electron transfer reaction. During catalysis the Mo at the active site cycles between the Mo(VI) and Mo(IV) states. The DMSO reductase family of molybdopterin-containing enzymes all contain a bis(molybdopterin guanine dinucleotide)Mo cofactor and over thirty examples have now been described. Over the last five years crystal structures of dimethylsulfoxide (DMSO) reductase and four other enzymes of the DMSO reductase family have revealed that enzymes of this family have a similar tertiary structure. The Mo atom at the active site is coordinated by four thiolate ligands provided by the dithiolene side chains of the two MGD molecules of the bis(MGD)Mo cofactor as well as a ligand provided by an amino acid side chain. In addition, an oxygen atom in the form of an oxo, hydroxo or aqua group is also coordinated to the Mo atom. In the case of dimethylsulfoxide reductase X-ray crystallography of the product-reduced species and Raman spectroscopy has demonstrated that the enzyme contains a single exchangeable oxo group that is H-bonded to W116

  4. Thioredoxin reductase 1 upregulates MCP-1 release in human endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Zhen-Bo [Institute of Biophysics, Chinese Academy of Sciences, and Graduate School of the Chinese Academy of Sciences, Beijing (China); Shen, Xun, E-mail: shenxun@sun5.ibp.ac.cn [Institute of Biophysics, Chinese Academy of Sciences, and Graduate School of the Chinese Academy of Sciences, Beijing (China)

    2009-09-04

    To know if thioredoxin reductase 1 (TrxR1) plays a role in antioxidant defense mechanisms against atherosclerosis, effect of TrxR1 on expression/release of monocyte chemoattractant protein (MCP-1) was investigated in activated human endothelial-like EAhy926 cells. The MCP-1 release and expression, cellular generation of reactive oxygen species (ROS), nuclear translocation and DNA-binding activity of NF-{kappa}B subunit p65 were assayed in cells either overexpressing recombinant TrxR1 or having their endogenous TrxR1 knocked down. It was found that overexpression of TrxR1 enhanced, while knockdown of TrxR1 reduced MCP-1 release and expression. Upregulation of MCP-1 by TrxR1 was associated with increasing generation of intracellular ROS generation, enhanced nuclear translocation and DNA-binding activity of NF-{kappa}B. Assay using NF-{kappa}B reporter revealed that TrxR1 upregulated transcriptional activity of NF-{kappa}B. This study suggests that TrxR1 enhances ROS generation, NF-{kappa}B activity and subsequent MCP-1 expression in endothelial cells, and may promote rather than prevent vascular endothelium from forming atherosclerotic plaque.

  5. Recombinant pinoresinol/lariciresinol reductase, recombinant dirigent protein, and methods of use

    Science.gov (United States)

    Lewis, Norman G.; Davin, Laurence B.; Dinkova-Kostova, Albena T.; Fujita, Masayuki; Gang, David R.; Sarkanen, Simo; Ford, Joshua D.

    2001-04-03

    Dirigent proteins and pinoresinol/lariciresinol reductases have been isolated, together with cDNAs encoding dirigent proteins and pinoresinol/lariciresinol reductases. Accordingly, isolated DNA sequences are provided which code for the expression of dirigent proteins and pinoresinol/lariciresinol reductases. In other aspects, replicable recombinant cloning vehicles are provided which code for dirigent proteins or pinoresinol/lariciresinol reductases or for a base sequence sufficiently complementary to at least a portion of dirigent protein or pinoresinol/lariciresinol reductase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding dirigent protein or pinoresinol/lariciresinol reductase. Thus, systems and methods are provided for the recombinant expression of dirigent proteins and/or pinoresinol/lariciresinol reductases.

  6. Inhibition of ligand exchange kinetics via active-site trapping with an antibody fragment.

    Science.gov (United States)

    Oyen, David; Steyaert, Jan; Barlow, John N

    2014-04-01

    We describe the first example of an inhibitory antibody fragment (nanobody ca1697) that binds simultaneously to an enzyme (the enzyme dihydrofolate reductase from Escherichia coli) and its bound substrate (folate). Binding of the antibody to the substrate causes a 20-fold reduction in the rate of folate exchange kinetics. This work opens up the prospect of designing new types of antibody-based inhibitors of enzymes and receptors through suitable design of immunogens.

  7. An Enterobacter Plasmid as a New Genetic Background for the Transposon Tn1331

    Science.gov (United States)

    2011-11-25

    determined to be 99% similar to E. cloacae by both 16S rDNA and Phoenix analysis and was designated Enterobacter sp W001. Enterobacter sp W001 was...adolescents. JAMA. 2002;287(23):3096–3102. 9. Foster TJ. Plasmid- determined resistance to antimicrobial drugs and toxic metal ions in bacteria. Microbiol...mediated type II dihydrofolate reductase gene among trimethoprim -resistant urinary pathogens in Greek hospitals. J Antimicrob Chemother. 1992;29

  8. Bioinformatics approach of three partial polyprenol reductase genes in Kandelia obovata

    Science.gov (United States)

    Basyuni, M.; Wati, R.; Sagami, H.; Oku, H.; Baba, S.

    2018-03-01

    This present study describesthe bioinformatics approach to analyze three partial polyprenol reductase genes from mangrove plant, Kandeliaobovataas well aspredictedphysical and chemical properties, potential peptide, subcellular localization, and phylogenetic. The diversity was noted in the physical and chemical properties of three partial polyprenol reductase genes. The values of chloroplast were relatively high, showed that chloroplast transit peptide occurred in mangrove polyprenol reductase. The target peptide value of mitochondria varied from 0.088 to 0.198 indicated it was possible to be present. These results suggested the importance of understanding the diversity of physicochemical properties of the different amino acids in polyprenol reductase. The subcellular localization of two partial genes located in the plasma membrane. To confirm the homology among the polyprenol reductase in the database, a dendrogram was drawn. The phylogenetic tree depicts that there are three clusters, the partial genes of K. obovata joined the largest one: C23157 was close to Ricinus communis polyprenol reductase. Whereas, C23901 and C24171 were grouped with Ipomoea nil polyprenol reductase, suggested that these polyprenol reductase genes form distinct separation into tropical habitat plants.

  9. Recominant Pinoresino-Lariciresinol Reductase, Recombinant Dirigent Protein And Methods Of Use

    Science.gov (United States)

    Lewis, Norman G.; Davin, Laurence B.; Dinkova-Kostova, Albena T.; Fujita, Masayuki , Gang; David R. , Sarkanen; Simo , Ford; Joshua D.

    2003-10-21

    Dirigent proteins and pinoresinol/lariciresinol reductases have been isolated, together with cDNAs encoding dirigent proteins and pinoresinol/lariciresinol reductases. Accordingly, isolated DNA sequences are provided from source species Forsythia intermedia, Thuja plicata, Tsuga heterophylla, Eucommia ulmoides, Linum usitatissimum, and Schisandra chinensis, which code for the expression of dirigent proteins and pinoresinol/lariciresinol reductases. In other aspects, replicable recombinant cloning vehicles are provided which code for dirigent proteins or pinoresinol/lariciresinol reductases or for a base sequence sufficiently complementary to at least a portion of dirigent protein or pinoresinol/lariciresinol reductase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding dirigent protein or pinoresinol/lariciresinol reductase. Thus, systems and methods are provided for the recombinant expression of dirigent proteins and/or pinoresinol/lariciresinol reductases.

  10. E2F1 promote the aggressiveness of human colorectal cancer by activating the ribonucleotide reductase small subunit M2

    Energy Technology Data Exchange (ETDEWEB)

    Fang, Zejun [Sanmen People' s Hospital of Zhejiang, Sanmen, Zhejiang, 317100 (China); Gong, Chaoju [Department of Pathology and Pathophysiology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310058 (China); Liu, Hong [Zhejiang Normal University – Jinhua People' s Hospital Joint Center for Biomedical Research, Jinhua, Zhejiang, 321004 (China); Zhang, Xiaomin; Mei, Lingming [Sanmen People' s Hospital of Zhejiang, Sanmen, Zhejiang, 317100 (China); Song, Mintao [Department of Pathophysiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (CAMS), School of Basic Medicine, Peking Union Medical College (PUMC), Beijing, 100005 (China); Qiu, Lanlan; Luo, Shuchai; Zhu, Zhihua; Zhang, Ronghui; Gu, Hongqian [Sanmen People' s Hospital of Zhejiang, Sanmen, Zhejiang, 317100 (China); Chen, Xiang, E-mail: sychenxiang@126.com [Sanmen People' s Hospital of Zhejiang, Sanmen, Zhejiang, 317100 (China)

    2015-08-21

    As the ribonucleotide reductase small subunit, the high expression of ribonucleotide reductase small subunit M2 (RRM2) induces cancer and contributes to tumor growth and invasion. In several colorectal cancer (CRC) cell lines, we found that the expression levels of RRM2 were closely related to the transcription factor E2F1. Mechanistic studies were conducted to determine the molecular basis. Ectopic overexpression of E2F1 promoted RRM2 transactivation while knockdown of E2F1 reduced the levels of RRM2 mRNA and protein. To further investigate the roles of RRM2 which was activated by E2F1 in CRC, CCK-8 assay and EdU incorporation assay were performed. Overexpression of E2F1 promoted cell proliferation in CRC cells, which was blocked by RRM2 knockdown attenuation. In the migration and invasion tests, overexpression of E2F1 enhanced the migration and invasion of CRC cells which was abrogated by silencing RRM2. Besides, overexpression of RRM2 reversed the effects of E2F1 knockdown partially in CRC cells. Examination of clinical CRC specimens demonstrated that both RRM2 and E2F1 were elevated in most cancer tissues compared to the paired normal tissues. Further analysis showed that the protein expression levels of E2F1 and RRM2 were parallel with each other and positively correlated with lymph node metastasis (LNM), TNM stage and distant metastasis. Consistently, the patients with low E2F1 and RRM2 levels have a better prognosis than those with high levels. Therefore, we suggest that E2F1 can promote CRC proliferation, migration, invasion and metastasis by regulating RRM2 transactivation. Understanding the role of E2F1 in activating RRM2 transcription will help to explain the relationship between E2F1 and RRM2 in CRC and provide a novel predictive marker for diagnosis and prognosis of the disease. - Highlights: • E2F1 promotes RRM2 transactivation in CRC cells. • E2F1 promotes the proliferation of CRC cells by activating RRM2. • E2F1 promotes the migration and

  11. Ionic liquid mediated stereoselective synthesis of alanine linked hybrid quinazoline-4(3H)-one derivatives perturbing the malarial reductase activity in folate pathway.

    Science.gov (United States)

    Patel, Tarosh S; Bhatt, Jaimin D; Vanparia, Satish F; Patel, Urmila H; Dixit, Ritu B; Chudasama, Chaitanya J; Patel, Bhavesh D; Dixit, Bharat C

    2017-12-15

    Grimmel's method was optimized as well as modified leading to the cyclization and incorporation of alanine linked sulphonamide in 4-quinazolin-(3H)-ones. Further, the generation of heterocyclic motif at position-3 of 4-quinazolinones was explored by synthesis of imines, which unfortunately led to an isomeric mixture of stereoisomers. The hurdle of diastereomers encountered on the path was eminently rectified by development of new rapid and reproducible methodology involving the use of imidazolium based ionic liquid as solvents as well as catalyst for cyclization as well as synthesis of imines in situ at position-3 leading to procurement of single E-isomer as the target hybrid heterocyclic molecules. The purity and presence of single isomer was also confirmed by HPLC and spectroscopic techniques. Further, the synthesized sulphonamide linked 4-quinazolin-(3H)-ones hybrids were screened for their antimalarial potency rendering potent entities (4b, 4c, 4 l, 4 t and 4u). The active hybrids were progressively screened for enzyme inhibitory efficacy against presumed receptor Pf-DHFR and h-DHFR computationally as well as in vitro, proving their potency as dihydrofolate reductase inhibitors. The ADME properties of these active molecules were also predicted to enhance the knowhow of the oral bioavailability, indicating good bioavailability of the active entities. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Sucrose mimics the light induction of Arabidopsis nitrate reductase gene transcription

    DEFF Research Database (Denmark)

    Cheng, Chi-Lien; Acedo, Gregoria N; Kristensen, Michael

    1992-01-01

    can replace light in eliciting an increase of nitrate reductase mRNA accumulation in dark-adapted green Arabidopsis plants. We show further that sucrose alone is sufficient for the full expression of nitrate reductase genes in etiolated Arabidopsis plants. Finally, using a reporter gene, we show......Nitrate reductase, the first enzyme in nitrate assimilation, is located at the crossroad of two energy-consuming pathways: nitrate assimilation and carbon fixation. Light, which regulates the expression of many higher-plant carbon fixation genes, also regulates nitrate reductase gene expression....... Located in the cytosol, nitrate reductase obtains its reductant not from photosynthesis but from carbohydrate catabolism. This relationship prompted us to investigate the indirect role that light might play, via photosynthesis, in the regulation of nitrate reductase gene expression. We show that sucrose...

  13. Damaged DNA binding protein 2 plays a role in breast cancer cell growth.

    Directory of Open Access Journals (Sweden)

    Zilal Kattan

    Full Text Available The Damaged DNA binding protein 2 (DDB2, is involved in nucleotide excision repair as well as in other biological processes in normal cells, including transcription and cell cycle regulation. Loss of DDB2 function may be related to tumor susceptibility. However, hypothesis of this study was that DDB2 could play a role in breast cancer cell growth, resulting in its well known interaction with the proliferative marker E2F1 in breast neoplasia. DDB2 gene was overexpressed in estrogen receptor (ER-positive (MCF-7 and T47D, but not in ER-negative breast cancer (MDA-MB231 and SKBR3 or normal mammary epithelial cell lines. In addition, DDB2 expression was significantly (3.0-fold higher in ER-positive than in ER-negative tumor samples (P = 0.0208 from 16 patients with breast carcinoma. Knockdown of DDB2 by small interfering RNA in MCF-7 cells caused a decrease in cancer cell growth and colony formation. Inversely, introduction of the DDB2 gene into MDA-MB231 cells stimulated growth and colony formation. Cell cycle distribution and 5 Bromodeoxyuridine incorporation by flow cytometry analysis showed that the growth-inhibiting effect of DDB2 knockdown was the consequence of a delayed G1/S transition and a slowed progression through the S phase of MCF-7 cells. These results were supported by a strong decrease in the expression of S phase markers (Proliferating Cell Nuclear Antigen, cyclin E and dihydrofolate reductase. These findings demonstrate for the first time that DDB2 can play a role as oncogene and may become a promising candidate as a predictive marker in breast cancer.

  14. Knockdown of p53 suppresses Nanog expression in embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Abdelalim, Essam Mohamed, E-mail: emohamed@qf.org.qa [Qatar Biomedical Research Institute, Qatar Foundation, Doha 5825 (Qatar); Molecular Neuroscience Research Center, Shiga University of Medical Science, Setatsukinowa-cho, Otsu, Shiga 520-2192 (Japan); Department of Cytology and Histology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia (Egypt); Tooyama, Ikuo [Molecular Neuroscience Research Center, Shiga University of Medical Science, Setatsukinowa-cho, Otsu, Shiga 520-2192 (Japan)

    2014-01-10

    Highlights: •We investigate the role of p53 in ESCs in the absence of DNA damage. •p53 knockdown suppresses ESC proliferation. •p53 knockdown downregulates Nanog expression. •p53 is essential for mouse ESC self-renewal. -- Abstract: Mouse embryonic stem cells (ESCs) express high levels of cytoplasmic p53. Exposure of mouse ESCs to DNA damage leads to activation of p53, inducing Nanog suppression. In contrast to earlier studies, we recently reported that chemical inhibition of p53 suppresses ESC proliferation. Here, we confirm that p53 signaling is involved in the maintenance of mouse ESC self-renewal. RNA interference-mediated knockdown of p53 induced downregulation of p21 and defects in ESC proliferation. Furthermore, p53 knockdown resulted in a significant downregulation in Nanog expression at 24 and 48 h post-transfection. p53 knockdown also caused a reduction in Oct4 expression at 48 h post-transfection. Conversely, exposure of ESCs to DNA damage caused a higher reduction of Nanog expression in control siRNA-treated cells than in p53 siRNA-treated cells. These data show that in the absence of DNA damage, p53 is required for the maintenance of mouse ESC self-renewal by regulating Nanog expression.

  15. Chemotherapy of Leishmaniasis.

    Science.gov (United States)

    1979-09-01

    active at a high dose in vivo against the two parasites against which it has been tested, i.e. L. donovani s.1. and L. major. i(i) Nystatin is highly...possess trypanocidal action (nifurti-ox, benznidazole), two metronidazole analogues (LIV/1319 and 1320), and two compounds with activity against...sion1ficantly blocked by certain dihydrofolate reductase inhibitors. A-3 S. *-h.." . "’" V" W2. Nitroreductase-linked pathways. Metronidazole has been shown

  16. Salvage trial of trimetrexate-leucovorin for the treatment of cerebral toxoplasmosis in patients with AIDS

    DEFF Research Database (Denmark)

    Masur, H; Polis, M A; Tuazon, C U

    1993-01-01

    The clinical efficacy of trimetrexate, a dihydrofolate reductase inhibitor with potent in vitro antitoxoplasma activity, was assessed in 9 sulfonamide-intolerant patients with AIDS and biopsy-proven cerebral toxoplasmosis. The 9 patients were treated for 28-149 days with trimetrexate (30-280 mg/m2...... doses for extended periods was not associated with serious toxicity. Trimetrexate alone had dramatic but transient activity in sulfonamide-intolerant patients and thus is not adequate as single-agent therapy for AIDS-associated toxoplasmosis....

  17. Cloning and nitrate induction of nitrate reductase mRNA

    OpenAIRE

    Cheng, Chi-Lien; Dewdney, Julia; Kleinhofs, Andris; Goodman, Howard M.

    1986-01-01

    Nitrate is the major source of nitrogen taken from the soil by higher plants but requires reduction to ammonia prior to incorporation into amino acids. The first enzyme in the reducing pathway is a nitrate-inducible enzyme, nitrate reductase (EC 1.6.6.1). A specific polyclonal antiserum raised against purified barley nitrate reductase has been used to immunoprecipitate in vivo labeled protein and in vitro translation products, demonstrating that nitrate induction increases nitrate reductase p...

  18. 5α-reductase activity in rat adipose tissue

    International Nuclear Information System (INIS)

    Zyirek, M.; Flood, C.; Longcope, C.

    1987-01-01

    We measured the 5 α-reductase activity in isolated cell preparations of rat adipose tissue using the formation of [ 3 H] dihydrotestosterone from [ 3 H] testosterone as an endpoint. Stromal cells were prepared from the epididymal fat pad, perinephric fat, and subcutaneous fat of male rats and from perinephric fat of female rats. Adipocytes were prepared from the epididymal fat pad and perinephric fat of male rats. Stromal cells from the epididymal fat pad and perinephric fat contained greater 5α-reductase activity than did the adipocytes from these depots. Stromal cells from the epididymal fat pad contained greater activity than those from perinephric and subcutaneous depots. Perinephric stromal cells from female rats were slightly more active than those from male rats. Estradiol (10 -8 M), when added to the medium, caused a 90% decrease in 5α-reductase activity. Aromatase activity was minimal, several orders of magnitude less than 5α-reductase activity in each tissue studied

  19. Characterization of mitochondrial thioredoxin reductase from C. elegans

    International Nuclear Information System (INIS)

    Lacey, Brian M.; Hondal, Robert J.

    2006-01-01

    Thioredoxin reductase catalyzes the NADPH-dependent reduction of the catalytic disulfide bond of thioredoxin. In mammals and other higher eukaryotes, thioredoxin reductases contain the rare amino acid selenocysteine at the active site. The mitochondrial enzyme from Caenorhabditis elegans, however, contains a cysteine residue in place of selenocysteine. The mitochondrial C. elegans thioredoxin reductase was cloned from an expressed sequence tag and then produced in Escherichia coli as an intein-fusion protein. The purified recombinant enzyme has a k cat of 610 min -1 and a K m of 610 μM using E. coli thioredoxin as substrate. The reported k cat is 25% of the k cat of the mammalian enzyme and is 43-fold higher than a cysteine mutant of mammalian thioredoxin reductase. The enzyme would reduce selenocysteine, but not hydrogen peroxide or insulin. The flanking glycine residues of the GCCG motif were mutated to serine. The mutants improved substrate binding, but decreased the catalytic rate

  20. Role of Mutations in Dihydrofolate Reductase DfrA (Rv2763c) and Thymidylate Synthase ThyA (Rv2764c) in Mycobacterium tuberculosis Drug Resistance

    KAUST Repository

    Koser, C. U.

    2010-09-17

    We would like to comment on a number of recent reports in this journal (6, 8, 12, 18) concerning Mycobacterium tuberculosis dihydrofolate reductase (DHFR), encoded by dfrA (Rv2763c). Around 36% of phenotypically para-aminosalicylic acid (PAS)-resistant M. tuberculosis strains harbor mutations in thyA (Rv2764c), which encodes a thymidylate synthase (20). In their effort to elucidate the remaining unknown resistance mechanism(s), Mathys et al. extended their sequence analysis to a number of additional genes, including dfrA (12). It was unclear whether the three dfrA mutations they identified in the PAS-resistant strains P-693 and P-3158 could contribute to PAS resistance on their own. Nonetheless, these findings are notable for two reasons. First, isoniazid (INH) has been shown to inhibit M. tuberculosis DHFR in vitro (1). Whether the same holds true for ethionamide, which shares a number of common resistance mechanisms with INH, was not tested (J. Blanchard, personal communication). In any case, the clinical relevance of DHFR-mediated INH resistance remains enigmatic. To date, only Ho et al. have addressed this question, but they did not identify any dfrA mutations in a screen of 127 INH-resistant clinical isolates (8). Consequently, Mathys et al. remain the first to describe mutations in this target (12). However, given that isolates with mutated DHFR are members of a cluster with baseline INH resistance, the importance of these mutations with respect to INH resistance remains unclear. Irrespective of their relevance in INH resistance, these dfrA mutations are noteworthy for a second reason. Contrary to previous wisdom, Forgacs et al. recently showed that M. tuberculosis is sensitive to the drug combination trimethoprim-sulfamethoxazole (TMP-SMX) (6, 18). DHFR is competitively inhibited by TMP, and consequently, mutations therein lead to resistance in a variety of organisms (9, 16, 19). The crystal structures of the wild-type M. tuberculosis DHFR in complex with

  1. Dietary sources of aldose reductase inhibitors: prospects for alleviating diabetic complications.

    Science.gov (United States)

    Saraswat, Megha; Muthenna, P; Suryanarayana, P; Petrash, J Mark; Reddy, G Bhanuprakash

    2008-01-01

    Activation of polyol pathway due to increased aldose reductase activity is one of the several mechanisms that have been implicated in the development of various secondary complications of diabetes. Though numerous synthetic aldose reductase inhibitors have been tested, these have not been very successful clinically. Therefore, a number of common plant/ natural products used in Indian culinary have been evaluated for their aldose reductase inhibitory potential in the present study. The aqueous extracts of 22 plant-derived materials were prepared and evaluated for the inhibitory property against rat lens and human recombinant aldose reductase. Specificity of these extracts towards aldose reductase was established by testing their ability to inhibit a closely related enzyme viz, aldehyde reductase. The ex vivo incubation of erythrocytes in high glucose containing medium was used to underscore the significance in terms of prevention of intracellular sorbitol accumulation. Among the 22 dietary sources tested, 10 showed considerable inhibitory potential against both rat lens and human recombinant aldose reductase. Prominent inhibitory property was found in spinach, cumin, fennel, lemon, basil and black pepper with an approximate IC50 of 0.2 mg/mL with an excellent selectivity towards aldose reductase. As against this, 10 to 20 times higher concentrations were required for 50% inhibition of aldehyde reductase. Reduction in the accumulation of intracellular sorbitol by the dietary extracts further substantiated their in vivo efficacy. The findings reported here indicate the scope of adapting life-style modifications in the form of inclusion of certain common sources in the diet for the management of diabetic complications.

  2. 5α-reductases in human physiology: an unfolding story.

    Science.gov (United States)

    Traish, Abdulmaged M

    2012-01-01

    5α-reductases are a family of isozymes expressed in a wide host of tissues including the central nervous system (CNS) and play a pivotal role in male sexual differentiation, development and physiology. A comprehensive literature search from 1970 to 2011 was made through PubMed and the relevant information was summarized. 5α reductases convert testosterone, progesterone, deoxycorticosterone, aldosterone and corticosterone into their respective 5α-dihydro-derivatives, which serve as substrates for 3α-hydroxysteroid dehydrogenase enzymes. The latter transforms these 5α-reduced metabolites into a subclass of neuroactive steroid hormones with distinct physiological functions. The neuroactive steroid hormones modulate a multitude of functions in human physiology encompassing regulation of sexual differentiation, neuroprotection, memory enhancement, anxiety, sleep and stress, among others. In addition, 5α -reductase type 3 is also implicated in the N-glycosylation of proteins via formation of dolichol phosphate. The family of 5α-reductases was targeted for drug development to treat pathophysiological conditions, such as benign prostatic hyperplasia and androgenetic alopecia. While the clinical use of 5α-reductase inhibitors was well established, the scope and the magnitude of the adverse side effects of such drugs, especially on the CNS, is still unrecognized due to lack of knowledge of the various physiological functions of this family of enzymes, especially in the CNS. There is an urgent need to better understand the function of 5α-reductases and the role of neuroactive steroids in human physiology in order to minimize the potential adverse side effects of inhibitors targeting 5α-reductases to treat benign prostatic hyperplasia and androgenic alopecia.

  3. An in-silico investigation of anti-Chagas phytochemicals.

    Science.gov (United States)

    McCulley, Stephanie F; Setzer, William N

    2014-01-01

    Over 18 million people in tropical and subtropical America are afflicted by American trypanosomiasis or Chagas disease. In humans, symptoms of the disease include fever, swelling, and heart and brain damage, usually leading to death. There is currently no effective treatment for this disease. Plant products continue to be rich sources of clinically useful drugs, and the biodiversity of the Neotropics suggests great phytomedicinal potential. Screening programs have revealed numerous plant species and phytochemical agents that have shown in-vitro or in-vivo antitrypanosomal activity, but the biochemical targets of these phytochemicals are not known. In this work, we present a molecular docking analysis of Neotropical phytochemicals, which have already demonstrated antiparasitic activity against Trypanosoma cruzi, with potential druggable protein targets of the parasite. Several protein targets showed in-silico selectivity for trypanocidal phytochemicals, including trypanothione reductase, pteridine reductase 2, lipoamide dehydrogenase, glucokinase, dihydroorotate dehydrogenase, cruzain, dihydrofolate-reductase/thymidylate-synthase, and farnesyl diphosphate synthase. Some of the phytochemical ligands showed notable docking preference for trypanothione reductase, including flavonoids, fatty-acid-derived oxygenated hydrocarbons, geranylgeraniol and the lignans ganschisandrine and eupomatenoid-6.

  4. Histochemical Localization of Glutathione Dependent NBT-Reductase in Mouse Skin

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective Localization of the glutathione dependent Nitroblue tetrazolium (NBT) reductase in fresh frozen sections of mouse skin and possible dependence of NBT reductase on tissue thiol levels has been investigated. Methods The fresh frozen tissue sections (8m thickness) were prepared and incubated in medium containing NBT, reduced glutathione (GSH) and phosphate buffer. The staining for GSH was performed with mercury orange. Results  The activity of the NBT-reductase in mouse skin has been found to be localized in the areas rich in glutathione and actively proliferating area of the skin. Conclusion The activity of the NBT-reductase seems to be dependent on the glutathione contents.

  5. DJ-1 KNOCK-DOWN IMPAIRS ASTROCYTE MITOCHONDRIAL FUNCTION

    Science.gov (United States)

    LARSEN, N. J.; AMBROSI, G.; MULLETT, S. J.; BERMAN, S. B.; HINKLE, D. A.

    2012-01-01

    Mitochondrial dysfunction has long been implicated in the pathogenesis of Parkinson’s disease (PD). PD brain tissues show evidence for mitochondrial respiratory chain Complex I deficiency. Pharmacological inhibitors of Complex I, such as rotenone, cause experimental parkinsonism. The cytoprotective protein DJ-1, whose deletion is sufficient to cause genetic PD, is also known to have mitochondria-stabilizing properties. We have previously shown that DJ-1 is over-expressed in PD astrocytes, and that DJ-1 deficiency impairs the capacity of astrocytes to protect co-cultured neurons against rotenone. Since DJ-1 modulated, astrocyte-mediated neuroprotection against rotenone may depend upon proper astrocytic mitochondrial functioning, we hypothesized that DJ-1 deficiency would impair astrocyte mitochondrial motility, fission/fusion dynamics, membrane potential maintenance, and respiration, both at baseline and as an enhancement of rotenone-induced mitochondrial dysfunction. In astrocyte-enriched cultures, we observed that DJ-1 knock-down reduced mitochondrial motility primarily in the cellular processes of both untreated and rotenone treated cells. In these same cultures, DJ-1 knock-down did not appreciably affect mitochondrial fission, fusion, or respiration, but did enhance rotenone-induced reductions in the mitochondrial membrane potential. In neuron–astrocyte co-cultures, astrocytic DJ-1 knock-down reduced astrocyte process mitochondrial motility in untreated cells, but this effect was not maintained in the presence of rotenone. In the same co-cultures, astrocytic DJ-1 knock-down significantly reduced mitochondrial fusion in the astrocyte cell bodies, but not the processes, under the same conditions of rotenone treatment in which DJ-1 deficiency is known to impair astrocyte-mediated neuroprotection. Our studies therefore demonstrated the following new findings: (i) DJ-1 deficiency can impair astrocyte mitochondrial physiology at multiple levels, (ii) astrocyte

  6. Crystallization and diffraction analysis of thioredoxin reductase from Streptomyces coelicolor

    International Nuclear Information System (INIS)

    Koháryová, Michaela; Brynda, Jiří; Řezáčová, Pavlína; Kollárová, Marta

    2011-01-01

    Thioredoxin reductase from S. coelicolor was crystallized and diffraction data were collected to 2.4 Å resolution. Thioredoxin reductases are homodimeric flavoenzymes that catalyze the transfer of electrons from NADPH to oxidized thioredoxin substrate. Bacterial thioredoxin reductases represent a promising target for the development of new antibiotics. Recombinant thioredoxin reductase TrxB from Streptomyces coelicolor was crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from cryocooled crystals to 2.4 Å resolution using a synchrotron-radiation source. The crystals belonged to the primitive monoclinic space group P2 1 , with unit-cell parameters a = 82.9, b = 60.6, c = 135.4 Å, α = γ = 90.0, β = 96.5°

  7. Light Sensitivity of Lactococcus lactis Thioredoxin Reductase

    DEFF Research Database (Denmark)

    Skjoldager, Nicklas

    The thioredoxin system has evolved in all kingdoms of life acting as a key antioxidant system in the defense against oxidative stress. The thioredoxin system utilizes reducing equivalents from NADPH to reduce protein disulfide targets. The reducing equivalents are shuttled via a flavin and redox...... active dithiol motif in thioredoxin reductase (TrxR) to reduce the small ubiquitous thioredoxin (Trx). Trx in turn regulates the protein dithiol/disulfide balance by reduction of protein disulfide targets in e.g. ribonucleotide reductase, peroxiredoxins and methionine sulfoxide reductase. The glutathione......, thus expected to rely mainly on the Trx system for thiol-disulfide control. L. lactis is an important industrial microorganism used as starter culture in the dairy production of cheese, buttermilk etc. and known to be sensitive to oxidative stress. The L. lactis TrxR (LlTrxR) is a homodimeric...

  8. Synthetic Folic Acid Supplementation during Pregnancy Increases the Risk of Neonatal Jaundice in Newborns

    OpenAIRE

    Somayeh Zaminpira; Sorush Niknamian

    2017-01-01

    Folate is easily and naturally absorbed and utilized by the body when it is metabolized in the small intestines. On the other hand, folic acid, which was first introduced around the 1940s and added to the prenatal supplements of pregnant women, requires the presence of a specific enzyme named dihydrofolate reductase, which is relatively rare in the body especially during pregnancy. The reason why synthetic form of folic acid supplementation by pregnant women should be replaced by natural fola...

  9. Stable SET knockdown in breast cell carcinoma inhibits cell migration and invasion

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jie [Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou (China); Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, Shenzhen (China); Yang, Xi-fei [Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, Shenzhen (China); Ren, Xiao-hu [Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou (China); Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, Shenzhen (China); Meng, Xiao-jing [Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou (China); Huang, Hai-yan [Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, Shenzhen (China); Zhao, Qiong-hui [Shenzhen Entry-Exit Inspection and Quarantine Bureau, Shenzhen (China); Yuan, Jian-hui; Hong, Wen-xu; Xia, Bo; Huang, Xin-feng; Zhou, Li [Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, Shenzhen (China); Liu, Jian-jun, E-mail: bio-research@hotmail.com [Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, Shenzhen (China); Zou, Fei, E-mail: zoufei616@163.com [Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou (China)

    2014-10-10

    Highlights: • We employed RNA interference to knockdown SET expression in breast cancer cells. • Knockdown of SET expression inhibits cell proliferation, migration and invasion. • Knockdown of SET expression increases the activity and expression of PP2A. • Knockdown of SET expression decreases the expression of MMP-9. - Abstract: Breast cancer is the most malignant tumor for women, however, the mechanisms underlying this devastating disease remain unclear. SET is an endogenous inhibitor of protein phosphatase 2A (PP2A) and involved in many physiological and pathological processes. SET could promote the occurrence of tumor through inhibiting PP2A. In this study, we explore the role of SET in the migration and invasion of breast cancer cells MDA-MB-231 and ZR-75-30. The stable suppression of SET expression through lentivirus-mediated RNA interference (RNAi) was shown to inhibit the growth, migration and invasion of breast cancer cells. Knockdown of SET increases the activity and expression of PP2Ac and decrease the expression of matrix metalloproteinase 9 (MMP-9). These data demonstrate that SET may be involved in the pathogenic processes of breast cancer, indicating that SET can serve as a potential therapeutic target for the treatment of breast cancer.

  10. Stable SET knockdown in breast cell carcinoma inhibits cell migration and invasion

    International Nuclear Information System (INIS)

    Li, Jie; Yang, Xi-fei; Ren, Xiao-hu; Meng, Xiao-jing; Huang, Hai-yan; Zhao, Qiong-hui; Yuan, Jian-hui; Hong, Wen-xu; Xia, Bo; Huang, Xin-feng; Zhou, Li; Liu, Jian-jun; Zou, Fei

    2014-01-01

    Highlights: • We employed RNA interference to knockdown SET expression in breast cancer cells. • Knockdown of SET expression inhibits cell proliferation, migration and invasion. • Knockdown of SET expression increases the activity and expression of PP2A. • Knockdown of SET expression decreases the expression of MMP-9. - Abstract: Breast cancer is the most malignant tumor for women, however, the mechanisms underlying this devastating disease remain unclear. SET is an endogenous inhibitor of protein phosphatase 2A (PP2A) and involved in many physiological and pathological processes. SET could promote the occurrence of tumor through inhibiting PP2A. In this study, we explore the role of SET in the migration and invasion of breast cancer cells MDA-MB-231 and ZR-75-30. The stable suppression of SET expression through lentivirus-mediated RNA interference (RNAi) was shown to inhibit the growth, migration and invasion of breast cancer cells. Knockdown of SET increases the activity and expression of PP2Ac and decrease the expression of matrix metalloproteinase 9 (MMP-9). These data demonstrate that SET may be involved in the pathogenic processes of breast cancer, indicating that SET can serve as a potential therapeutic target for the treatment of breast cancer

  11. Evidence that steroid 5alpha-reductase isozyme genes are differentially methylated in human lymphocytes.

    Science.gov (United States)

    Rodríguez-Dorantes, M; Lizano-Soberón, M; Camacho-Arroyo, I; Calzada-León, R; Morimoto, S; Téllez-Ascencio, N; Cerbón, M A

    2002-03-01

    The synthesis of dihydrotestosterone (DHT) is catalyzed by steroid 5alpha-reductase isozymes 1 and 2, and this function determines the development of the male phenotype during embriogenesis and the growth of androgen sensitive tissues during puberty. The aim of this study was to determine the cytosine methylation status of 5alpha-reductase isozymes types 1 and 2 genes in normal and in 5alpha-reductase deficient men. Genomic DNA was obtained from lymphocytes of both normal subjects and patients with primary 5alpha-reductase deficiency due to point mutations in 5alpha-reductase 2 gene. Southern blot analysis of 5alpha-reductase types 1 and 2 genes from DNA samples digested with HpaII presented a different cytosine methylation pattern compared to that observed with its isoschizomer MspI, indicating that both genes are methylated in CCGG sequences. The analysis of 5alpha-reductase 1 gene from DNA samples digested with Sau3AI and its isoschizomer MboI which recognize methylation in GATC sequences showed an identical methylation pattern. In contrast, 5alpha-reductase 2 gene digested with Sau3AI presented a different methylation pattern to that of the samples digested with MboI, indicating that steroid 5alpha-reductase 2 gene possess methylated cytosines in GATC sequences. Analysis of exon 4 of 5alpha-reductase 2 gene after metabisulfite PCR showed that normal and deficient subjects present a different methylation pattern, being more methylated in patients with 5alpha-reductase 2 mutated gene. The overall results suggest that 5alpha-reductase genes 1 and 2 are differentially methylated in lymphocytes from normal and 5alpha-reductase deficient patients. Moreover, the extensive cytosine methylation pattern observed in exon 4 of 5alpha-reductase 2 gene in deficient patients, points out to an increased rate of mutations in this gene.

  12. Knockdown of Pnpla6 protein results in motor neuron defects in zebrafish

    Directory of Open Access Journals (Sweden)

    Yang Song

    2013-03-01

    Mutations in patatin-like phospholipase domain containing 6 (PNPLA6, also known as neuropathy target esterase (NTE or SPG39, cause hereditary spastic paraplegia (HSP. Although studies on animal models, including mice and Drosophila, have extended our understanding of PNPLA6, its roles in neural development and in HSP are not clearly understood. Here, we describe the generation of a vertebrate model of PNPLA6 insufficiency using morpholino oligonucleotide knockdown in zebrafish (Danio rerio. Pnpla6 knockdown resulted in developmental abnormalities and motor neuron defects, including axon truncation and branching. The phenotypes in pnpla6 knockdown morphants were rescued by the introduction of wild-type, but not mutant, human PNPLA6 mRNA. Our results also revealed the involvement of BMP signaling in pnpla6 knockdown phenotypes. Taken together, these results demonstrate an important role of PNPLA6 in motor neuron development and implicate overexpression of BMP signaling as a possible mechanism underlying the developmental defects in pnpla6 morphants.

  13. Hepatocyte Hyperproliferation upon Liver-Specific Co-disruption of Thioredoxin-1, Thioredoxin Reductase-1, and Glutathione Reductase

    Directory of Open Access Journals (Sweden)

    Justin R. Prigge

    2017-06-01

    Full Text Available Energetic nutrients are oxidized to sustain high intracellular NADPH/NADP+ ratios. NADPH-dependent reduction of thioredoxin-1 (Trx1 disulfide and glutathione disulfide by thioredoxin reductase-1 (TrxR1 and glutathione reductase (Gsr, respectively, fuels antioxidant systems and deoxyribonucleotide synthesis. Mouse livers lacking both TrxR1 and Gsr sustain these essential activities using an NADPH-independent methionine-consuming pathway; however, it remains unclear how this reducing power is distributed. Here, we show that liver-specific co-disruption of the genes encoding Trx1, TrxR1, and Gsr (triple-null causes dramatic hepatocyte hyperproliferation. Thus, even in the absence of Trx1, methionine-fueled glutathione production supports hepatocyte S phase deoxyribonucleotide production. Also, Trx1 in the absence of TrxR1 provides a survival advantage to cells under hyperglycemic stress, suggesting that glutathione, likely via glutaredoxins, can reduce Trx1 disulfide in vivo. In triple-null livers like in many cancers, deoxyribonucleotide synthesis places a critical yet relatively low-volume demand on these reductase systems, thereby favoring high hepatocyte turnover over sustained hepatocyte integrity.

  14. Kinetics of carbonyl reductase from human brain.

    OpenAIRE

    Bohren, K M; von Wartburg, J P; Wermuth, B

    1987-01-01

    Initial-rate analysis of the carbonyl reductase-catalysed reduction of menadione by NADPH gave families of straight lines in double-reciprocal plots consistent with a sequential mechanism being obeyed. The fluorescence of NADPH was increased up to 7-fold with a concomitant shift of the emission maximum towards lower wavelength in the presence of carbonyl reductase, and both NADPH and NADP+ caused quenching of the enzyme fluorescence, indicating formation of a binary enzyme-coenzyme complex. D...

  15. Respiratory arsenate reductase as a bidirectional enzyme

    Science.gov (United States)

    Richey, C.; Chovanec, P.; Hoeft, S.E.; Oremland, R.S.; Basu, P.; Stolz, J.F.

    2009-01-01

    The haloalkaliphilic bacterium Alkalilimnicola ehrlichii is capable of anaerobic chemolithoautotrophic growth by coupling the oxidation of arsenite (As(III)) to the reduction of nitrate and carbon dioxide. Analysis of its complete genome indicates that it lacks a conventional arsenite oxidase (Aox), but instead possesses two operons that each encode a putative respiratory arsenate reductase (Arr). Here we show that one homolog is expressed under chemolithoautotrophic conditions and exhibits both arsenite oxidase and arsenate reductase activity. We also demonstrate that Arr from two arsenate respiring bacteria, Alkaliphilus oremlandii and Shewanella sp. strain ANA-3, is also biochemically reversible. Thus Arr can function as a reductase or oxidase. Its physiological role in a specific organism, however, may depend on the electron potentials of the molybdenum center and [Fe–S] clusters, additional subunits, or constitution of the electron transfer chain. This versatility further underscores the ubiquity and antiquity of microbial arsenic metabolism.

  16. Nitrate reductase activity and its relationship with applied nitrogen in soybean

    International Nuclear Information System (INIS)

    Ge Wenting; Jin Xijun; Ma Chunmei; Dong Shoukun; Gong Zhenping; Zhang Lei

    2011-01-01

    Field experiments were conducted to study the nitrate reductase activity and its relationship to nitrogen by using frame tests (pot without bottom), sand culture and 15 N-urea at transplanting in soybean variety Suinong 14. Results showed that the activity of nitrate reductase in leaf changed as a signal peak curve with the soybean growth, lower in vegetative growth phase, higher in reproductive growth period and reached the peak in blooming period, then decreased gradually. Nitrogen application showed obvious effect on the nitrate reductase activity. The activities of nitrate reductase in leaves followed the order of N 135 > N 90 > N 45 > N 0 in vegetative growth stage, no clear regularity was found during the whole reproductive growth period. The activities of nitrate reductase in leaves were accorded with the order of upper leaves > mid leaves > lower leaves, and it was very significant differences (P 15 N labeling method during beginning seed stage and full seed stage shown that 15 N abundance in various organs at different node position also followed the same order, suggesting that high level of nitrate reductase activity at upper leaves of soybean promoted the assimilation of NO 3 - . (authors)

  17. Differential expression of disulfide reductase enzymes in a free-living platyhelminth (Dugesia dorotocephala.

    Directory of Open Access Journals (Sweden)

    Alberto Guevara-Flores

    Full Text Available A search of the disulfide reductase activities expressed in the adult stage of the free-living platyhelminth Dugesia dorotocephala was carried out. Using GSSG or DTNB as substrates, it was possible to obtain a purified fraction containing both GSSG and DTNB reductase activities. Through the purification procedure, both disulfide reductase activities were obtained in the same chromatographic peak. By mass spectrometry analysis of peptide fragments obtained after tryptic digestion of the purified fraction, the presence of glutathione reductase (GR, thioredoxin-glutathione reductase (TGR, and a putative thioredoxin reductase (TrxR was detected. Using the gold compound auranofin to selectively inhibit the GSSG reductase activity of TGR, it was found that barely 5% of the total GR activity in the D. dorotocephala extract can be assigned to GR. Such strategy did allow us to determine the kinetic parameters for both GR and TGR. Although It was not possible to discriminate DTNB reductase activity due to TrxR from that of TGR, a chromatofocusing experiment with a D. dorotocephala extract resulted in the obtention of a minor protein fraction enriched in TrxR, strongly suggesting its presence as a functional protein. Thus, unlike its parasitic counterparts, in the free-living platyhelminth lineage the three disulfide reductases are present as functional proteins, albeit TGR is still the major disulfide reductase involved in the reduction of both Trx and GSSG. This fact suggests the development of TGR in parasitic flatworms was not linked to a parasitic mode of life.

  18. Knockdown of ZFR suppresses cell proliferation and invasion of human pancreatic cancer

    Directory of Open Access Journals (Sweden)

    Xiaolan Zhao

    Full Text Available BACKGROUND: Zinc finger RNA binding protein (ZFR is involved in the regulation of growth and cancer development. However, little is known about ZFR function in pancreatic cancer. METHODS: Herein, to investigate whether ZFR is involved in tumor growth, Oncomine microarray data was firstly used to evaluate ZFR gene expression in human pancreatic tumors. Then short hairpin RNA (shRNA targeting ZFR was designed and delivered into PANC-1 pancreatic cancer cells to knock down ZFR expression. Cell viability, cell proliferation and cell cycle analysis after ZFR knockdown were determined by MTT, colony forming and FACS, respectively. In addition, cell migration and invasion were assessed using the Transwell system. RESULTS: The expression of ZFR was significantly higher in pancreatic tumors than normal pancreas tissues by Oncomine database analysis. Knockdown of ZFR by shRNA-expressing lentivirus significantly decreased the viability and invasion ability of pancreatic cancer cells. Moreover, FACS analysis showed that knockdown of ZFR in PANC-1 cells caused a significant cell cycle arrest at G0/G1 phase. Furthermore, knockdown of ZFR decreased the levels of CDK2, CDK4, CyclinA and CyclinD1 and enhanced the expression of p27, which has evidenced by qRT-PCR and Western blot analysis. CONCLUSIONS: Knockdown of ZFR might provide a novel alternative to targeted therapy of pancreatic cancer and deserves further investigation.

  19. Immunological comparison of the NADH:nitrate reductase from different cucumber tissues

    Directory of Open Access Journals (Sweden)

    Jolanta Marciniak

    2014-01-01

    Full Text Available Soluble nitrate reductase from cucumber roots (Cucumis sativus L. was isolated and purified with blue-Sepharose 4B. Specific antibodies against the NR protein were raised by immunization of a goat. Using polyclonal antibodies anti-NR properties of the nitrate reductase from various cucumber tissues were examined. Experiments showed difference in immuno-logical properties of nitrate reductase (NR from cotyledon roots and leaves.

  20. Ribosomal protein gene knockdown causes developmental defects in zebrafish.

    Directory of Open Access Journals (Sweden)

    Tamayo Uechi

    Full Text Available The ribosomal proteins (RPs form the majority of cellular proteins and are mandatory for cellular growth. RP genes have been linked, either directly or indirectly, to various diseases in humans. Mutations in RP genes are also associated with tissue-specific phenotypes, suggesting a possible role in organ development during early embryogenesis. However, it is not yet known how mutations in a particular RP gene result in specific cellular changes, or how RP genes might contribute to human diseases. The development of animal models with defects in RP genes will be essential for studying these questions. In this study, we knocked down 21 RP genes in zebrafish by using morpholino antisense oligos to inhibit their translation. Of these 21, knockdown of 19 RPs resulted in the development of morphants with obvious deformities. Although mutations in RP genes, like other housekeeping genes, would be expected to result in nonspecific developmental defects with widespread phenotypes, we found that knockdown of some RP genes resulted in phenotypes specific to each gene, with varying degrees of abnormality in the brain, body trunk, eyes, and ears at about 25 hours post fertilization. We focused further on the organogenesis of the brain. Each knocked-down gene that affected the morphogenesis of the brain produced a different pattern of abnormality. Among the 7 RP genes whose knockdown produced severe brain phenotypes, 3 human orthologs are located within chromosomal regions that have been linked to brain-associated diseases, suggesting a possible involvement of RP genes in brain or neurological diseases. The RP gene knockdown system developed in this study could be a powerful tool for studying the roles of ribosomes in human diseases.

  1. Rapid, radiochemical-ligand binding assay for methotrexate

    International Nuclear Information System (INIS)

    Caston, J.D.

    1976-01-01

    A radiochemical ligand binding assay for methotrexate is provided. A binder factor comprising a partially purified dihydrofolic acid reductase preparation is employed. The binder factor is conveniently prepared by homogenizing a factor containing animal organ such as liver, and extracting with isotonic saline and ammonium sulfate. A binder cofactor, NADPH 2 , is also employed in the binding reaction. The procedure contemplates both direct and sequential assay techniques, and it is not interfered with by vast excesses of many natural folate derivatives. 12 claims, 6 drawing figures

  2. Clonal origin of aminoglycoside-resistant Citrobacter freundii isolates in a Danish county

    DEFF Research Database (Denmark)

    Norskov-Lauritsen, N.; Sandvang, Dorthe; Hedegaard, J.

    2001-01-01

    During 1997, attention was drawn to an increased frequency of aminoglycoside-resistant Citrobacter freundii in a Danish county, when a total of 24 resistant C. freundii isolates was detected. In this study, 15 such isolates were typed by pulsed-field gel electrophoresis, riboprinting and partial...... with a dihydrofolate reductase gene in a class I integron. The source of the strain remains unresolved. Representative isolates were obtained from various specimens from hospitals and general practice throughout the county, with no evidence of patient-to-patient transmission....

  3. Carbonyl Reduction of NNK by Recombinant Human Lung Enzymes. Identification of HSD17β12 as the Reductase important in (R)-NNAL formation in Human Lung.

    Science.gov (United States)

    Ashmore, Joseph H; Luo, Shaman; Watson, Christy J W; Lazarus, Philip

    2018-05-17

    4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most abundant and carcinogenic tobacco-specific nitrosamine in tobacco and tobacco smoke. The major metabolic pathway for NNK is carbonyl reduction to form the (R) and (S) enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) which, like NNK, is a potent lung carcinogen. The goal of the present study was to characterize NNAL enantiomer formation in human lung and identify the enzymes responsible for this activity. While (S)-NNAL was the major enantiomer of NNAL formed in incubations with NNK in lung cytosolic fractions, (R)-NNAL comprised ~60 and ~95% of the total NNAL formed in lung whole cell lysates and microsomes, respectively. In studies examining the role of individual recombinant reductase enzymes in lung NNAL enantiomer formation, AKR1C1, AKR1C2, AKR1C3, AKR1C4 and CBR1 all exhibited (S)-NNAL formation activity. To identify the microsomal enzymes responsible for (R)-NNAL formation, 28 microsomal reductase enzymes were screened for expression by real-time PCR in normal human lung. HSD17β6, HSD17β12, KDSR, NSDHL, RDH10, RDH11 and SDR16C5 were all expressed at levels >HSD11β1, the only previously reported microsomal reductase enzyme with NNK-reducing activity, with HSD17β12 the most highly expressed. Of these lung-expressing enzymes, only HSD17β12 exhibited activity against NNK, forming primarily (>95%) (R)-NNAL, a pattern consistent with that observed in lung microsomes. siRNA knockdown of HSD17β12 resulted in significant decreases in (R)-NNAL formation activity in HEK293 cells. These data suggest that both cytosolic and microsomal enzymes are active against NNK and that HSD17β12 is the major active microsomal reductase that contributes to (R)-NNAL formation in human lung.

  4. In vivo photoinactivation of Escherichia coli ribonucleoside reductase by near-ultraviolet light

    International Nuclear Information System (INIS)

    Peters, J.

    1977-01-01

    Some experimental work is described showing that near-U.V. irradiation of E.coli cells selectively destroys RDP-reductase (ribonucleoside diphosphate reductase) activity in vivo are providing evidence relating the loss of RDP-reductase to loss of cellular visibility and the inactivity of irrdiated cells to support the replication of DNA phages. The data are consistent with the interpretation that the principal cause in the killing of exponentially growing E.coli cells by near-U.V., and the loss of ability of irradiated host cells to support the replication of DNA phages, is the photoinactivation of the RDP-reductase complex. (U.K.)

  5. In vivo photoinactivation of Escherichia coli ribonucleoside reductase by near-ultraviolet light

    Energy Technology Data Exchange (ETDEWEB)

    Peters, J [California Univ., Irvine (USA)

    1977-06-09

    Some experimental work is described showing that near-uv irradiation of E.coli cells selectively destroys RDP-reductase (ribonucleoside diphosphate reductase) activity in vivo are providing evidence relating the loss of RDP-reductase to loss of cellular visibility and the inactivity of irrdiated cells to support the replication of DNA phages. The data are consistent with the interpretation that the principal cause in the killing of exponentially growing E.coli cells by near-uv, and the loss of ability of irradiated host cells to support the replication of DNA phages, is the photoinactivation of the RDP-reductase complex.

  6. Association between methylenetetrahydrofolate reductase (MTHFR ...

    African Journals Online (AJOL)

    Association between methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism and risk of ischemic stroke in North Indian population: A hospital based case–control study. Amit Kumar, Shubham Misra, Anjali Hazarika, Pradeep Kumar, Ram Sagar, Abhishek Pathak, Kamalesh Chakravarty, Kameshwar ...

  7. Mitochondria-Targeted Antioxidant Prevents Cardiac Dysfunction Induced by Tafazzin Gene Knockdown in Cardiac Myocytes

    Directory of Open Access Journals (Sweden)

    Quan He

    2014-01-01

    Full Text Available Tafazzin, a mitochondrial acyltransferase, plays an important role in cardiolipin side chain remodeling. Previous studies have shown that dysfunction of tafazzin reduces cardiolipin content, impairs mitochondrial function, and causes dilated cardiomyopathy in Barth syndrome. Reactive oxygen species (ROS have been implicated in the development of cardiomyopathy and are also the obligated byproducts of mitochondria. We hypothesized that tafazzin knockdown increases ROS production from mitochondria, and a mitochondria-targeted antioxidant prevents tafazzin knockdown induced mitochondrial and cardiac dysfunction. We employed cardiac myocytes transduced with an adenovirus containing tafazzin shRNA as a model to investigate the effects of the mitochondrial antioxidant, mito-Tempo. Knocking down tafazzin decreased steady state levels of cardiolipin and increased mitochondrial ROS. Treatment of cardiac myocytes with mito-Tempo normalized tafazzin knockdown enhanced mitochondrial ROS production and cellular ATP decline. Mito-Tempo also significantly abrogated tafazzin knockdown induced cardiac hypertrophy, contractile dysfunction, and cell death. We conclude that mitochondria-targeted antioxidant prevents cardiac dysfunction induced by tafazzin gene knockdown in cardiac myocytes and suggest mito-Tempo as a potential therapeutic for Barth syndrome and other dilated cardiomyopathies resulting from mitochondrial oxidative stress.

  8. Enzyme structure and interaction with inhibitors

    International Nuclear Information System (INIS)

    London, R.E.

    1983-01-01

    This article reviews some of the results of studies on the 13 C-labeled enzyme dihydrofolate reductase (DHFR). Nuclear magnetic resonance (NMR) techniques are used in combination with isotopic labeling to learn about the structure and dynamics of this enzyme. 13 C-labeling is used for the purpose of studying enzyme/substrate and enzyme/inhibitor interactions. A second set of studies with DHFR was designed to investigate the basis for the high affinity between the inhibitor methotrexate and DHFR. The label was placed on the inhibitor, rather than the enzyme

  9. The effect of ionic and non-ionic surfactants on the growth, nitrate reductase and nitrite reductase activities of Spirodela polyrrhiza (L. Schleiden

    Directory of Open Access Journals (Sweden)

    Józef Buczek

    2014-01-01

    Full Text Available Inclusion into the medium of 5 mg•dm-3 of non-ionic (ENF or ionic (DBST surfactant caused 50-60% inhibition of nitrite reductase MR activity in S. polyrrhiza. At the same time, increased accumulation of NO2- in the plant tissues and lowering of the total and soluble protein contents were found. DBST also lowered the nitrate reductase (NR activity and the dry mass of the plants.

  10. Enhanced toxic cloud knockdown spray system for decontamination applications

    Science.gov (United States)

    Betty, Rita G [Rio Rancho, NM; Tucker, Mark D [Albuquerque, NM; Brockmann, John E [Albuquerque, NM; Lucero, Daniel A [Albuquerque, NM; Levin, Bruce L [Tijeras, NM; Leonard, Jonathan [Albuquerque, NM

    2011-09-06

    Methods and systems for knockdown and neutralization of toxic clouds of aerosolized chemical or biological warfare (CBW) agents and toxic industrial chemicals using a non-toxic, non-corrosive aqueous decontamination formulation.

  11. Expression, purification, crystallization and preliminary X-ray analysis of perakine reductase, a new member of the aldo-keto reductase enzyme superfamily from higher plants

    Energy Technology Data Exchange (ETDEWEB)

    Rosenthal, Cindy [Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudinger Weg 5, D-55099 Mainz (Germany); Mueller, Uwe [Berliner Elektronenspeicherring-Gesellschaft für Synchrotronstrahlung mbH, Albert-Einstein-Strasse 15, D-12489 Berlin (Germany); Panjikar, Santosh [European Molecular Biology Laboratory Hamburg, Outstation Deutsches Elektronen-Synchrotron, Notkestrasse 85, D-22603 Hamburg (Germany); Sun, Lianli [Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudinger Weg 5, D-55099 Mainz (Germany); Department of TCM and Natural Drug Research, College of Pharmaceutical Sciences, 513 Zijingang Campus, Zhejiang University, 310058 Hangzhou (China); Ruppert, Martin [Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudinger Weg 5, D-55099 Mainz (Germany); Zhao, Yu [Department of TCM and Natural Drug Research, College of Pharmaceutical Sciences, 513 Zijingang Campus, Zhejiang University, 310058 Hangzhou (China); Stöckigt, Joachim [Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudinger Weg 5, D-55099 Mainz (Germany); Department of TCM and Natural Drug Research, College of Pharmaceutical Sciences, 513 Zijingang Campus, Zhejiang University, 310058 Hangzhou (China)

    2006-12-01

    Perakine reductase, a novel member of the aldo-keto reductase enzyme superfamily of higher plants, is involved in the biosynthesis of monoterpenoid indole alkaloids in the Indian medicinal plant Rauvolfia serpentina. The enzyme has been crystallized in C-centered orthorhombic space group and diffracts to 2.0 Å resolution. Perakine reductase (PR) is a novel member of the aldo-keto reductase enzyme superfamily from higher plants. PR from the plant Rauvolfia serpentina is involved in the biosynthesis of monoterpenoid indole alkaloids by performing NADPH-dependent reduction of perakine, yielding raucaffrinoline. However, PR can also reduce cinnamic aldehyde and some of its derivatives. After heterologous expression of a triple mutant of PR in Escherichia coli, crystals of the purified and methylated enzyme were obtained by the hanging-drop vapour-diffusion technique at 293 K with 100 mM sodium citrate pH 5.6 and 27% PEG 4000 as precipitant. Crystals belong to space group C222{sub 1} and diffract to 2.0 Å, with unit-cell parameters a = 58.9, b = 93.0, c = 143.4 Å.

  12. Expression, purification, crystallization and preliminary X-ray analysis of perakine reductase, a new member of the aldo-keto reductase enzyme superfamily from higher plants

    International Nuclear Information System (INIS)

    Rosenthal, Cindy; Mueller, Uwe; Panjikar, Santosh; Sun, Lianli; Ruppert, Martin; Zhao, Yu; Stöckigt, Joachim

    2006-01-01

    Perakine reductase, a novel member of the aldo-keto reductase enzyme superfamily of higher plants, is involved in the biosynthesis of monoterpenoid indole alkaloids in the Indian medicinal plant Rauvolfia serpentina. The enzyme has been crystallized in C-centered orthorhombic space group and diffracts to 2.0 Å resolution. Perakine reductase (PR) is a novel member of the aldo-keto reductase enzyme superfamily from higher plants. PR from the plant Rauvolfia serpentina is involved in the biosynthesis of monoterpenoid indole alkaloids by performing NADPH-dependent reduction of perakine, yielding raucaffrinoline. However, PR can also reduce cinnamic aldehyde and some of its derivatives. After heterologous expression of a triple mutant of PR in Escherichia coli, crystals of the purified and methylated enzyme were obtained by the hanging-drop vapour-diffusion technique at 293 K with 100 mM sodium citrate pH 5.6 and 27% PEG 4000 as precipitant. Crystals belong to space group C222 1 and diffract to 2.0 Å, with unit-cell parameters a = 58.9, b = 93.0, c = 143.4 Å

  13. Identification of the 7-Hydroxymethyl Chlorophyll a Reductase of the Chlorophyll Cycle in Arabidopsis[W

    Science.gov (United States)

    Meguro, Miki; Ito, Hisashi; Takabayashi, Atsushi; Tanaka, Ryouichi; Tanaka, Ayumi

    2011-01-01

    The interconversion of chlorophyll a and chlorophyll b, referred to as the chlorophyll cycle, plays a crucial role in the processes of greening, acclimation to light intensity, and senescence. The chlorophyll cycle consists of three reactions: the conversions of chlorophyll a to chlorophyll b by chlorophyllide a oxygenase, chlorophyll b to 7-hydroxymethyl chlorophyll a by chlorophyll b reductase, and 7-hydroxymethyl chlorophyll a to chlorophyll a by 7-hydroxymethyl chlorophyll a reductase. We identified 7-hydroxymethyl chlorophyll a reductase, which is the last remaining unidentified enzyme of the chlorophyll cycle, from Arabidopsis thaliana by genetic and biochemical methods. Recombinant 7-hydroxymethyl chlorophyll a reductase converted 7-hydroxymethyl chlorophyll a to chlorophyll a using ferredoxin. Both sequence and biochemical analyses showed that 7-hydroxymethyl chlorophyll a reductase contains flavin adenine dinucleotide and an iron-sulfur center. In addition, a phylogenetic analysis elucidated the evolution of 7-hydroxymethyl chlorophyll a reductase from divinyl chlorophyllide vinyl reductase. A mutant lacking 7-hydroxymethyl chlorophyll a reductase was found to accumulate 7-hydroxymethyl chlorophyll a and pheophorbide a. Furthermore, this accumulation of pheophorbide a in the mutant was rescued by the inactivation of the chlorophyll b reductase gene. The downregulation of pheophorbide a oxygenase activity is discussed in relation to 7-hydroxymethyl chlorophyll a accumulation. PMID:21934147

  14. Heritable and lineage-specific gene knockdown in zebrafish embryo.

    Directory of Open Access Journals (Sweden)

    Mei Dong

    Full Text Available BACKGROUND: Reduced expression of developmentally important genes and tumor suppressors due to haploinsufficiency or epigenetic suppression has been shown to contribute to the pathogenesis of various malignancies. However, methodology that allows spatio-temporally knockdown of gene expression in various model organisms such as zebrafish has not been well established, which largely limits the potential of zebrafish as a vertebrate model of human malignant disorders. PRINCIPAL FINDING: Here, we report that multiple copies of small hairpin RNA (shRNA are expressed from a single transcript that mimics the natural microRNA-30e precursor (mir-shRNA. The mir-shRNA, when microinjected into zebrafish embryos, induced an efficient knockdown of two developmentally essential genes chordin and alpha-catenin in a dose-controllable fashion. Furthermore, we designed a novel cassette vector to simultaneously express an intronic mir-shRNA and a chimeric red fluorescent protein driven by lineage-specific promoter, which efficiently reduced the expression of a chromosomally integrated reporter gene and an endogenously expressed gata-1 gene in the developing erythroid progenitors and hemangioblasts, respectively. SIGNIFICANCE: This methodology provides an invaluable tool to knockdown developmental important genes in a tissue-specific manner or to establish animal models, in which the gene dosage is critically important in the pathogenesis of human disorders. The strategy should be also applicable to other model organisms.

  15. Genome sequence analysis of predicted polyprenol reductase gene from mangrove plant kandelia obovata

    Science.gov (United States)

    Basyuni, M.; Sagami, H.; Baba, S.; Oku, H.

    2018-03-01

    It has been previously reported that dolichols but not polyprenols were predominated in mangrove leaves and roots. Therefore, the occurrence of larger amounts of dolichol in leaves of mangrove plants implies that polyprenol reductase is responsible for the conversion of polyprenol to dolichol may be active in mangrove leaves. Here we report the early assessment of probably polyprenol reductase gene from genome sequence of mangrove plant Kandelia obovata. The functional assignment of the gene was based on a homology search of the sequences against the non-redundant (nr) peptide database of NCBI using Blastx. The degree of sequence identity between DNA sequence and known polyprenol reductase was confirmed using the Blastx probability E-value, total score, and identity. The genome sequence data resulted in three partial sequences, termed c23157 (700 bp), c23901 (960 bp), and c24171 (531 bp). The c23157 gene showed the highest similarity (61%) to predicted polyprenol reductase 2- like from Gossypium raimondii with E-value 2e-100. The second gene was c23901 to exhibit high similarity (78%) to the steroid 5-alpha-reductase Det2 from J. curcas with E-value 2e-140. Furthermore, the c24171 gene depicted highest similarity (79%) to the polyprenol reductase 2 isoform X1 from Jatropha curcas with E- value 7e-21.The present study suggested that the c23157, c23901, and c24171, genes may encode predicted polyprenol reductase. The c23157, c23901, c24171 are therefore the new type of predicted polyprenol reductase from K. obovata.

  16. Effect of specific enzyme inhibitors on replication, total genome DNA repair and on gene-specific DNA repair after UV irradiation in CHO cells

    Energy Technology Data Exchange (ETDEWEB)

    Jones, J.C.; Stevsner, Tinna; Bohr, Vilhelm A. (National Cancer Institute, NIH, Bethesda, MD (USA). Division of Cancer Treatment, Laboratory of Molecular Pharmacology); Mattern, M.R. (Smith Kline Beecham Pharmaceuticals, King of Prussia, PA (USA). Department of Biomolecular Discovery)

    1991-09-01

    The effects were studied of some specific enzyme inhibitors on DNA repair and replication after UV damage in Chinese hamster ovary cells. The DNA repair was studied at the level of the average, overall genome and also in the active dihydrofolate reductase gene. Replication was measured in the overall genome. The inhibitors were tested of DNA poly-merase {alpha} and {delta} (aphidicolin), of poly(ADPr) polymerase (3-aminobenzamide), of ribonucleotide reductase (hydroxyurea), of topo-isomerase I (camptothecin), and of topoisomerase II (merbarone, VP-16). In addition, the effects were tested of the potential topoisomerase I activator, {beta}-lapachone. All of these compounds inhibited genome replication and all topoisomerase inhibitors affected the overall genome repair; {beta}-lapachone stimulated it. None of these compounds had any effect on the gene-specific repair. (author). 36 refs.; 3 figs.; 2 tabs.

  17. Sucrose mimics the light induction of Arabidopsis nitrate reductase gene transcription

    DEFF Research Database (Denmark)

    Cheng, Chi-Lien; Acedo, Gregoria N; Kristensen, Michael

    1992-01-01

    Nitrate reductase, the first enzyme in nitrate assimilation, is located at the crossroad of two energy-consuming pathways: nitrate assimilation and carbon fixation. Light, which regulates the expression of many higher-plant carbon fixation genes, also regulates nitrate reductase gene expression. ...

  18. Bioinformatics analysis of the predicted polyprenol reductase genes in higher plants

    Science.gov (United States)

    Basyuni, M.; Wati, R.

    2018-03-01

    The present study evaluates the bioinformatics methods to analyze twenty-four predicted polyprenol reductase genes from higher plants on GenBank as well as predicted the structure, composition, similarity, subcellular localization, and phylogenetic. The physicochemical properties of plant polyprenol showed diversity among the observed genes. The percentage of the secondary structure of plant polyprenol genes followed the ratio order of α helix > random coil > extended chain structure. The values of chloroplast but not signal peptide were too low, indicated that few chloroplast transit peptide in plant polyprenol reductase genes. The possibility of the potential transit peptide showed variation among the plant polyprenol reductase, suggested the importance of understanding the variety of peptide components of plant polyprenol genes. To clarify this finding, a phylogenetic tree was drawn. The phylogenetic tree shows several branches in the tree, suggested that plant polyprenol reductase genes grouped into divergent clusters in the tree.

  19. Identification of novel Mycobacterium tuberculosis dihydrofolate reductase inhibitors through rational drug design

    Directory of Open Access Journals (Sweden)

    Mymoona Akhter

    2016-01-01

    Conclusion: Structure based drug design can be used as an effective tool for the design of new cheiocal entity. Number of novel agents have been identified as antitubercular agents whose mechanism of action needs to be ascertained.

  20. Gene cloning and overexpression of two conjugated polyketone reductases, novel aldo-keto reductase family enzymes, of Candida parapsilosis.

    Science.gov (United States)

    Kataoka, M; Delacruz-Hidalgo, A-R G; Akond, M A; Sakuradani, E; Kita, K; Shimizu, S

    2004-04-01

    The genes encoding two conjugated polyketone reductases (CPR-C1, CPR-C2) of Candida parapsilosis IFO 0708 were cloned and sequenced. The genes encoded a total of 304 and 307 amino acid residues for CPR-C1 and CPR-C2, respectively. The deduced amino acid sequences of the two enzymes showed high similarity to each other and to several proteins of the aldo-keto reductase (AKR) superfamily. However, several amino acid residues in putative active sites of AKRs were not conserved in CPR-C1 and CPR-C2. The two CPR genes were overexpressed in Escherichia coli. The E. coli transformant bearing the CPR-C2 gene almost stoichiometrically reduced 30 mg ketopantoyl lactone/ml to D-pantoyl lactone.

  1. Identification of 5α-reductase isoenzymes in canine skin.

    Science.gov (United States)

    Bernardi de Souza, Lucilene; Paradis, Manon; Zamberlam, Gustavo; Benoit-Biancamano, Marie-Odile; Price, Christopher

    2015-10-01

    Alopecia X in dogs is a noninflammatory alopecia that may be caused by a hormonal dysfunction. It may be similar to androgenic alopecia in men that is caused by the effect of dihydrotestosterone (DHT). The 5α-reductase isoenzymes, 5αR1 and 5αR2, and a recently described 5αR3, are responsible for the conversion of testosterone into DHT. However, which 5α-reductases are present in canine skin has not yet been described. The main objective of this study was to determine the pattern of expression of 5α-reductase genes in canine skin. Skin biopsies were obtained from healthy, intact young-mature beagles (three males, four females) at three anatomical sites normally affected by alopecia X (dorsal neck, back of thighs and base of tail) and two sites generally unaffected (dorsal head and ventral thorax). Prostate samples (n = 3) were collected as positive controls for 5α-reductase mRNA abundance measurement by real-time PCR. We detected mRNA encoding 5αR1 and 5αR3 but not 5αR2. There were no significant differences in 5αR1 and 5αR3 mRNA levels between the different anatomical sites, irrespective of gender (P > 0.05). Moreover, the mean mRNA abundance in each anatomical site did not differ between males and females (P > 0.05). To the best of the authors' knowledge, this is the first study demonstrating the expression of 5α-reductases in canine skin and the expression of 5αR3 in this tissue. These results may help to elucidate the pathogenesis of alopecia X and to determine more appropriate treatments for this disorder. © 2015 ESVD and ACVD.

  2. Gamma-irradiation activates biochemical systems: induction of nitrate reductase activity in plant callus.

    OpenAIRE

    Pandey, K N; Sabharwal, P S

    1982-01-01

    Gamma-irradiation induced high levels of nitrate reductase activity (NADH:nitrate oxidoreductase, EC 1.6.6.1) in callus of Haworthia mirabilis Haworth. Subcultures of gamma-irradiated tissues showed autonomous growth on minimal medium. We were able to mimic the effects of gamma-irradiation by inducing nitrate reductase activity in unirradiated callus with exogenous auxin and kinetin. These results revealed that induction of nitrate reductase activity by gamma-irradiation is mediated through i...

  3. Brain gene expression changes elicited by peripheral vitellogenin knockdown in the honey bee.

    Science.gov (United States)

    Wheeler, M M; Ament, S A; Rodriguez-Zas, S L; Robinson, G E

    2013-10-01

    Vitellogenin (Vg) is best known as a yolk protein precursor. Vg also functions to regulate behavioural maturation in adult honey bee workers, but the underlying molecular mechanisms by which it exerts this novel effect are largely unknown. We used abdominal vitellogenin (vg) knockdown with RNA interference (RNAi) and brain transcriptomic profiling to gain insights into how Vg influences honey bee behavioural maturation. We found that vg knockdown caused extensive gene expression changes in the bee brain, with much of this transcriptional response involving changes in central biological functions such as energy metabolism. vg knockdown targeted many of the same genes that show natural, maturation-related differences, but the direction of change for the genes in these two contrasts was not correlated. By contrast, vg knockdown targeted many of the same genes that are regulated by juvenile hormone (JH) and there was a significant correlation for the direction of change for the genes in these two contrasts. These results indicate that the tight coregulatory relationship that exists between JH and Vg in the regulation of honey bee behavioural maturation is manifest at the genomic level and suggest that these two physiological factors act through common pathways to regulate brain gene expression and behaviour. © 2013 Royal Entomological Society.

  4. TET1 knockdown inhibits the odontogenic differentiation potential of human dental pulp cells.

    Science.gov (United States)

    Rao, Li-Jia; Yi, Bai-Cheng; Li, Qi-Meng; Xu, Qiong

    2016-06-30

    Human dental pulp cells (hDPCs) possess the capacity to differentiate into odontoblast-like cells and generate reparative dentin in response to exogenous stimuli or injury. Ten-eleven translocation 1 (TET1) is a novel DNA methyldioxygenase that plays an important role in the promotion of DNA demethylation and transcriptional regulation in several cell lines. However, the role of TET1 in the biological functions of hDPCs is unknown. To investigate the effect of TET1 on the proliferation and odontogenic differentiation potential of hDPCs, a recombinant shRNA lentiviral vector was used to knock down TET1 expression in hDPCs. Following TET1 knockdown, TET1 was significantly downregulated at both the mRNA and protein levels. Proliferation of the hDPCs was suppressed in the TET1 knockdown groups. Alkaline phosphatase activity, the formation of mineralized nodules, and the expression levels of DSPP and DMP1 were all reduced in the TET1-knockdown hDPCs undergoing odontogenic differentiation. Based on these results, we concluded that TET1 knockdown can prevent the proliferation and odontogenic differentiation of hDPCs, which suggests that TET1 may play an important role in dental pulp repair and regeneration.

  5. In Vivo Testing of MicroRNA-Mediated Gene Knockdown in Zebrafish

    Directory of Open Access Journals (Sweden)

    Ivone Un San Leong

    2012-01-01

    Full Text Available The zebrafish (Danio rerio has become an attractive model for human disease modeling as there are a large number of orthologous genes that encode similar proteins to those found in humans. The number of tools available to manipulate the zebrafish genome is limited and many currently used techniques are only effective during early development (such as morpholino-based antisense technology or it is phenotypically driven and does not offer targeted gene knockdown (such as chemical mutagenesis. The use of RNA interference has been met with controversy as off-target effects can make interpreting phenotypic outcomes difficult; however, this has been resolved by creating zebrafish lines that contain stably integrated miRNA constructs that target the desired gene of interest. In this study, we show that a commercially available miRNA vector system with a mouse-derived miRNA backbone is functional in zebrafish and is effective in causing eGFP knockdown in a transient in vivo eGFP sensor assay system. We chose to apply this system to the knockdown of transcripts that are implicated in the human cardiac disorder, Long QT syndrome.

  6. Transcripts of Anthocyanidin Reductase and Leucoanthocyanidin Reductase and Measurement of Catechin and Epicatechin in Tartary Buckwheat

    Directory of Open Access Journals (Sweden)

    Yeon Bok Kim

    2014-01-01

    Full Text Available Anthocyanidin reductase (ANR and leucoanthocyanidin reductase (LAR play an important role in the monomeric units biosynthesis of proanthocyanidins (PAs such as catechin and epicatechin in several plants. The aim of this study was to clone ANR and LAR genes involved in PAs biosynthesis and examine the expression of these two genes in different organs under different growth conditions in two tartary buckwheat cultivars, Hokkai T8 and T10. Gene expression was carried out by quantitative real-time RT-PCR, and catechin and epicatechin content was analyzed by high performance liquid chromatography. The expression pattern of ANR and LAR did not match the accumulation pattern of PAs in different organs of two cultivars. Epicatechin content was the highest in the flowers of both cultivars and it was affected by light in only Hokkai T8 sprouts. ANR and LAR levels in tartary buckwheat might be regulated by different mechanisms for catechin and epicatechin biosynthesis under light and dark conditions.

  7. Comparative modelling and molecular docking of nitrate reductase from Bacillus weihenstephanensis (DS45

    Directory of Open Access Journals (Sweden)

    R. Seenivasagan

    2016-07-01

    Full Text Available Nitrate reductase catalyses the oxidation of NAD(PH and the reduction of nitrate to nitrite. NR serves as a central point for the integration of metabolic pathways by governing the flux of reduced nitrogen through several regulatory mechanisms in plants, algae and fungi. Bacteria express nitrate reductases that convert nitrate to nitrite, but mammals lack these specific enzymes. The microbial nitrate reductase reduces toxic compounds to nontoxic compounds with the help of NAD(PH. In the present study, our results revealed that Bacillus weihenstephanensis expresses a nitrate reductase enzyme, which was made to generate the 3D structure of the enzyme. Six different modelling servers, namely Phyre2, RaptorX, M4T Server, HHpred, SWISS MODEL and Mod Web, were used for comparative modelling of the structure. The model was validated with standard parameters (PROCHECK and Verify 3D. This study will be useful in the functional characterization of the nitrate reductase enzyme and its docking with nitrate molecules, as well as for use with autodocking.

  8. Transcription factor Runx2 knockdown regulates colon cancer transplantation tumor growth in vitro: an experimental study

    Directory of Open Access Journals (Sweden)

    Bin Xu1

    2017-05-01

    Full Text Available Objective: To study the effect of transcription factor Runx2 knockdown on colon cancer transplantation tumor growth in vitro. Methods: Colon cancer cell lines HT29 were cultured and transfected with negative control (NC - shRNA plasmids and Runx2-shRNA plasmids respectively, the colon cancer cells transfected with shRNA were subcutaneously injected into C57 nude mice, and they were included in NC group and Runx2 knockdown group respectively. 1 week, 2 weeks and 3 weeks after model establishment, serum was collected to determine the contents of tumor markers, and tumor lesions were collected to determine proliferation and apoptosis gene expression. Results: CCSA-2, CEA and CA19-9 levels in serum as well as Rac1, Wnt3a, PLD2 and FAM96B protein expression in transplantation tumor lesions of Runx2 knockdown group were significantly lower than those of NC group while MS4A12, ASPP2 and Fas protein expression in transplantation tumor lesions of Runx2 knockdown group were significantly higher than those of NC group. Conclusion: Transcription factor Runx2 knockdown could inhibit the colon cancer transplantation tumor growth in vitro.

  9. [Knockdown of dopamine receptor D2 upregulates the expression of adiogenic genes in mouse primary mesencephalic neurons].

    Science.gov (United States)

    Ding, Jiaqi; Chen, Xiaoli; Lin, Jiaji; Zhu, Junling; Li, Zhuyi

    2018-01-01

    Objective To study the effects of dopamine receptor D2 (DRD2) on the adipogenesis genes in mouse primary mesencephalic neurons. Methods The lentiviral vectors which expressed specific shRNA targeting DRD2 were constructed to decrease DRD2 expression in mouse primary mesencephalic neurons. High throughput sequencing (HTS) analysis was used to investigate gene expression changes between the DRD2 knock-down group and the negative control group. Real-time quantitative PCR (qRT-PCR) and Western blot analysis were applied to verify the differently expressed genes. Fatty acids were measured by fatty acid detection kit. Results DRD2 expression was effectively down-regulated in mouse primary mesencephalic neurons by lentiviral vectors. HTS revealed adipogenesis genes were significantly up-regulated after DRD2 down-regulation, mainly including delta(14)-sterol reductase, acetyl-coenzyme A synthetase, insulin-induced gene 1 protein and especially stearoyl-coenzyme A desaturase 1 (SCD1, 4-fold upregulated). The qRT-PCR and Western blot analysis verified that SCD1 was upregulated 2.6 folds and 2 folds respectively by lentiviral DRD2-shRNA vectors. Moreover, the SCD1-related free fatty acids were significantly more increased than the negative control group. Conclusion DRD2 in primary mesencephalic neurons had a significant regulative effect on the adipogenesis genes. The up-regulation of SCD1 can accelerate the conversion of saturated fatty acids to monounsaturated fatty acids and prevent the damage of lipid toxicity to cells.

  10. Glutathione reductase: solvent equilibrium and kinetic isotope effects

    International Nuclear Information System (INIS)

    Wong, K.K.; Vanoni, M.A.; Blanchard, J.S.

    1988-01-01

    Glutathione reductase catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG). The kinetic mechanism is ping-pong, and we have investigated the rate-limiting nature of proton-transfer steps in the reactions catalyzed by the spinach, yeast, and human erythrocyte glutathione reductases using a combination of alternate substrate and solvent kinetic isotope effects. With NADPH or GSSG as the variable substrate, at a fixed, saturating concentration of the other substrate, solvent kinetic isotope effects were observed on V but not V/K. Plots of Vm vs mole fraction of D 2 O (proton inventories) were linear in both cases for the yeast, spinach, and human erythrocyte enzymes. When solvent kinetic isotope effect studies were performed with DTNB instead of GSSG as an alternate substrate, a solvent kinetic isotope effect of 1.0 was observed. Solvent kinetic isotope effect measurements were also performed on the asymmetric disulfides GSSNB and GSSNP by using human erythrocyte glutathione reductase. The Km values for GSSNB and GSSNP were 70 microM and 13 microM, respectively, and V values were 62 and 57% of the one calculated for GSSG, respectively. Both of these substrates yield solvent kinetic isotope effects greater than 1.0 on both V and V/K and linear proton inventories, indicating that a single proton-transfer step is still rate limiting. These data are discussed in relationship to the chemical mechanism of GSSG reduction and the identity of the proton-transfer step whose rate is sensitive to solvent isotopic composition. Finally, the solvent equilibrium isotope effect measured with yeast glutathione reductase is 4.98, which allows us to calculate a fractionation factor for the thiol moiety of GSH of 0.456

  11. Short Hairpin RNA (shRNA): Design, Delivery, and Assessment of Gene Knockdown

    Science.gov (United States)

    Moore, Chris B.; Guthrie, Elizabeth H.; Huang, Max Tze-Han; Taxman, Debra J.

    2013-01-01

    Shortly after the cellular mechanism of RNA interference (RNAi) was first described, scientists began using this powerful technique to study gene function. This included designing better methods for the successful delivery of small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) into mammalian cells. While the simplest method for RNAi is the cytosolic delivery of siRNA oligonucleotides, this technique is limited to cells capable of transfection and is primarily utilized during transient in vitro studies. The introduction of shRNA into mammalian cells through infection with viral vectors allows for stable integration of shRNA and long-term knockdown of the targeted gene; however, several challenges exist with the implementation of this technology. Here we describe some well-tested protocols which should increase the chances of successful design, delivery, and assessment of gene knockdown by shRNA. We provide suggestions for designing shRNA targets and controls, a protocol for sequencing through the secondary structure of the shRNA hairpin structure, and protocols for packaging and delivery of shRNA lentiviral particles. Using real-time PCR and functional assays we demonstrate the successful knockdown of ASC, an inflammatory adaptor molecule. These studies demonstrate the practicality of including two shRNAs with different efficacies of knockdown to provide an additional level of control and to verify dose dependency of functional effects. Along with the methods described here, as new techniques and algorithms are designed in the future, shRNA is likely to include further promising application and continue to be a critical component of gene discovery. PMID:20387148

  12. RNA interference of NADPH-cytochrome P450 reductase results in reduced insecticide resistance in the bed bug, Cimex lectularius.

    Science.gov (United States)

    Zhu, Fang; Sams, Sarah; Moural, Tim; Haynes, Kenneth F; Potter, Michael F; Palli, Subba R

    2012-01-01

    NADPH-cytochrome P450 reductase (CPR) plays a central role in cytochrome P450 action. The genes coding for P450s are not yet fully identified in the bed bug, Cimex lectularius. Hence, we decided to clone cDNA and knockdown the expression of the gene coding for CPR which is suggested to be required for the function of all P450s to determine whether or not P450s are involved in resistance of bed bugs to insecticides. The full length Cimex lectularius CPR (ClCPR) cDNA was isolated from a deltamethrin resistant bed bug population (CIN-1) using a combined PCR strategy. Bioinformatics and in silico modeling were employed to identify three conserved binding domains (FMN, FAD, NADP), a FAD binding motif, and the catalytic residues. The critical amino acids involved in FMN, FAD, NADP binding and their putative functions were also analyzed. No signal peptide but a membrane anchor domain with 21 amino acids which facilitates the localization of ClCPR on the endoplasmic reticulum was identified in ClCPR protein. Phylogenetic analysis showed that ClCPR is closer to the CPR from the body louse, Pediculus humanus corporis than to the CPRs from the other insect species studied. The ClCPR gene was ubiquitously expressed in all tissues tested but showed an increase in expression as immature stages develop into adults. We exploited the traumatic insemination mechanism of bed bugs to inject dsRNA and successfully knockdown the expression of the gene coding for ClCPR. Suppression of the ClCPR expression increased susceptibility to deltamethrin in resistant populations but not in the susceptible population of bed bugs. These data suggest that P450-mediated metabolic detoxification may serve as one of the resistance mechanisms in bed bugs.

  13. Substrate and cofactor binding to nitrile reductase : A mass spectrometry based study

    NARCIS (Netherlands)

    Gjonaj, L.; Pinkse, M.W.H.; Fernandez Fueyo, E.; Hollmann, F.; Hanefeld, U.

    2016-01-01

    Nitrile reductases catalyse a two-step reduction of nitriles to amines. This requires the binding of two NADPH molecules during one catalytic cycle. For the nitrile reductase from E. coli (EcoNR) mass spectrometry studies of the catalytic mechanism were performed. EcoNR is dimeric and has no Rossman

  14. MADD knock-down enhances doxorubicin and TRAIL induced apoptosis in breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Andrea Turner

    Full Text Available The Map kinase Activating Death Domain containing protein (MADD isoform of the IG20 gene is over-expressed in different types of cancer tissues and cell lines and it functions as a negative regulator of apoptosis. Therefore, we speculated that MADD might be over-expressed in human breast cancer tissues and that MADD knock-down might synergize with chemotherapeutic or TRAIL-induced apoptosis of breast cancer cells. Analyses of breast tissue microarrays revealed over-expression of MADD in ductal and invasive carcinomas relative to benign tissues. MADD knockdown resulted in enhanced spontaneous apoptosis in human breast cancer cell lines. Moreover, MADD knockdown followed by treatment with TRAIL or doxorubicin resulted in increased cell death compared to either treatment alone. Enhanced cell death was found to be secondary to increased caspase-8 activation. These data indicate that strategies to decrease MADD expression or function in breast cancer may be utilized to increase tumor cell sensitivity to TRAIL and doxorubicin induced apoptosis.

  15. N-terminus determines activity and specificity of styrene monooxygenase reductases.

    Science.gov (United States)

    Heine, Thomas; Scholtissek, Anika; Westphal, Adrie H; van Berkel, Willem J H; Tischler, Dirk

    2017-12-01

    Styrene monooxygenases (SMOs) are two-enzyme systems that catalyze the enantioselective epoxidation of styrene to (S)-styrene oxide. The FADH 2 co-substrate of the epoxidase component (StyA) is supplied by an NADH-dependent flavin reductase (StyB). The genome of Rhodococcus opacus 1CP encodes two SMO systems. One system, which we define as E1-type, displays homology to the SMO from Pseudomonas taiwanensis VLB120. The other system, originally reported as a fused system (RoStyA2B), is defined as E2-type. Here we found that E1-type RoStyB is inhibited by FMN, while RoStyA2B is known to be active with FMN. To rationalize the observed specificity of RoStyB for FAD, we generated an artificial reductase, designated as RoStyBart, in which the first 22 amino acid residues of RoStyB were joined to the reductase part of RoStyA2B, while the oxygenase part (A2) was removed. RoStyBart mainly purified as apo-protein and mimicked RoStyB in being inhibited by FMN. Pre-incubation with FAD yielded a turnover number at 30°C of 133.9±3.5s -1 , one of the highest rates observed for StyB reductases. RoStyBart holo-enzyme switches to a ping-pong mechanism and fluorescence analysis indicated for unproductive binding of FMN to the second (co-substrate) binding site. In summary, it is shown for the first time that optimization of the N-termini of StyB reductases allows the evolution of their activity and specificity. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Prevalence of methylenetetrahydrofolate reductase ( MTHFR ) and ...

    African Journals Online (AJOL)

    Methylenetetrahydrofolate reductase (MTHFR) and Cytosolic serine hydroxymethyltransferase (cSHMT) are enzymes involve in folate regulation in human. The C to T transition of the cSHMT and MTHFR genes at the 1420 as well as 677 nucleotides both carries TT genotype respectively. These enzymes have direct and ...

  17. Neurobasal media facilitates increased specificity of siRNA-mediated knockdown in primary cerebellar cultures

    DEFF Research Database (Denmark)

    Gustafsson, Julie Ry; Katsioudi, Georgia; Issazadeh-Navikas, Shohreh

    2016-01-01

    be effectively grown in Neurobasal™ media. NEW METHOD: We tested the efficiency of siRNA from the Accell range from Dharmacon™ when delivered in Neurobasal™ media in contrast to the recommended Accell Delivery media provided by the manufacturer. RESULTS: We observed a more specific knockdown of target...... in Neurobasal™ media, than in Accell Delivery media when using cerebellar granule neurons. Transfection efficiency and cell viability was comparable between the two media. COMPARISON WITH EXISTING METHODS: Delivery of siRNA in Neurobasal™ media facilitates increased specificity of the knockdown compared...... to delivery in Accell Delivery media. The off-target effect observed in Accell Delivery media was not a secondary biological response to downregulation of target, but rather a mixture of specific and non-specific off-target effects. CONCLUSIONS: Specific knockdown of target can be achieved in primary...

  18. Methemoglobin reductase activity in intact fish red blood cells

    DEFF Research Database (Denmark)

    Jensen, Frank B; Nielsen, Karsten

    2018-01-01

    RBCs in physiological saline at normal Pco2 and pH. After initial loading of oxygenated RBCs with nitrite (partly oxidizing Hb to metHb), the nitrite is removed by three washes of the RBCs in nitrite-free physiological saline to enable the detection of RBC metHb reductase activity in the absence......Hb reductase activity in fish offsets their higher Hb autoxidation and higher likelihood of encountering elevated nitrite. Deoxygenation significantly raised the rates of RBC metHb reduction, and more so in rainbow trout than in carp. The temperature sensitivity of metHb reduction in rainbow trout RBCs...

  19. Molecular mechanisms of drug resistance and tumor promotion involving mammalian ribonucleotide reductase

    Energy Technology Data Exchange (ETDEWEB)

    Choy, B.B.K.

    1991-01-01

    Mammalian ribonucleotide reductase is a highly regulated, rate-limiting activity responsible for converting ribonucleoside diphosphates to the deoxyribonucleotide precursors of DNA. The enzyme consists of two nonidentical proteins called M1 and M2, both of which are required for activity. Hydroxyurea is an antitumor agent which inhibits ribonucleotide reductase by interacting with the M2 component specifically at a unique tyrosyl free radical. Studies were conducted on a series of drug resistant mouse cell lines, selected by a step-wise procedure for increasing levels of resistance to the cytotoxic effects of hydroxyurea. Each successive drug selection step leading to the isolation of highly resistant cells was accompanied by stable elevations in cellular resistance and ribonucleotide reductase activity. The drug resistant cell lines exhibited gene amplification of the M2 gene, elevated M2 mRNA, and M2 protein. In addition to M2 gene amplification, posttranscriptional modulation also occurred during the drug selection. Studies of the biosynthesis rates with exogenously added iron suggest a role for iron in regulating the level of M2 protein when cells are cultured in the presence of hydroxyurea. The hydroxyurea-inactivated ribonucleotide reductase protein M2 has a destabilized iron centre, which readily releases iron. Altered expression of ferritin appears to be required for the development of hydroxyurea resistance in nammalian cells. The results show an interesting relationship between the expressions of ribonucleotide reductase and ferritin. The phorbol ester tumor promoter, TPA, is also able to alter the expression of M2. TPA was able to induce M2 mRNA levels transiently up to 18-fold within 1/2 hour. This rapid and large elevation of ribonucleotide reductase suggests that the enzyme may play a role in tumor promotion. Studies of the M2 promoter region were undertaken to better understand the mechanism of TPA induction of M2.

  20. BIOLOGICAL ROLE OF ALDO-KETO REDUCTASES IN RETINOIC ACID BIOSYNTHESIS AND SIGNALING

    Directory of Open Access Journals (Sweden)

    F. Xavier eRuiz

    2012-04-01

    Full Text Available Several aldo-keto reductase (AKR enzymes from subfamilies 1B and 1C show retinaldehyde reductase activity, having low Km and kcat values. Only AKR1B10 and 1B12, with all-trans-retinaldehyde, and AKR1C3, with 9-cis-retinaldehyde, display high catalytic efficiency. Major structural determinants for retinaldehyde isomer specificity are located in the external loops (A and C for AKR1B10, and B for AKR1C3, as assessed by site-directed mutagenesis and molecular dynamics. Cellular models have shown that AKR1B and 1C enzymes are well suited to work in vivo as retinaldehyde reductases and to regulate retinoic acid (RA biosynthesis at hormone pre-receptor level. An additional physiological role for the retinaldehyde reductase activity of these enzymes, consistent with their tissue localization, is their participation in β-carotene absorption. Retinaldehyde metabolism may be subjected to subcellular compartmentalization, based on enzyme localization. While retinaldehyde oxidation to RA takes place in the cytosol, reduction to retinol could take place in the cytosol by AKRs or in the membranes of endoplasmic reticulum by microsomal retinaldehyde reductases. Upregulation of some AKR1 enzymes in different cancer types may be linked to their induction by oxidative stress and to their participation in different signaling pathways related to cell proliferation. AKR1B10 and AKR1C3, through their retinaldehyde reductase activity, trigger a decrease in the RA biosynthesis flow, resulting in RA deprivation and consequently lower differentiation, with an increased cancer risk in target tissues. Rational design of selective AKR inhibitors could lead to development of novel drugs for cancer treatment as well as reduction of chemotherapeutic drug resistance.

  1. Identification of Multiple Soluble Fe(III Reductases in Gram-Positive Thermophilic Bacterium Thermoanaerobacter indiensis BSB-33

    Directory of Open Access Journals (Sweden)

    Subrata Pal

    2014-01-01

    Full Text Available Thermoanaerobacter indiensis BSB-33 has been earlier shown to reduce Fe(III and Cr(VI anaerobically at 60°C optimally. Further, the Gram-positive thermophilic bacterium contains Cr(VI reduction activity in both the membrane and cytoplasm. The soluble fraction prepared from T. indiensis cells grown at 60°C was found to contain the majority of Fe(III reduction activity of the microorganism and produced four distinct bands in nondenaturing Fe(III reductase activity gel. Proteins from each of these bands were partially purified by chromatography and identified by mass spectrometry (MS with the help of T. indiensis proteome sequences. Two paralogous dihydrolipoamide dehydrogenases (LPDs, thioredoxin reductase (Trx, NADP(H-nitrite reductase (Ntr, and thioredoxin disulfide reductase (Tdr were determined to be responsible for Fe(III reductase activity. Amino acid sequence and three-dimensional (3D structural similarity analyses of the T. indiensis Fe(III reductases were carried out with Cr(VI reducing proteins from other bacteria. The two LPDs and Tdr showed very significant sequence and structural identity, respectively, with Cr(VI reducing dihydrolipoamide dehydrogenase from Thermus scotoductus and thioredoxin disulfide reductase from Desulfovibrio desulfuricans. It appears that in addition to their iron reducing activity T. indiensis LPDs and Tdr are possibly involved in Cr(VI reduction as well.

  2. Genetic and chemical knockdown: a complementary strategy for evaluating an anti-infective target

    Directory of Open Access Journals (Sweden)

    Ramachandran V

    2013-02-01

    Full Text Available Vasanthi Ramachandran,1,* Ragini Singh,2,* Xiaoyu Yang,1 Ragadeepthi Tunduguru,1 Subrat Mohapatra,2 Swati Khandelwal,2 Sanjana Patel,2 Santanu Datta21AstraZeneca India R&D, Bangalore, India; 2Cellworks India, Bangalore, India *These authors contributed equally to this workAbstract: The equity of a drug target is principally evaluated by its genetic vulnerability with tools ranging from antisense- and microRNA-driven knockdowns to induced expression of the target protein. In order to upgrade the process of antibacterial target identification and discern its most effective type of inhibition, an in silico toolbox that evaluates its genetic and chemical vulnerability leading either to stasis or cidal outcome was constructed and validated. By precise simulation and careful experimentation using enolpyruvyl shikimate-3-phosphate synthase and its specific inhibitor glyphosate, it was shown that genetic knockdown is distinct from chemical knockdown. It was also observed that depending on the particular mechanism of inhibition, viz competitive, uncompetitive, and noncompetitive, the antimicrobial potency of an inhibitor could be orders of magnitude different. Susceptibility of Escherichia coli to glyphosate and the lack of it in Mycobacterium tuberculosis could be predicted by the in silico platform. Finally, as predicted and simulated in the in silico platform, the translation of growth inhibition to a cidal effect was able to be demonstrated experimentally by altering the carbon source from sorbitol to glucose.Keywords: knockdown, inhibition, in silico, vulnerability

  3. Knockdown of TFIIS by RNA silencing inhibits cancer cell proliferation and induces apoptosis

    International Nuclear Information System (INIS)

    Hubbard, Kyle; Catalano, Jennifer; Puri, Raj K; Gnatt, Averell

    2008-01-01

    A common element among cancer cells is the presence of improperly controlled transcription. In these cells, the degree of specific activation of some genes is abnormal, and altering the aberrant transcription may therefore directly target cancer. TFIIS is a transcription elongation factor, which directly binds the transcription motor, RNA Polymerase II and allows it to read through various transcription arrest sites. We report on RNA interference of TFIIS, a transcription elongation factor, and its affect on proliferation of cancer cells in culture. RNA interference was performed by transfecting siRNA to specifically knock down TFIIS expression in MCF7, MCF10A, PL45 and A549 cells. Levels of TFIIS expression were determined by the Quantigene method, and relative protein levels of TFIIS, c-myc and p53 were determined by C-ELISA. Induction of apoptosis was determined by an enzymatic Caspase 3/7 assay, as well as a non-enzymatic assay detecting cytoplasmic mono- and oligonucleosomes. A gene array analysis was conducted for effects of TFIIS siRNA on MCF7 and MCF10A cell lines. Knockdown of TFIIS reduced cancer cell proliferation in breast, lung and pancreatic cancer cell lines. More specifically, TFIIS knockdown in the MCF7 breast cancer cell line induced cancer cell death and increased c-myc and p53 expression whereas TFIIS knockdown in the non-cancerous breast cell line MCF10A was less affected. Differential effects of TFIIS knockdown in MCF7 and MCF10A cells included the estrogenic, c-myc and p53 pathways, as observed by C-ELISA and gene array, and were likely involved in MCF7 cell-death. Although transcription is a fundamental process, targeting select core transcription factors may provide for a new and potent avenue for cancer therapeutics. In the present study, knockdown of TFIIS inhibited cancer cell proliferation, suggesting that TFIIS could be studied as a potential cancer target within the transcription machinery

  4. Metabolic enzyme expression highlights a key role for MTHFD2 and the mitochondrial folate pathway in cancer

    Science.gov (United States)

    Nilsson, Roland; Jain, Mohit; Madhusudhan, Nikhil; Sheppard, Nina Gustafsson; Strittmatter, Laura; Kampf, Caroline; Huang, Jenny; Asplund, Anna; Mootha, Vamsi K.

    2014-01-01

    Metabolic remodeling is now widely regarded as a hallmark of cancer, but it is not clear whether individual metabolic strategies are frequently exploited by many tumours. Here we compare messenger RNA profiles of 1,454 metabolic enzymes across 1,981 tumours spanning 19 cancer types to identify enzymes that are consistently differentially expressed. Our meta-analysis recovers established targets of some of the most widely used chemotherapeutics, including dihydrofolate reductase, thymidylate synthase and ribonucleotide reductase, while also spotlighting new enzymes, such as the mitochondrial proline biosynthetic enzyme PYCR1. The highest scoring pathway is mitochondrial one-carbon metabolism and is centred on MTHFD2. MTHFD2 RNA and protein are markedly elevated in many cancers and correlated with poor survival in breast cancer. MTHFD2 is expressed in the developing embryo, but is absent in most healthy adult tissues, even those that are proliferating. Our study highlights the importance of mitochondrial compartmentalization of one-carbon metabolism in cancer and raises important therapeutic hypotheses.

  5. DNA amplification is rare in normal human cells

    International Nuclear Information System (INIS)

    Wright, J.A.; Watt, F.M.; Hudson, D.L.; Stark, G.R.; Smith, H.S.; Hancock, M.C.

    1990-01-01

    Three types of normal human cells were selected in tissue culture with three drugs without observing a single amplification event from a total of 5 x 10 8 cells. No drug-resistant colonies were observed when normal foreskin keratinocytes were selected with N-(phosphonacetyl)-L-aspartate or with hydroxyurea or when normal mammary epithelial cells were selected with methotrexate. Some slightly resistant colonies with limited potential for growth were obtained when normal diploid fibroblast cells derived from fetal lung were selected with methotrexate or hydroxyurea but careful copy-number analysis of the dihydrofolate reductase and ribonucleotide reductase genes revealed no evidence of amplification. The rarity of DNA amplification in normal human cells contrasts strongly with the situation in tumors and in established cell lines, where amplification of onogenes and of genes mediating drug resistance is frequent. The results suggest that tumors and cell lines have acquired the abnormal ability to amplify DNA with high frequency

  6. Vivo-morpholinos induced transient knockdown of physical activity related proteins.

    Directory of Open Access Journals (Sweden)

    David P Ferguson

    Full Text Available Physical activity is associated with disease prevention and overall wellbeing. Additionally there has been evidence that physical activity level is a result of genetic influence. However, there has not been a reliable method to silence candidate genes in vivo to determine causal mechanisms of physical activity regulation. Vivo-morpholinos are a potential method to transiently silence specific genes. Thus, the aim of this study was to validate the use of Vivo-morpholinos in a mouse model for voluntary physical activity with several sub-objectives. We observed that Vivo-morpholinos achieved between 60-97% knockdown of Drd1-, Vmat2-, and Glut4-protein in skeletal muscle, the delivery moiety of Vivo-morpholinos (scramble did not influence physical activity and that a cocktail of multiple Vivo-morpholinos can be given in a single treatment to achieve protein knockdown of two different targeted proteins in skeletal muscle simultaneously. Knocking down Drd1, Vmat2, or Glut4 protein in skeletal muscle did not affect physical activity. Vivo-morpholinos injected intravenously alone did not significantly knockdown Vmat2-protein expression in the brain (p = 0.28. However, the use of a bradykinin analog to increase blood-brain-barrier permeability in conjunction with the Vivo-morpholinos significantly (p = 0.0001 decreased Vmat2-protein in the brain with a corresponding later over-expression of Vmat2 coincident with a significant (p = 0.0016 increase in physical activity. We conclude that Vivo-morpholinos can be a valuable tool in determining causal gene-phenotype relationships in whole animal models.

  7. Differentiation of breast cancer stem cells by knockdown of CD44: promising differentiation therapy

    Directory of Open Access Journals (Sweden)

    Pham Phuc V

    2011-12-01

    Full Text Available Abstract Background Breast cancer stem cells (BCSCs are the source of breast tumors. Compared with other cancer cells, cancer stem cells show high resistance to both chemotherapy and radiotherapy. Targeting of BCSCs is thus a potentially promising and effective strategy for breast cancer treatment. Differentiation therapy represents one type of cancer stem-cell-targeting therapy, aimed at attacking the stemness of cancer stem cells, thus reducing their chemo- and radioresistance. In a previous study, we showed that down-regulation of CD44 sensitized BCSCs to the anti-tumor agent doxorubicin. This study aimed to determine if CD44 knockdown caused BCSCs to differentiate into breast cancer non-stem cells (non-BCSCs. Methods We isolated a breast cancer cell population (CD44+CD24- cells from primary cultures of malignant breast tumors. These cells were sorted into four sub-populations based on their expression of CD44 and CD24 surface markers. CD44 knockdown in the BCSC population was achieved using small hairpin RNA lentivirus particles. The differentiated status of CD44 knock-down BCSCs was evaluated on the basis of changes in CD44+CD24- phenotype, tumorigenesis in NOD/SCID mice, and gene expression in relation to renewal status, metastasis, and cell cycle in comparison with BCSCs and non-BCSCs. Results Knockdown of CD44 caused BCSCs to differentiate into non-BCSCs with lower tumorigenic potential, and altered the cell cycle and expression profiles of some stem cell-related genes, making them more similar to those seen in non-BCSCs. Conclusions Knockdown of CD44 is an effective strategy for attacking the stemness of BCSCs, resulting in a loss of stemness and an increase in susceptibility to chemotherapy or radiation. The results of this study highlight a potential new strategy for breast cancer treatment through the targeting of BCSCs.

  8. Knockdown of HSPA9 induces TP53-dependent apoptosis in human hematopoietic progenitor cells.

    Directory of Open Access Journals (Sweden)

    Tuoen Liu

    Full Text Available Myelodysplastic syndromes (MDS are the most common adult myeloid blood cancers in the US. Patients have increased apoptosis in their bone marrow cells leading to low peripheral blood counts. The full complement of gene mutations that contribute to increased apoptosis in MDS remains unknown. Up to 25% of MDS patients harbor and acquired interstitial deletion on the long arm of chromosome 5 [del(5q], creating haploinsufficiency for a large set of genes including HSPA9. Knockdown of HSPA9 in primary human CD34+ hematopoietic progenitor cells significantly inhibits growth and increases apoptosis. We show here that HSPA9 knockdown is associated with increased TP53 expression and activity, resulting in increased expression of target genes BAX and p21. HSPA9 protein interacts with TP53 in CD34+ cells and knockdown of HSPA9 increases nuclear TP53 levels, providing a possible mechanism for regulation of TP53 by HSPA9 haploinsufficiency in hematopoietic cells. Concurrent knockdown of TP53 and HSPA9 rescued the increased apoptosis observed in CD34+ cells following knockdown of HSPA9. Reduction of HSPA9 below 50% results in severe inhibition of cell growth, suggesting that del(5q cells may be preferentially sensitive to further reductions of HSPA9 below 50%, thus providing a genetic vulnerability to del(5q cells. Treatment of bone marrow cells with MKT-077, an HSPA9 inhibitor, induced apoptosis in a higher percentage of cells from MDS patients with del(5q compared to non-del(5q MDS patients and normal donor cells. Collectively, these findings indicate that reduced levels of HSPA9 may contribute to TP53 activation and increased apoptosis observed in del(5q-associated MDS.

  9. Knockdown of Heparanase Suppresses Invasion of Human Trophoblasts by Activating p38 MAPK Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Guanglu Che

    2018-01-01

    Full Text Available Preeclampsia is a pregnancy-related disease with increasing maternal and perinatal morbidity and mortality worldwide. Defective trophoblast invasion is considered to be a major factor in the pathophysiological mechanism of preeclampsia. Heparanase, the only endo-β-glucuronidase in mammalian cells, has been shown to be abnormally expressed in the placenta of preeclampsia patients in our previous study. The biological role and potential mechanism of heparanase in trophoblasts remain unclear. In the present study, stably transfected HTR8/SVneo cell lines with heparanase overexpression or knockdown were constructed. The effect of heparanase on cellular proliferation, apoptosis, invasion, tube formation, and potential pathways in trophoblasts was explored. Our results showed that overexpression of heparanase promoted proliferation and invasion. Knockdown of heparanase suppressed proliferation, invasion, and tube formation but induced apoptosis. These findings reveal that downregulation of heparanase may contribute to defective placentation and plays a crucial role in the pathogenesis of preeclampsia. Furthermore, increased activation of p38 MAPK in heparanase-knockdown HTR8/SVneo cell was shown by MAPK pathway phosphorylation array and Western blotting assay. After pretreatment with 3 specific p38 MAPK inhibitors (BMS582949, SB203580, or BIRB796, inadequate invasion in heparanase-knockdown HTR8/SVneo cell was rescued. That indicates that knockdown of heparanase decreases HTR8/SVneo cell invasion through excessive activation of the p38 MAPK signaling pathway. Our study suggests that heparanase can be a potential predictive biomarker for preeclampsia at an early stage of pregnancy and represents a promising therapeutic target for the treatment of preeclampsia.

  10. Optimization of Critical Hairpin Features Allows miRNA-based Gene Knockdown Upon Single-copy Transduction

    Directory of Open Access Journals (Sweden)

    Renier Myburgh

    2014-01-01

    Full Text Available Gene knockdown using micro RNA (miRNA-based vector constructs is likely to become a prominent gene therapy approach. It was the aim of this study to improve the efficiency of gene knockdown through optimizing the structure of miRNA mimics. Knockdown of two target genes was analyzed: CCR5 and green fluorescent protein. We describe here a novel and optimized miRNA mimic design called mirGE comprising a lower stem length of 13 base pairs (bp, positioning of the targeting strand on the 5′ side of the miRNA, together with nucleotide mismatches in upper stem positions 1 and 12 placed on the passenger strand. Our mirGE proved superior to miR-30 in four aspects: yield of targeting strand incorporation into RNA-induced silencing complex (RISC; incorporation into RISC of correct targeting strand; precision of cleavage by Drosha; and ratio of targeting strand over passenger strand. A triple mirGE hairpin cassette targeting CCR5 was constructed. It allowed CCR5 knockdown with an efficiency of over 90% upon single-copy transduction. Importantly, single-copy expression of this construct rendered transduced target cells, including primary human macrophages, resistant to infection with a CCR5-tropic strain of HIV. Our results provide new insights for a better knockdown efficiency of constructs containing miRNA. Our results also provide the proof-of-principle that cells can be rendered HIV resistant through single-copy vector transduction, rendering this approach more compatible with clinical applications.

  11. In silico docking studies of aldose reductase inhibitory activity of commercially available flavonoids

    Directory of Open Access Journals (Sweden)

    Arumugam Madeswaran

    2012-12-01

    Full Text Available The primary objective of this study was to investigate the aldose reductase inhibitory activity of flavonoids using in silico docking studies. In this perspective, flavonoids like biochanin, butein, esculatin, fisetin and herbacetin were selected. Epalrestat, a known aldose reductase inhibitor was used as the standard. In silico docking studies were carried out using AutoDock 4.2, based on the Lamarckian genetic algorithm principle. The results showed that all the selected flavonoids showed binding energy ranging between -9.33 kcal/mol to -7.23 kcal/mol when compared with that of the standard (-8.73 kcal/mol. Inhibition constant (144.13 µM to 4.98 µM and intermolecular energy (-11.42 kcal/mol to -7.83 kcal/mol of the flavonoids also coincide with the binding energy. All the selected flavonoids contributed aldose reductase inhibitory activity because of its structural properties. These molecular docking analyses could lead to the further development of potent aldose reductase inhibitors for the treatment of diabetes.

  12. Crystallization and preliminary X-ray diffraction analysis of maize aldose reductase

    Energy Technology Data Exchange (ETDEWEB)

    Kiyota, Eduardo [Laboratório de Biologia Estrutural, Instituto de Química, Universidade Estadual de Campinas, CP 6154, 13083-970 Campinas-SP (Brazil); Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas-SP (Brazil); Sousa, Sylvia Morais de [Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas-SP (Brazil); Santos, Marcelo Leite dos; Costa Lima, Aline da [Laboratório de Biologia Estrutural, Instituto de Química, Universidade Estadual de Campinas, CP 6154, 13083-970 Campinas-SP (Brazil); Menossi, Marcelo [Departamento de Genética e Evolução, Instituto de Biologia, Universidade Estadual de Campinas, Campinas-SP (Brazil); Yunes, José Andrés [Laboratório de Biologia Molecular, Centro Infantil Boldrini, Campinas-SP (Brazil); Aparicio, Ricardo, E-mail: aparicio@iqm.unicamp.br [Laboratório de Biologia Estrutural, Instituto de Química, Universidade Estadual de Campinas, CP 6154, 13083-970 Campinas-SP (Brazil)

    2007-11-01

    Preliminary X-ray diffraction studies of apo maize aldose reductase at 2.0 Å resolution are reported. Maize aldose reductase (AR) is a member of the aldo-keto reductase superfamily. In contrast to human AR, maize AR seems to prefer the conversion of sorbitol into glucose. The apoenzyme was crystallized in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 47.2, b = 54.5, c = 100.6 Å and one molecule in the asymmetric unit. Synchrotron X-ray diffraction data were collected and a final resolution limit of 2.0 Å was obtained after data reduction. Phasing was carried out by an automated molecular-replacement procedure and structural refinement is currently in progress. The refined structure is expected to shed light on the functional/enzymatic mechanism and the unusual activities of maize AR.

  13. Crystallization and preliminary X-ray diffraction analysis of maize aldose reductase

    International Nuclear Information System (INIS)

    Kiyota, Eduardo; Sousa, Sylvia Morais de; Santos, Marcelo Leite dos; Costa Lima, Aline da; Menossi, Marcelo; Yunes, José Andrés; Aparicio, Ricardo

    2007-01-01

    Preliminary X-ray diffraction studies of apo maize aldose reductase at 2.0 Å resolution are reported. Maize aldose reductase (AR) is a member of the aldo-keto reductase superfamily. In contrast to human AR, maize AR seems to prefer the conversion of sorbitol into glucose. The apoenzyme was crystallized in space group P2 1 2 1 2 1 , with unit-cell parameters a = 47.2, b = 54.5, c = 100.6 Å and one molecule in the asymmetric unit. Synchrotron X-ray diffraction data were collected and a final resolution limit of 2.0 Å was obtained after data reduction. Phasing was carried out by an automated molecular-replacement procedure and structural refinement is currently in progress. The refined structure is expected to shed light on the functional/enzymatic mechanism and the unusual activities of maize AR

  14. Purification of nitrate reductase from Nicotiana plumbaginifolia by affinity chromatography using 5'AMP-sepharose and monoclonal antibodies.

    Science.gov (United States)

    Moureaux, T; Leydecker, M T; Meyer, C

    1989-02-15

    Nitrate reductase was purified from leaves of Nicotiana plumbaginifolia using either 5'AMP-Sepharose chromatography or two steps of immunoaffinity chromatography involving monoclonal antibodies directed against nitrate reductase from maize and against ribulose-1,5-bisphosphate carboxylase from N. plumbaginifolia. Nitrate reductase obtained by the first method was purified 1000-fold to a specific activity of 9 units/mg protein. The second method produced an homogenous enzyme, purified 21,000-fold to a specific activity of 80 units/mg protein. SDS/PAGE of nitrate reductase always resulted in two bands of 107 and 99.5 kDa. The 107-kDa band was the nitrate reductase subunit of N. plumbaginifolia; the smaller one of 99.5 kDa is thought, as commonly reported, to result from proteolysis of the larger protein. The molecular mass of 107 kDa is close to the values calculated from the coding sequences of the two nitrate reductase genes recently cloned from tobacco (Nicotiana tabacum cv Xanthi).

  15. ETV4 and Myeov knockdown impairs colon cancer cell line proliferation and invasion

    International Nuclear Information System (INIS)

    Moss, Alan C.; Lawlor, Garrett; Murray, David; Tighe, Donal; Madden, Stephen F.; Mulligan, Anne-Marie; Keane, Conor O.; Brady, Hugh R.; Doran, Peter P.; MacMathuna, Padraic

    2006-01-01

    We have identified novel colorectal cancer-associated genes using NCBI's UNIGENE cDNA libraries. Colon cancer libraries were examined using Digital Differential Display and disease-associated genes were selected. Among these were ETV4 and MYEOV, novel colorectal cancer-associated genes. Samples of matched normal and neoplastic colon were obtained from human subjects and gene expression was quantified using real-time PCR. ETV4 gene expression was significantly increased in colonic neoplasia in comparison to matched normal colonic tissue (p < 0.05). Myeov expression was also increased in colon neoplasia in comparison to matched normal tissue. The effect of siRNA-mediated knockdown of ETV4 and Myeov on cell proliferation and invasion was assessed. ETV4 knockdown resulted in a 90% decrease in cell proliferation (p < 0.05) and a 67% decrease in cell invasion. Myeov knockdown resulted in a 48% decrease in cell proliferation (p < 0.05) and a 36% decrease in cell invasion. These data suggest that ETV4 and Myeov may provide novel targets for therapeutic intervention

  16. Constitutive non-inducible expression of the Arabidopsis thaliana Nia 2 gene in two nitrate reductase mutants of Nicotiana plumbaginifolia.

    Science.gov (United States)

    Kaye, C; Crawford, N M; Malmberg, R L

    1997-04-01

    We have isolated a haploid cell line of N. plumbaginifolia, hNP 588, that is constitutive and not inducible for nitrate reductase. Nitrate reductase mutants were isolated from hNP 588 protoplasts upon UV irradiation. Two of these nitrate reductase-deficient cell lines, nia 3 and nia 25, neither of which contained any detectable nitrate reductase activity, were selected for complementation studies. A cloned Arabidopsis thaliana nitrate reductase gene Nia 2 was introduced into each of the two mutants resulting in 56 independent kanamycin-resistant cell lines. Thirty of the 56 kanamycin-resistant cell lines were able to grow on nitrate as the sole nitrogen source. Eight of these were further analyzed for nitrate reductase enzyme activity and nitrate reductase mRNA production. All eight lines had detectable nitrate reductase activity ranging from 7% to 150% of wild-type hNP 588 callus. The enzyme activity levels were not influenced by the nitrogen source in the medium. The eight lines examined expressed a constitutive, non-inducible 3.2 kb mRNA species that was not present in untransformed controls.

  17. Dihydrofolate reductase amplification and sensitization to methotrexate of methotrexate-resistant colon cancer cells

    DEFF Research Database (Denmark)

    Morales Torres, Christina; García, Maria J; Ribas, Maria

    2009-01-01

    Gene amplification is one of the most frequent manifestations of genomic instability in human tumors and plays an important role in tumor progression and acquisition of drug resistance. To better understand the factors involved in acquired resistance to cytotoxic drugs via gene amplification, we ...

  18. Transfection of Babesia bovis by Double Selection with WR99210 and Blasticidin-S and Its Application for Functional Analysis of Thioredoxin Peroxidase-1.

    Directory of Open Access Journals (Sweden)

    Masahito Asada

    Full Text Available Genetic manipulation is an essential technique to analyze gene function; however, limited methods are available for Babesia bovis, a causative pathogen of the globally important cattle disease, bovine babesiosis. To date, two stable transfection systems have been developed for B. bovis, using selectable markers blasticidin-S deaminase (bsd or human dihydrofolate reductase (hdhfr. In this work, we combine these two selectable markers in a sequential transfection system. Specifically, a parent transgenic B. bovis line which episomally expresses green fluorescent protein (GFP and human dihydrofolate reductase (hDHFR, was transfected with a plasmid encoding a fusion protein consisting of red fluorescent protein (RFP and blasticidin-S deaminase (BSD. Selection with WR99210 and blasticidin-S resulted in the emergence of parasites double positive for GFP and RFP. We then applied this method to complement gene function in a parasite line in which thioredoxin peroxidase-1 (Bbtpx-1 gene was knocked out using hDHFR as a selectable marker. A plasmid was constructed harboring both RFP-BSD and Bbtpx-1 expression cassettes, and transfected into a Bbtpx-1 knockout (KO parasite. Transfectants were independently obtained by two transfection methods, episomal transfection and genome integration. Complementation of Bbtpx-1 resulted in full recovery of resistance to nitrosative stress, via the nitric oxide donor sodium nitroprusside, which was impaired in the Bbtpx-1 KO parasites. In conclusion, we developed a sequential transfection method in B. bovis and subsequently applied this technique in a gene complementation study. This method will enable broader genetic manipulation of Babesia toward enhancing our understanding of the biology of this parasite.

  19. Factors That Cause Trimethoprim Resistance in Streptococcus pyogenes

    Science.gov (United States)

    Bergmann, René; van der Linden, Mark; Chhatwal, Gursharan S.

    2014-01-01

    The use of trimethoprim in treatment of Streptococcus pyogenes infections has long been discouraged because it has been widely believed that this pathogen is resistant to this antibiotic. To gain more insight into the extent and molecular basis of trimethoprim resistance in S. pyogenes, we tested isolates from India and Germany and sought the factors that conferred the resistance. Resistant isolates were identified in tests for trimethoprim or trimethoprim-sulfamethoxazole (SXT) susceptibility. Resistant isolates were screened for the known horizontally transferable trimethoprim-insensitive dihydrofolate reductase (dfr) genes dfrG, dfrF, dfrA, dfrD, and dfrK. The nucleotide sequence of the intrinsic dfr gene was determined for resistant isolates lacking the horizontally transferable genes. Based on tentative criteria, 69 out of 268 isolates (25.7%) from India were resistant to trimethoprim. Occurring in 42 of the 69 resistant isolates (60.9%), dfrF appeared more frequently than dfrG (23 isolates; 33.3%) in India. The dfrF gene was also present in a collection of SXT-resistant isolates from Germany, in which it was the only detected trimethoprim resistance factor. The dfrF gene caused resistance in 4 out of 5 trimethoprim-resistant isolates from the German collection. An amino acid substitution in the intrinsic dihydrofolate reductase known from trimethoprim-resistant Streptococcus pneumoniae conferred resistance to S. pyogenes isolates of emm type 102.2, which lacked other aforementioned dfr genes. Trimethoprim may be more useful in treatment of S. pyogenes infections than previously thought. However, the factors described herein may lead to the rapid development and spread of resistance of S. pyogenes to this antibiotic agent. PMID:24492367

  20. Knockdown of Pokemon protein expression inhibits hepatocellular carcinoma cell proliferation by suppression of AKT activity.

    Science.gov (United States)

    Zhu, Xiaosan; Dai, Yichen; Chen, Zhangxin; Xie, Junpei; Zeng, Wei; Lin, Yuanyuan

    2013-01-01

    Overexpression of Pokemon, which is an erythroid myeloid ontogenic factor protein, occurs in different cancers, including hepatocellular carcinoma (HCC). Pokemon is also reported to have an oncogenic activity in various human cancers. This study investigated the effect of Pokemon knockdown on the regulation of HCC growth. POK shRNA suppressed the expression of Pokemon protein in HepG2 cells compared to the negative control vector-transfected HCC cells. Pokemon knockdown also reduced HCC cell viability and enhanced cisplatin-induced apoptosis in HCC cells. AKT activation and the expression of various cell cycle-related genes were inhibited following Pokemon knockdown. These data demonstrate that Pokemon may play a role in HCC progression, suggesting that inhibition of Pokemon expression using Pokemon shRNA should be further evaluated as a novel target for the control of HCC.

  1. Overexpression of chloroplast NADPH-dependent thioredoxin reductase in Arabidopsis enhances leaf growth and elucidates in-vivo function of reductase and thioredoxin domains

    Directory of Open Access Journals (Sweden)

    Jouni eToivola

    2013-10-01

    Full Text Available Plant chloroplasts have versatile thioredoxin systems including two thioredoxin reductases and multiple types of thioredoxins. Plastid-localized NADPH-dependent thioredoxin reductase (NTRC contains both reductase (NTRd and thioredoxin (TRXd domains in a single polypeptide and forms homodimers. To study the action of NTRC and NTRC domains in vivo, we have complemented the ntrc knockout line of Arabidopsis with the wild type and full-length NTRC genes, in which 2-Cys motifs either in NTRd, or in TRXd were inactivated. The ntrc line was also transformed either with the truncated NTRd or TRXd alone. Overexpression of wild-type NTRC promoted plant growth by increasing leaf size and biomass yield of the rosettes. Complementation of the ntrc line with the full-length NTRC gene containing an active reductase but an inactive thioredoxin domain, or vice versa, recovered wild-type chloroplast phenotype and, partly, rosette biomass production, indicating that the NTRC domains are capable of interacting with other chloroplast thioredoxin systems. Overexpression of truncated NTRd or TRXd in ntrc background did not restore wild-type phenotype. Modelling of the 3-dimensional structure of the NTRC dimer indicates extensive interactions between the NTR domains and the TRX domains further stabilize the dimeric structure. The long linker region between the NTRd and TRXd, however, allows flexibility for the position of the TRXd in the dimer. Supplementation of the TRXd in the NTRC homodimer model by free chloroplast thioredoxins indicated that TRXf is the most likely partner to interact with NTRC. We propose that overexpression of NTRC promotes plant biomass yield both directly by stimulation of chloroplast biosynthetic and protected pathways controlled by NTRC and indirectly via free chloroplast thioredoxins. Our data indicate that overexpression of chloroplast thiol redox-regulator has a potential to increase biofuel yield in plant and algal species suitable for

  2. The structure of Lactococcus lactis thioredoxin reductase reveals molecular features of photo-oxidative damage

    DEFF Research Database (Denmark)

    Skjoldager, Nicklas; Bang, Maria Blanner; Rykær, Martin

    2017-01-01

    The NADPH-dependent homodimeric flavoenzyme thioredoxin reductase (TrxR) provides reducing equivalents to thioredoxin, a key regulator of various cellular redox processes. Crystal structures of photo-inactivated thioredoxin reductase (TrxR) from the Gram-positive bacterium Lactococcus lactis have...

  3. Characterization and regulation of Leishmania major 3-hydroxy-3-methylglutaryl-CoA reductase

    DEFF Research Database (Denmark)

    Montalvetti, A; Pena Diaz, Javier; Hurtado, R

    2000-01-01

    In eukaryotes the enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyses the synthesis of mevalonic acid, a common precursor to all isoprenoid compounds. Here we report the isolation and overexpression of the gene coding for HMG-CoA reductase from Leishmania major. The protein from L...

  4. Methylenetetrahydrofolate reductase gene polymorphism in type 1 ...

    African Journals Online (AJOL)

    In patients with type-I diabetes mellitus folate deficiency is associated with endothelial dysfunction. So, polymorphism in genes involved in folate metabolism may have a role in vascular disease. This study was designed to evaluate the relationship between methylenetetrahydrofolate reductase (MTHFR) gene polymorphism ...

  5. Characterization of human warfarin reductase

    OpenAIRE

    Sokolová, Simona

    2016-01-01

    Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Simona Sokolová Supervisor: PharmDr. Petra Malátková, Ph.D. Title of diploma thesis: Characterization of human warfarin reductase Warfarin is widely used anticoagulant drug. Considering the narrow therapeutic window of warfarin, it is important to fully understand its metabolism in human body. Oxidative, reductive and conjugation reactions are involved in warfarin metabolism. Howev...

  6. Synthesis, antimalarial activity and molecular docking of hybrid 4-aminoquinoline-1,3,5-triazine derivatives.

    Science.gov (United States)

    Bhat, Hans Raj; Singh, Udaya Pratap; Thakur, Anjali; Kumar Ghosh, Surajit; Gogoi, Kabita; Prakash, Anil; Singh, Ramendra K

    2015-10-01

    A series of novel hybrid 4-aminoquinoline 1,3,5-triazine derivatives was synthesized in a five-steps reaction and evaluated for their in vitro antimalarial activity against chloroquine-sensitive (3D7) and chloroquine-resistant (RKL-2) strains of Plasmodium falciparum. Entire synthetic derivatives showed higher antimalarial activity on the sensitive strain while two compounds, viz., 9a and 9c displayed good activity against both the strains of P. falciparum. The observed activity was further substantiated by docking study on both wild and qradruple mutant type P. falciparum dihydrofolate reductase-thymidylate synthase (pf-DHFR-TS). Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Comparison of molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) and molecular mechanics-three-dimensional reference interaction site model (MM-3D-RISM) method to calculate the binding free energy of protein-ligand complexes: Effect of metal ion and advance statistical test

    Science.gov (United States)

    Pandey, Preeti; Srivastava, Rakesh; Bandyopadhyay, Pradipta

    2018-03-01

    The relative performance of MM-PBSA and MM-3D-RISM methods to estimate the binding free energy of protein-ligand complexes is investigated by applying these to three proteins (Dihydrofolate Reductase, Catechol-O-methyltransferase, and Stromelysin-1) differing in the number of metal ions they contain. None of the computational methods could distinguish all the ligands based on their calculated binding free energies (as compared to experimental values). The difference between the two comes from both polar and non-polar part of solvation. For charged ligand case, MM-PBSA and MM-3D-RISM give a qualitatively different result for the polar part of solvation.

  8. Dual knockdown of N-ras and epiregulin synergistically suppressed the growth of human hepatoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Meng; He, Hong-wei; Sun, Huan-xing; Ren, Kai-huan [Department of Oncology, Institute of Medicinal Biotechnology, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100050 (China); Shao, Rong-guang, E-mail: shaor@bbn.cn [Department of Oncology, Institute of Medicinal Biotechnology, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100050 (China)

    2009-09-18

    Hepatocellular carcinoma (HCC) is a major challenge because of its resistance to conventional cytotoxic chemotherapy and radiotherapy. Multi-targeted therapy might be a new option for HCC treatment. Our previous study showed that N-ras gene was activated in HCC and was inhibited by RNA interference. In the present study, we investigated the alternation of gene expression by microarray in N-Ras-siRNA-treated HepG2 cells. The results revealed that the EREG gene, encoding epiregulin, was dramatically up-regulated in response to silence of N-ras. We speculated that the up-regulation of epiregulin was involved in the compensatory mechanism of N-ras knockdown for cell growth. Therefore, we evaluated whether dual silence of N-ras and epiregulin display a greater suppression of cell growth. The results confirmed that dual knockdown of N-ras and epiregulin synergistically inhibited cell growth. Our results also showed that dual knockdown of N-ras and epiregulin significantly induced cell arrest at G0/G1 phase. Furthermore, Western blot assay showed that dual knockdown of N-ras and epiregulin markedly reduced the phosphorylations of ERK1/2, Akt and Rb, and inhibited the expression of cyclin D1. Our findings imply that multi-targeted silence of oncogenes might be an effective treatment for HCC.

  9. Inhibitory effect of rhetsinine isolated from Evodia rutaecarpa on aldose reductase activity.

    Science.gov (United States)

    Kato, A; Yasuko, H; Goto, H; Hollinshead, J; Nash, R J; Adachi, I

    2009-03-01

    Aldose reductase inhibitors have considerable potential for the treatment of diabetic complications, without increased risk of hypoglycemia. Search for components inhibiting aldose reductase led to the discovery of active compounds contained in Evodia rutaecarpa Bentham (Rutaceae), which is the one of the component of Kampo-herbal medicine. The hot water extract from the E. rutaecarpa was subjected to distribution or gel filtration chromatography to give an active compound, N2-(2-methylaminobenzoyl)tetrahydro-1H-pyrido[3,4-b]indol-1-one (rhetsinine). It inhibited aldose reductase with IC(50) values of 24.1 microM. Furthermore, rhetsinine inhibited sorbitol accumulation by 79.3% at 100 microM. These results suggested that the E. rutaecarpa derived component, rhetsinine, would be potentially useful in the treatment of diabetic complications.

  10. Stable knockdown of Kif5b in MDCK cells leads to epithelial–mesenchymal transition

    International Nuclear Information System (INIS)

    Cui, Ju; Jin, Guoxiang; Yu, Bin; Wang, Zai; Lin, Raozhou; Huang, Jian-Dong

    2015-01-01

    Polarization of epithelial cells requires vectorial sorting and transport of polarity proteins to apical or basolateral domains. Kif5b is the mouse homologue of the human ubiquitous Kinesin Heavy Chain (uKHC). To investigate the function of Kif5b in epithelial cells, we examined the phenotypes of Kif5b-deficient MDCK cells. Stable knockdown of Kif5b in MDCK cells resulted in reduced cell proliferation rate, profound changes in cell morphology, loss of epithelial cell marker, and gain of mesenchymal marker, as well as increased cell migration, invasion, and tumorigenesis abilities. E-cadherin and NMMIIA could interact with Kif5b in polarized MDCK cells, and their expression levels were decreased in Kif5b-deficient MDCK cells. Overexpression of E-cadherin and NMMIIA in Kif5b depleted MDCK cells could decrease mesenchymal marker expression and cell migration ability. These results indicate that stable knockdown of Kif5b in MDCK cells can lead to epithelial–mesenchymal transition, which is mediated by defective E-cadherin and NMMIIA expression. - Highlights: • Knockdown of Kif5b in MDCK cells resulted in reduced cell proliferation rate. • Kif5b deficient MDCK cells underwent epithelial–mesenchymal transition. • E-cadherin and NMMIIA could interact with Kif5b in polarized MDCK cells. • Decreased E-cadherin and NMMIIA levels mediate EMT in Kif5b deficient MDCK cells. • Overexpression of E-cadherin and NMMIIA reverse the effects of Kif5b knockdown

  11. Stable knockdown of Kif5b in MDCK cells leads to epithelial–mesenchymal transition

    Energy Technology Data Exchange (ETDEWEB)

    Cui, Ju, E-mail: juzi.cui@gmail.com [The Key Laboratory of Geriatrics, Beijing Hospital & Beijing Institute of Geriatrics, Ministry of Health, Beijing (China); Department of Biochemistry, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR (China); Jin, Guoxiang; Yu, Bin [Department of Biochemistry, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR (China); Wang, Zai [Department of Biochemistry, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR (China); Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Beijing (China); Lin, Raozhou [Department of Biochemistry, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR (China); Huang, Jian-Dong, E-mail: jdhuang@hku.hk [Department of Biochemistry, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR (China); The Centre for Synthetic Biology Engineering Research, Shenzhen Institutes of Advanced Technology, Shenzhen (China)

    2015-07-17

    Polarization of epithelial cells requires vectorial sorting and transport of polarity proteins to apical or basolateral domains. Kif5b is the mouse homologue of the human ubiquitous Kinesin Heavy Chain (uKHC). To investigate the function of Kif5b in epithelial cells, we examined the phenotypes of Kif5b-deficient MDCK cells. Stable knockdown of Kif5b in MDCK cells resulted in reduced cell proliferation rate, profound changes in cell morphology, loss of epithelial cell marker, and gain of mesenchymal marker, as well as increased cell migration, invasion, and tumorigenesis abilities. E-cadherin and NMMIIA could interact with Kif5b in polarized MDCK cells, and their expression levels were decreased in Kif5b-deficient MDCK cells. Overexpression of E-cadherin and NMMIIA in Kif5b depleted MDCK cells could decrease mesenchymal marker expression and cell migration ability. These results indicate that stable knockdown of Kif5b in MDCK cells can lead to epithelial–mesenchymal transition, which is mediated by defective E-cadherin and NMMIIA expression. - Highlights: • Knockdown of Kif5b in MDCK cells resulted in reduced cell proliferation rate. • Kif5b deficient MDCK cells underwent epithelial–mesenchymal transition. • E-cadherin and NMMIIA could interact with Kif5b in polarized MDCK cells. • Decreased E-cadherin and NMMIIA levels mediate EMT in Kif5b deficient MDCK cells. • Overexpression of E-cadherin and NMMIIA reverse the effects of Kif5b knockdown.

  12. Promising Noninvasive Cellular Phenotype in Prostate Cancer Cells Knockdown of Matrix Metalloproteinase 9

    Directory of Open Access Journals (Sweden)

    Aditi Gupta

    2013-01-01

    Full Text Available Cell surface interaction of CD44 and MMP9 increases migration and invasion of PC3 cells. We show here that stable knockdown of MMP9 in PC3 cells switches CD44 isoform expression from CD44s to CD44v6 which is more glycosylated. These cells showed highly adhesive morphology with extensive cell spreading which is due to the formation of focal adhesions and well organized actin-stress fibers. MMP9 knockdown blocks invadopodia formation and matrix degradation activity as well. However, CD44 knockdown PC3 cells failed to develop focal adhesions and stress fibers; hence these cells make unstable adhesions. A part of the reason for these changes could be caused by silencing of CD44v6 as well. Immunostaining of prostate tissue microarray sections illustrated significantly lower levels of CD44v6 in adenocarcinoma than normal tissue. Our results suggest that interaction between CD44 and MMP9 is a potential mechanism of invadopodia formation. CD44v6 expression may be essential for the protection of non-invasive cellular phenotype. CD44v6 decrease may be a potential marker for prognosis and therapeutics.

  13. 21 CFR 864.7375 - Glutathione reductase assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375 Glutathione...

  14. Knockdown of CDK2AP1 in human embryonic stem cells reduces the threshold of differentiation.

    Directory of Open Access Journals (Sweden)

    Khaled N Alsayegh

    Full Text Available Recent studies have suggested a role for the Cyclin Dependent Kinase-2 Associated Protein 1 (CDK2AP1 in stem cell differentiation and self-renewal. In studies with mouse embryonic stem cells (mESCs derived from generated mice embryos with targeted deletion of the Cdk2ap1 gene, CDK2AP1 was shown to be required for epigenetic silencing of Oct4 during differentiation, with deletion resulting in persistent self-renewal and reduced differentiation potential. Differentiation capacity was restored in these cells following the introduction of a non-phosphorylatible form of the retinoblastoma protein (pRb or exogenous Cdk2ap1. In this study, we investigated the role of CDK2AP1 in human embryonic stem cells (hESCs. Using a shRNA to reduce its expression in hESCs, we found that CDK2AP1 knockdown resulted in a significant reduction in the expression of the pluripotency genes, OCT4 and NANOG. We also found that CDK2AP1 knockdown increased the number of embryoid bodies (EBs formed when differentiation was induced. In addition, the generated EBs had significantly higher expression of markers of all three germ layers, indicating that CDK2AP1 knockdown enhanced differentiation. CDK2AP1 knockdown also resulted in reduced proliferation and reduced the percentage of cells in the S phase and increased cells in the G2/M phase of the cell cycle. Further investigation revealed that a higher level of p53 protein was present in the CDK2AP1 knockdown hESCs. In hESCs in which p53 and CDK2AP1 were simultaneously downregulated, OCT4 and NANOG expression was not affected and percentage of cells in the S phase of the cell cycle was not reduced. Taken together, our results indicate that the knockdown of CDK2AP1 in hESCs results in increased p53 and enhances differentiation and favors it over a self-renewal fate.

  15. Characterisation of a desmosterol reductase involved in phytosterol dealkylation in the silkworm, Bombyx mori.

    Directory of Open Access Journals (Sweden)

    Leonora F Ciufo

    Full Text Available Most species of invertebrate animals cannot synthesise sterols de novo and many that feed on plants dealkylate phytosterols (mostly C(29 and C(28 yielding cholesterol (C(27. The final step of this dealkylation pathway involves desmosterol reductase (DHCR24-catalysed reduction of desmosterol to cholesterol. We now report the molecular characterisation in the silkworm, Bombyx mori, of such a desmosterol reductase involved in production of cholesterol from phytosterol, rather than in de novo synthesis of cholesterol. Phylogenomic analysis of putative desmosterol reductases revealed the occurrence of various clades that allowed for the identification of a strong reductase candidate gene in Bombyx mori (BGIBMGA 005735. Following PCR-based cloning of the cDNA (1.6 kb and its heterologous expression in Saccharomyces cerevisae, the recombinant protein catalysed reduction of desmosterol to cholesterol in an NADH- and FAD-dependent reaction.Conceptual translation of the cDNA, that encodes a 58.9 kDa protein, and database searching, revealed that the enzyme belongs to an FAD-dependent oxidoreductase family. Western blotting revealed reductase protein expression exclusively in the microsomal subcellular fraction and primarily in the gut. The protein is peripherally associated with microsomal membranes. 2D-native gel and PAGE analysis revealed that the reductase is part of a large complex with molecular weight approximately 250 kDa. The protein occurs in midgut microsomes at a fairly constant level throughout development in the last two instars, but is drastically reduced during the wandering stage in preparation for metamorphosis. Putative Broad Complex transcription factor-binding sites detectable upstream of the DHCR24 gene may play a role in this down-regulation.

  16. Characterisation of a Desmosterol Reductase Involved in Phytosterol Dealkylation in the Silkworm, Bombyx mori

    Science.gov (United States)

    Ciufo, Leonora F.; Murray, Patricia A.; Thompson, Anu; Rigden, Daniel J.; Rees, Huw H.

    2011-01-01

    Most species of invertebrate animals cannot synthesise sterols de novo and many that feed on plants dealkylate phytosterols (mostly C29 and C28) yielding cholesterol (C27). The final step of this dealkylation pathway involves desmosterol reductase (DHCR24)-catalysed reduction of desmosterol to cholesterol. We now report the molecular characterisation in the silkworm, Bombyx mori, of such a desmosterol reductase involved in production of cholesterol from phytosterol, rather than in de novo synthesis of cholesterol. Phylogenomic analysis of putative desmosterol reductases revealed the occurrence of various clades that allowed for the identification of a strong reductase candidate gene in Bombyx mori (BGIBMGA 005735). Following PCR-based cloning of the cDNA (1.6 kb) and its heterologous expression in Saccharomyces cerevisae, the recombinant protein catalysed reduction of desmosterol to cholesterol in an NADH- and FAD- dependent reaction. Conceptual translation of the cDNA, that encodes a 58.9 kDa protein, and database searching, revealed that the enzyme belongs to an FAD-dependent oxidoreductase family. Western blotting revealed reductase protein expression exclusively in the microsomal subcellular fraction and primarily in the gut. The protein is peripherally associated with microsomal membranes. 2D-native gel and PAGE analysis revealed that the reductase is part of a large complex with molecular weight approximately 250kDa. The protein occurs in midgut microsomes at a fairly constant level throughout development in the last two instars, but is drastically reduced during the wandering stage in preparation for metamorphosis. Putative Broad Complex transcription factor-binding sites detectable upstream of the DHCR24 gene may play a role in this down-regulation. PMID:21738635

  17. Inhibition of human anthracycline reductases by emodin — A possible remedy for anthracycline resistance

    International Nuclear Information System (INIS)

    Hintzpeter, Jan; Seliger, Jan Moritz; Hofman, Jakub; Martin, Hans-Joerg; Wsol, Vladimir; Maser, Edmund

    2016-01-01

    The clinical application of anthracyclines, like daunorubicin and doxorubicin, is limited by two factors: dose-related cardiotoxicity and drug resistance. Both have been linked to reductive metabolism of the parent drug to their metabolites daunorubicinol and doxorubicinol, respectively. These metabolites show significantly less anti-neoplastic properties as their parent drugs and accumulate in cardiac tissue leading to chronic cardiotoxicity. Therefore, we aimed to identify novel and potent natural inhibitors for anthracycline reductases, which enhance the anticancer effect of anthracyclines by preventing the development of anthracycline resistance. Human enzymes responsible for the reductive metabolism of daunorubicin were tested for their sensitivity towards anthrachinones, in particular emodin and anthraflavic acid. Intense inhibition kinetic data for the most effective daunorubicin reductases, including IC 50 - and K i -values, the mode of inhibition, as well as molecular docking, were compiled. Subsequently, a cytotoxicity profile and the ability of emodin to reverse daunorubicin resistance were determined using multiresistant A549 lung cancer and HepG2 liver cancer cells. Emodin potently inhibited the four main human daunorubicin reductases in vitro. Further, we could demonstrate that emodin is able to synergistically sensitize human cancer cells towards daunorubicin at clinically relevant concentrations. Therefore, emodin may yield the potential to enhance the therapeutic effectiveness of anthracyclines by preventing anthracycline resistance via inhibition of the anthracycline reductases. In symphony with its known pharmacological properties, emodin might be a compound of particular interest in the management of anthracycline chemotherapy efficacy and their adverse effects. - Highlights: • Natural and synthetic compounds were identified as inhibitors for human daunorubicin reductases. • Emodin is a potent inhibitor for human daunorubicin reductases.

  18. Colour formation in fermented sausages by meat-associated staphylococci with different nitrite- and nitrate-reductase activities

    DEFF Research Database (Denmark)

    Gøtterup, Jacob; Olsen, Karsten; Knøchel, Susanne

    2008-01-01

    nitrate depended on the specific Staphylococcus strain. Strains with high nitrate-reductase activity showed a significantly faster rate of pigment formation, but other factors were of influence as well. Product stability for the sliced, packaged sausage was evaluated as surface colour and oxidation......Three Staphylococcus strains, S. carnosus, S. simulans and S. saprophyticus, selected due to their varying nitrite and/or nitrate-reductase activities, were used to initiate colour formation during sausage fermentation. During fermentation of sausages with either nitrite or nitrate added, colour...... with hexanal content, and may be used as predictive tools. Overall, nitrite- and nitrate-reductase activities of Staphylococcus strains in nitrite-cured sausages were of limited importance regarding colour development, while in nitrate-cured sausages strains with higher nitrate reductase activity were crucial...

  19. Knockdown of angiopoietin-like 2 mimics the benefits of intermittent fasting on insulin responsiveness and weight loss.

    Science.gov (United States)

    Martel, Cécile; Pinçon, Anthony; Bélanger, Alexandre Maxime; Luo, Xiaoyan; Gillis, Marc-Antoine; de Montgolfier, Olivia; Thorin-Trescases, Nathalie; Thorin, Éric

    2018-01-01

    Angiopoietin-like 2 (ANGPTL2) is an inflammatory adipokine linking obesity to insulin resistance. Intermittent fasting, on the other hand, is a lifestyle intervention able to prevent obesity and diabetes but difficult to implement and maintain. Our objectives were to characterize a link between ANGPTL2 and intermittent fasting and to investigate whether the knockdown of ANGPTL2 reproduces the benefits of intermittent fasting on weight gain and insulin responsiveness in knockdown and wild-type littermates mice. Intermittent fasting, access to food ad libitum once every other day, was initiated at the age of three months and maintained for four months. Intermittent fasting decreased by 63% (p < 0.05) gene expression of angptl2 in adipose tissue of wild-type mice. As expected, intermittent fasting improved insulin sensitivity (p < 0.05) and limited weight gain (p < 0.05) in wild-type mice. Knockdown mice fed ad libitum, however, were comparable to wild-type mice following the intermittent fasting regimen: insulin sensitivity and weight gain were identical, while intermittent fasting had no additional impact on these parameters in knockdown mice. Energy intake was similar between both wild-type fed intermittent fasting and ANGPTL2 knockdown mice fed ad libitum, suggesting that intermittent fasting and knockdown of ANGPTL2 equally lower feeding efficiency. These results suggest that the reduction of ANGPTL2 could be a useful and promising strategy to prevent obesity and insulin resistance, although further investigation of the mechanisms linking ANGPTL2 and intermittent fasting is warranted. Impact statement Intermittent fasting is an efficient diet pattern to prevent weight gain and improve insulin sensitivity. It is, however, a difficult regimen to follow and compliance is expected to be very low. In this work, we demonstrate that knockdown of ANGPTL2 in mice fed ad libitum mimics the beneficial effects of intermittent fasting on weight gain and insulin

  20. Aldose Reductase Inhibitory and Antiglycation Activities of Four ...

    African Journals Online (AJOL)

    Aldose Reductase Inhibitory and Antiglycation Activities of Four Medicinal Plant Standardized Extracts and Their Main Constituents for the Prevention of ... levels in galactosemic condition by using reverse phase high pressure liquid chromatography (RP-HPLC) and gas liquid chromatography (GLC) was determined.

  1. Xylose reductase from the thermophilic fungus Talaromyces emersonii

    Indian Academy of Sciences (India)

    Prakash

    Xylose reductase is involved in the first step of the fungal pentose catabolic pathway. The gene .... proteins with reversed coenzyme preference from NADPH to NADH ..... 399–404. Hasper A A, Visser J and de Graaff L H 2000 The Aspergillus.

  2. Lentivirus-mediated Knockdown of HDAC1 Uncovers Its Role in Esophageal Cancer Metastasis and Chemosensitivity

    OpenAIRE

    Song, Min; He, Gang; Wang, Yan; Pang, Xueli; Zhang, Bo

    2016-01-01

    Histone deacetylationase 1 (HDAC1) is ubiquitously expressed in various cell lines and tissues and play an important role of regulation gene expression. Overexpression of HDAC1 has been observed in various types of cancers, which indicated that it might be a target for cancer therapy. To test HDAC1 inhibition for cancer treatment, the gene expression of HDAC1 was knockdown mediated by a lentivirus system. Our data showed the gene expression of HDAC1 could be efficiently knockdown by RNAi medi...

  3. Regulation of schistosome egg production by HMG CoA reductase

    International Nuclear Information System (INIS)

    VandeWaa, E.A.; Bennett, J.L.

    1986-01-01

    Hydroxymethylglutaryl coenzyme A reductase (HMG CoA reductase) catalyzes the conversion of HMG CoA to mevalonate in the synthesis of steroids, isoprenoids and terpenes. Mevinolin, an inhibitor of this enzyme, decreased egg production in Schistosoma mansoni during in vitro incubations. This was associated with a reduction in the incorporation of 14 C-acetate into polyisoprenoids and a reduction in the formation of a lipid-linked oligosaccharide. In vivo, mevinolin in daily doses of 50 mg/kg (p.o., from days 30-48 post-infection) caused no change in gross liver pathology in S. mansoni infected mice. However, when parasites exposed to mevinolin or its vehicle in vivo were cultured in vitro, worms from mevinolin-treated mice produced six times more eggs than control parasites. When infected mice were dosed with 250 mg/kg mevinolin daily (p.o., from days 35-45 post-infection), liver pathology was reduced in comparison to control mice. Thus, during in vivo exposure to a high dose of the drug egg production is decreased, while at a lower dose it appears unaffected until the parasites are cultured in a drug-free in vitro system wherein egg production is stimulated to extraordinarily high levels. It may be that at low doses mevinolin, by inhibiting the enzyme, is blocking the formation of a product (such as an isoprenoid) which normally acts to down-regulate enzyme synthesis, resulting in enzyme induction. Induction of HMG CoA reductase is then expressed as increased egg production when the worms are removed from the drug. These data suggest that HMG CoA reductase plays a role in schistosome egg production

  4. Goat activin receptor type IIB knockdown by muscle specific promoter driven artificial microRNAs.

    Science.gov (United States)

    Patel, Amrutlal K; Shah, Ravi K; Patel, Utsav A; Tripathi, Ajai K; Joshi, Chaitanya G

    2014-10-10

    Activin receptor type IIB (ACVR2B) is a transmembrane receptor which mediates signaling of TGF beta superfamily ligands known to function in regulation of muscle mass, embryonic development and reproduction. ACVR2B antagonism has shown to enhance the muscle growth in several disease and transgenic models. Here, we show ACVR2B knockdown by RNA interference using muscle creatine kinase (MCK) promoter driven artificial microRNAs (amiRNAs). Among the various promoter elements tested, the ∼1.26 kb MCK promoter region showed maximum transcriptional activity in goat myoblasts cells. We observed up to 20% silencing in non-myogenic 293T cells and up to 32% silencing in myogenic goat myoblasts by MCK directed amiRNAs by transient transfection. Goat myoblasts stably integrated with MCK directed amiRNAs showed merely 8% silencing in proliferating myoblasts which was increased to 34% upon induction of differentiation at transcript level whereas up to 57% silencing at protein level. Knockdown of ACVR2B by 5'-UTR derived amiRNAs resulted in decreased SMAD2/3 signaling, increased expression of myogenic regulatory factors (MRFs) and enhanced proliferation and differentiation of myoblasts. Unexpectedly, knockdown of ACVR2B by 3'-UTR derived amiRNAs resulted in increased SMAD2/3 signaling, reduced expression of MRFs and suppression of myogenesis. Our study offers muscle specific knockdown of ACVR2B as a potential strategy to enhance muscle mass in the farm animal species. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. RNAi-mediated double gene knockdown and gustatory perception measurement in honey bees (Apis mellifera).

    Science.gov (United States)

    Wang, Ying; Baker, Nicholas; Amdam, Gro V

    2013-07-25

    This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception. RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species. The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.

  6. Inhibition of aldose reductase activity by Cannabis sativa chemotypes extracts with high content of cannabidiol or cannabigerol.

    Science.gov (United States)

    Smeriglio, Antonella; Giofrè, Salvatore V; Galati, Enza M; Monforte, Maria T; Cicero, Nicola; D'Angelo, Valeria; Grassi, Gianpaolo; Circosta, Clara

    2018-02-07

    Aldose reductase (ALR2) is a key enzyme involved in diabetic complications and the search for new aldose reductase inhibitors (ARIs) is currently very important. The synthetic ARIs are often associated with deleterious side effects and medicinal and edible plants, containing compounds with aldose reductase inhibitory activity, could be useful for prevention and therapy of diabetic complications. Non-psychotropic phytocannabinoids exert multiple pharmacological effects with therapeutic potential in many diseases such as inflammation, cancer, diabetes. Here, we have investigated the inhibitory effects of extracts and their fractions from two Cannabis sativa L. chemotypes with high content of cannabidiol (CBD)/cannabidiolic acid (CBDA) and cannabigerol (CBG)/cannabigerolic acid (CBGA), respectively, on human recombinant and pig kidney aldose reductase activity in vitro. A molecular docking study was performed to evaluate the interaction of these cannabinoids with the active site of ALR2 compared to known ARIs. The extracts showed significant dose-dependent aldose reductase inhibitory activity (>70%) and higher than fractions. The inhibitory activity of the fractions was greater for acidic cannabinoid-rich fractions. Comparative molecular docking results have shown a higher stability of the ALR2-cannabinoid acids complex than the other inhibitors. The extracts of Cannabis with high content of non-psychotropic cannabinoids CBD/CBDA or CBG/CBGA significantly inhibit aldose reductase activity. These results may have some relevance for the possible use of C. sativa chemotypes based preparations as aldose reductase inhibitors. Copyright © 2018 Elsevier B.V. All rights reserved.

  7. Mitochondrial fumarate reductase as a target of chemotherapy: from parasites to cancer cells.

    Science.gov (United States)

    Sakai, Chika; Tomitsuka, Eriko; Esumi, Hiroyasu; Harada, Shigeharu; Kita, Kiyoshi

    2012-05-01

    Recent research on respiratory chain of the parasitic helminth, Ascaris suum has shown that the mitochondrial NADH-fumarate reductase system (fumarate respiration), which is composed of complex I (NADH-rhodoquinone reductase), rhodoquinone and complex II (rhodoquinol-fumarate reductase) plays an important role in the anaerobic energy metabolism of adult parasites inhabiting hosts. The enzymes in these parasite-specific pathways are potential target for chemotherapy. We isolated a novel compound, nafuredin, from Aspergillus niger, which inhibits NADH-fumarate reductase in helminth mitochondria at nM order. It competes for the quinone-binding site in complex I and shows high selective toxicity to the helminth enzyme. Moreover, nafuredin exerts anthelmintic activity against Haemonchus contortus in in vivo trials with sheep indicating that mitochondrial complex I is a promising target for chemotherapy. In addition to complex I, complex II is a good target because its catalytic direction is reverse of succinate-ubiquionone reductase in the host complex II. Furthermore, we found atpenin and flutolanil strongly and specifically inhibit mitochondrial complex II. Interestingly, fumarate respiration was found not only in the parasites but also in some types of human cancer cells. Analysis of the mitochondria from the cancer cells identified an anthelminthic as a specific inhibitor of the fumarate respiration. Role of isoforms of human complex II in the hypoxic condition of cancer cells and fetal tissues is a challenge. This article is part of a Special Issue entitled Biochemistry of Mitochondria, Life and Intervention 2010. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. Inhibition of human anthracycline reductases by emodin — A possible remedy for anthracycline resistance

    Energy Technology Data Exchange (ETDEWEB)

    Hintzpeter, Jan, E-mail: hintzpeter@toxi.uni-kiel.de [Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, Campus Kiel, Brunswiker Str. 10, 24105 Kiel (Germany); Seliger, Jan Moritz [Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, Campus Kiel, Brunswiker Str. 10, 24105 Kiel (Germany); Hofman, Jakub [Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 50005 Hradec Kralove (Czech Republic); Martin, Hans-Joerg [Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, Campus Kiel, Brunswiker Str. 10, 24105 Kiel (Germany); Wsol, Vladimir [Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 50005 Hradec Kralove (Czech Republic); Maser, Edmund [Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, Campus Kiel, Brunswiker Str. 10, 24105 Kiel (Germany)

    2016-02-15

    The clinical application of anthracyclines, like daunorubicin and doxorubicin, is limited by two factors: dose-related cardiotoxicity and drug resistance. Both have been linked to reductive metabolism of the parent drug to their metabolites daunorubicinol and doxorubicinol, respectively. These metabolites show significantly less anti-neoplastic properties as their parent drugs and accumulate in cardiac tissue leading to chronic cardiotoxicity. Therefore, we aimed to identify novel and potent natural inhibitors for anthracycline reductases, which enhance the anticancer effect of anthracyclines by preventing the development of anthracycline resistance. Human enzymes responsible for the reductive metabolism of daunorubicin were tested for their sensitivity towards anthrachinones, in particular emodin and anthraflavic acid. Intense inhibition kinetic data for the most effective daunorubicin reductases, including IC{sub 50}- and K{sub i}-values, the mode of inhibition, as well as molecular docking, were compiled. Subsequently, a cytotoxicity profile and the ability of emodin to reverse daunorubicin resistance were determined using multiresistant A549 lung cancer and HepG2 liver cancer cells. Emodin potently inhibited the four main human daunorubicin reductases in vitro. Further, we could demonstrate that emodin is able to synergistically sensitize human cancer cells towards daunorubicin at clinically relevant concentrations. Therefore, emodin may yield the potential to enhance the therapeutic effectiveness of anthracyclines by preventing anthracycline resistance via inhibition of the anthracycline reductases. In symphony with its known pharmacological properties, emodin might be a compound of particular interest in the management of anthracycline chemotherapy efficacy and their adverse effects. - Highlights: • Natural and synthetic compounds were identified as inhibitors for human daunorubicin reductases. • Emodin is a potent inhibitor for human daunorubicin

  9. Cloning and sequence of the human adrenodoxin reductase gene

    International Nuclear Information System (INIS)

    Lin, Dong; Shi, Y.; Miller, W.L.

    1990-01-01

    Adrenodoxin reductase is a flavoprotein mediating electron transport to all mitochondrial forms of cytochrome P450. The authors cloned the human adrenodoxin reductase gene and characterized it by restriction endonuclease mapping and DNA sequencing. The entire gene is approximately 12 kilobases long and consists of 12 exons. The first exon encodes the first 26 of the 32 amino acids of the signal peptide, and the second exon encodes the remainder of signal peptide and the apparent FAD binding site. The remaining 10 exons are clustered in a region of only 4.3 kilobases, separated from the first two exons by a large intron of about 5.6 kilobases. Two forms of human adrenodoxin reductase mRNA, differing by the presence or absence of 18 bases in the middle of the sequence, arise from alternate splicing at the 5' end of exon 7. This alternately spliced region is directly adjacent to the NADPH binding site, which is entirely contained in exon 6. The immediate 5' flanking region lacks TATA and CAAT boxes; however, this region is rich in G+C and contains six copies of the sequence GGGCGGG, resembling promoter sequences of housekeeping genes. RNase protection experiments show that transcription is initiated from multiple sites in the 5' flanking region, located about 21-91 base pairs upstream from the AUG translational initiation codon

  10. Structural and biochemical properties of cloned and expressed human and rat steroid 5α-reductases

    International Nuclear Information System (INIS)

    Andersson, S.; Russell, D.W.

    1990-01-01

    The microsomal enzyme steroid 5α-reductase is responsible for the conversion of testosterone into the more potent androgen dihydrotestosterone. In man, this steroid acts on a variety of androgen-responsive target tissues to mediate such diverse endocrine processes as male sexual differentiation in the fetus and prostatic growth in men. Here we describe the isolation, structure, and expression of a cDNA encoding the human steroid 5α-reductase. A rat cDNA was used as a hybridization probe to screen a human prostate cDNA library. A 2.1-kilobase cDNA was identified and DNA sequence analysis indicated that the human steroid 5α-reductase was a hydrophobic protein of 259 amino acids with a predicted molecular weight of 29,462. A comparison of the human and rat protein sequences revealed a 60% identity. Transfection of expression vectors containing the human and rat cDNAs into simian COS cells resulted in the synthesis of high levels of steroid 5α-reductase enzyme activity. Both enzymes expressed in COS cells showed similar substrate specificities for naturally occurring steroid hormones. However, synthetic 4-azasteroids demonstrated marked differences in their abilities to inhibit the human and rat steroid 5α-reductases

  11. Use of zebrafish and knockdown technology to define proprotein convertase activity.

    Science.gov (United States)

    Chitramuthu, Babykumari P; Bennett, Hugh P J

    2011-01-01

    The Zebrafish (Danio rerio) is a powerful and well-established tool used extensively for the study of early vertebrate development and as a model of human diseases. Zebrafish genes orthologous to their mammalian counterparts generally share conserved biological function. Protein knockdown or overexpression can be effectively achieved by microinjection of morpholino antisense oligonucleotides (MOs) or mRNA, respectively, into developing embryos at the one- to two-cell stage. Correlating gene expression patterns with the characterizing of phenotypes resulting from over- or underexpression can reveal the function of a particular protein. The microinjection technique is simple and results are reproducible. We defined the expression pattern of the proprotein convertase PCSK5 within the lateral line neuromasts and various organs including the liver, gut and otic vesicle by whole-mount in situ hybridization (ISH) and immunofluorescence (IF). MO-mediated knockdown of zebrafish PCSK5 expression generated embryos that display abnormal neuromast deposition within the lateral line system resulting in uncoordinated patterns of swimming.

  12. Overview of Catalytic Properties of Fungal Xylose Reductases and Molecular Engineering Approaches for Improved Xylose Utilisation in Yeast

    Directory of Open Access Journals (Sweden)

    Sk Amir Hossain

    2018-03-01

    Full Text Available Background and Objective: Xylose reductases belong to the aldo-keto reductase family of enzymes, which catalyse the conversion of xylose to xylitol. Yeast xylose reductases have been intensively studied in the last two decades due to their significance in biotechnological production of ethanol and xylitol from xylose. Due to its GRAS status and pronounced tolerance to harsh conditions, Saccharomyces cerevisiae is the ideal organism for industrial production of both xylitol and ethanol. However, Saccharomyces cerevisiae is unable to use xylose as the sole carbon source due to the lack of xylose specific transporters and insufficient activity of metabolic pathways for xylose utilisation. The aim of this paper is to give an overview of attempts in increasing biotechnological potential of xylose reductases and to highlight the prospective of this application. Results and Conclusion: In order to create strains with improved xylose utilization, different approaches were attempted including simultaneous overexpression of xylitol dehydrogenase, xylose reductase and pentose phosphate pathway enzymes, heterologous expression of putative xylose transporters or heterologous expression of genes coding for enzymes included in the xylose metabolism, respectively. Furthermore, number of attempts to genetically modify different xylose reductases is increasing. This review presents current knowledge about yeast xylose reductases and the different approaches applied in order to improve xylose metabolism in yeast.Conflict of interest: The authors declare no conflict of interest.

  13. Aldose Reductase Inhibitory Activity of Compounds from  Zea mays L.

    Science.gov (United States)

    Kim, Tae Hyeon; Kim, Jin Kyu; Kang, Young-Hee; Lee, Jae-Yong; Kang, Il Jun; Lim, Soon Sung

    2013-01-01

    Aldose reductase (AR) inhibitors have a considerable therapeutic potential against diabetes complications and do not increase the risk of hypoglycemia. Through bioassay-guided fractionation of an EtOH extract of the kernel from purple corn (Zea mays L.), 7 nonanthocyanin phenolic compounds (compound 1–7) and 5 anthocyanins (compound 8–12) were isolated. These compounds were investigated by rat lens aldose reductase (RLAR) inhibitory assays. Kinetic analyses of recombinant human aldose reductase (rhAR) were performed, and intracellular galactitol levels were measured. Hirsutrin, one of 12 isolated compounds, showed the most potent RLAR inhibitory activity (IC50, 4.78 μM). In the kinetic analyses using Lineweaver-Burk plots of 1/velocity and 1/substrate concentration, hirsutrin showed competitive inhibition against rhAR. Furthermore, hirsutrin inhibited galactitol formation in rat lens and erythrocytes sample incubated with a high concentration of galactose; this finding indicates that hirsutrin may effectively prevent osmotic stress in hyperglycemia. Therefore, hirsutrin derived from Zea mays L. may be a potential therapeutic agent against diabetes complications. PMID:23586057

  14. Characterization of a cultured human T-cell line with genetically altered ribonucleotide reductase activity. Model for immunodeficiency.

    Science.gov (United States)

    Waddell, D; Ullman, B

    1983-04-10

    From human CCRF-CEM T-cells growing in continuous culture, we have selected, isolated, and characterized a clonal cell line, APHID-D2, with altered ribonucleotide reductase activity. In comparative growth rate experiments, the APHID-D2 cell line is less sensitive than the parental cell line to growth inhibition by deoxyadenosine in the presence of 10 microM erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of adenosine deaminase. The APHID-D2 cell line has elevated levels of all four dNTPs. The resistance of the APHID-D2 cell line to growth inhibition by deoxyadenosine and the abnormal dNTP levels can be explained by the fact that the APHID-D2 ribonucleotide reductase, unlike the parental ribonucleotide reductase, is not normally sensitive to inhibition by dATP. These results suggest that the allosteric site of ribonucleotide reductase which binds both dATP and ATP is altered in the APHID-D2 line. The isolation of a mutant clone of human T-cells which contains a ribonucleotide reductase that has lost its normal sensitivity to dATP and which is resistant to deoxyadenosine-mediated growth inhibition suggests that a primary pathogenic target of accumulated dATP in lymphocytes from patients with adenosine deaminase deficiency may be the cellular ribonucleotide reductase.

  15. Plasmid-encoded diacetyl (acetoin) reductase in Leuconostoc pseudomesenteroides

    DEFF Research Database (Denmark)

    Rattray, Fergal P; Myling-Petersen, Dorte; Larsen, Dianna

    2003-01-01

    A plasmid-borne diacetyl (acetoin) reductase (butA) from Leuconostoc pseudomesenteroides CHCC2114 was sequenced and cloned. Nucleotide sequence analysis revealed an open reading frame encoding a protein of 257 amino acids which had high identity at the amino acid level to diacetyl (acetoin...

  16. Fascin-1 knock-down of human glioma cells reduces their microvilli/filopodia while improving their susceptibility to lymphocyte-mediated cytotoxicity

    Science.gov (United States)

    Hoa, Neil T; Ge, Lisheng; Erickson, Kate L; Kruse, Carol A; Cornforth, Andrew N; Kuznetsov, Yurii; McPherson, Alex; Martini, Filippo; Jadus, Martin R

    2015-01-01

    Cancer cells derived from Glioblastoma multiforme possess membranous protrusions allowing these cells to infiltrate surrounding tissue, while resisting lymphocyte cytotoxicity. Microvilli and filopodia are supported by actin filaments cross-linked by fascin. Fascin-1 was genetically silenced within human U251 glioma cells; these knock-down glioma cells lost their microvilli/filopodia. The doubling time of these fascin-1 knock-down cells was doubled that of shRNA control U251 cells. Fascin-1 knock-down cells lost their transmigratory ability responding to interleukin-6 or insulin-like growth factor-1. Fascin-1 silenced U251 cells were more easily killed by cytolytic lymphocytes. Fascin-1 knock-down provides unique opportunities to augment glioma immunotherapy by simultaneously targeting several key glioma functions: like cell transmigration, cell division and resisting immune responses. PMID:25901196

  17. Adipose-derived stem cells retain their regenerative potential after methotrexate treatment

    International Nuclear Information System (INIS)

    Beane, Olivia S.; Fonseca, Vera C.; Darling, Eric M.

    2014-01-01

    In musculoskeletal tissues like bone, chemotherapy can impair progenitor cell differentiation and proliferation, resulting in decreased bone growth and mineralization throughout a patient's lifetime. In the current study, we investigated the effects of chemotherapeutics on adipose-derived stem cell (ASC) function to determine whether this cell source could be a candidate for repairing, or even preventing, chemotherapy-induced tissue damage. Dose-dependent proliferation rates of ASCs and normal human fibroblasts (NHFs) were quantified after treatment with cytarabine (CY), etoposide (ETO), methotrexate (MTX), and vincristine (VIN) using a fluorescence-based assay. The influence of MTX on the multipotency of ASCs and freshly isolated stromal vascular fraction (SVF) cells was also evaluated using lineage-specific stains and spectrophotometry. ASC and NHF proliferation were equally inhibited by exposure to CY and ETO; however, when treated with MTX and VIN, ASCs exhibited greater resistance. This was especially apparent for MTX-treated samples, with ASC proliferation showing no inhibition for clinically relevant MTX doses ranging from 0.1 to 50 μM. Additional experiments revealed that the differentiation potential of ASCs was not affected by MTX treatment and that upregulation of dihydrofolate reductase possibly contributed to this response. Moreover, SVF cells, which include ASCs, exhibited similar resistance to MTX impairment, with respect to cellular proliferation, clonogenicity, and differentiation capability. Therefore, we have shown that the regenerative properties of ASCs resist the cytotoxicity of MTX, identifying these cells as a potential key for repairing musculoskeletal damage in patients undergoing chemotherapy. - Highlights: • Long-term effects of chemotherapeutics can include musculoskeletal dysfunction. • A screen of common drugs showed disparate effects on ASCs and fibroblasts. • One drug, methotrexate, did not impair ASC growth

  18. Functional Repertoire of Antibiotic Resistance Genes in Antibiotic Manufacturing Effluents and Receiving Freshwater Sediments

    Directory of Open Access Journals (Sweden)

    Juan J. González-Plaza

    2018-01-01

    Full Text Available Environments polluted by direct discharges of effluents from antibiotic manufacturing are important reservoirs for antibiotic resistance genes (ARGs, which could potentially be transferred to human pathogens. However, our knowledge about the identity and diversity of ARGs in such polluted environments remains limited. We applied functional metagenomics to explore the resistome of two Croatian antibiotic manufacturing effluents and sediments collected upstream of and at the effluent discharge sites. Metagenomic libraries built from an azithromycin-production site were screened for resistance to macrolide antibiotics, whereas the libraries from a site producing veterinary antibiotics were screened for resistance to sulfonamides, tetracyclines, trimethoprim, and beta-lactams. Functional analysis of eight libraries identified a total of 82 unique, often clinically relevant ARGs, which were frequently found in clusters and flanked by mobile genetic elements. The majority of macrolide resistance genes identified from matrices exposed to high levels of macrolides were similar to known genes encoding ribosomal protection proteins, macrolide phosphotransferases, and transporters. Potentially novel macrolide resistance genes included one most similar to a 23S rRNA methyltransferase from Clostridium and another, derived from upstream unpolluted sediment, to a GTPase HflX from Emergencia. In libraries deriving from sediments exposed to lower levels of veterinary antibiotics, we found 8 potentially novel ARGs, including dihydrofolate reductases and beta-lactamases from classes A, B, and D. In addition, we detected 7 potentially novel ARGs in upstream sediment, including thymidylate synthases, dihydrofolate reductases, and class D beta-lactamase. Taken together, in addition to finding known gene types, we report the discovery of novel and diverse ARGs in antibiotic-polluted industrial effluents and sediments, providing a qualitative basis for monitoring the

  19. Adipose-derived stem cells retain their regenerative potential after methotrexate treatment

    Energy Technology Data Exchange (ETDEWEB)

    Beane, Olivia S. [Center for Biomedical Engineering, Brown University, Providence, RI (United States); Fonseca, Vera C. [Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, RI (United States); Darling, Eric M., E-mail: Eric_Darling@brown.edu [Center for Biomedical Engineering, Brown University, Providence, RI (United States); Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, RI (United States); Department of Orthopaedics, Brown University, Providence, RI (United States); School of Engineering, Brown University, Providence, RI (United States)

    2014-10-01

    In musculoskeletal tissues like bone, chemotherapy can impair progenitor cell differentiation and proliferation, resulting in decreased bone growth and mineralization throughout a patient's lifetime. In the current study, we investigated the effects of chemotherapeutics on adipose-derived stem cell (ASC) function to determine whether this cell source could be a candidate for repairing, or even preventing, chemotherapy-induced tissue damage. Dose-dependent proliferation rates of ASCs and normal human fibroblasts (NHFs) were quantified after treatment with cytarabine (CY), etoposide (ETO), methotrexate (MTX), and vincristine (VIN) using a fluorescence-based assay. The influence of MTX on the multipotency of ASCs and freshly isolated stromal vascular fraction (SVF) cells was also evaluated using lineage-specific stains and spectrophotometry. ASC and NHF proliferation were equally inhibited by exposure to CY and ETO; however, when treated with MTX and VIN, ASCs exhibited greater resistance. This was especially apparent for MTX-treated samples, with ASC proliferation showing no inhibition for clinically relevant MTX doses ranging from 0.1 to 50 μM. Additional experiments revealed that the differentiation potential of ASCs was not affected by MTX treatment and that upregulation of dihydrofolate reductase possibly contributed to this response. Moreover, SVF cells, which include ASCs, exhibited similar resistance to MTX impairment, with respect to cellular proliferation, clonogenicity, and differentiation capability. Therefore, we have shown that the regenerative properties of ASCs resist the cytotoxicity of MTX, identifying these cells as a potential key for repairing musculoskeletal damage in patients undergoing chemotherapy. - Highlights: • Long-term effects of chemotherapeutics can include musculoskeletal dysfunction. • A screen of common drugs showed disparate effects on ASCs and fibroblasts. • One drug, methotrexate, did not impair ASC growth

  20. Functional Repertoire of Antibiotic Resistance Genes in Antibiotic Manufacturing Effluents and Receiving Freshwater Sediments

    Science.gov (United States)

    González-Plaza, Juan J.; Šimatović, Ana; Milaković, Milena; Bielen, Ana; Wichmann, Fabienne; Udiković-Kolić, Nikolina

    2018-01-01

    Environments polluted by direct discharges of effluents from antibiotic manufacturing are important reservoirs for antibiotic resistance genes (ARGs), which could potentially be transferred to human pathogens. However, our knowledge about the identity and diversity of ARGs in such polluted environments remains limited. We applied functional metagenomics to explore the resistome of two Croatian antibiotic manufacturing effluents and sediments collected upstream of and at the effluent discharge sites. Metagenomic libraries built from an azithromycin-production site were screened for resistance to macrolide antibiotics, whereas the libraries from a site producing veterinary antibiotics were screened for resistance to sulfonamides, tetracyclines, trimethoprim, and beta-lactams. Functional analysis of eight libraries identified a total of 82 unique, often clinically relevant ARGs, which were frequently found in clusters and flanked by mobile genetic elements. The majority of macrolide resistance genes identified from matrices exposed to high levels of macrolides were similar to known genes encoding ribosomal protection proteins, macrolide phosphotransferases, and transporters. Potentially novel macrolide resistance genes included one most similar to a 23S rRNA methyltransferase from Clostridium and another, derived from upstream unpolluted sediment, to a GTPase HflX from Emergencia. In libraries deriving from sediments exposed to lower levels of veterinary antibiotics, we found 8 potentially novel ARGs, including dihydrofolate reductases and beta-lactamases from classes A, B, and D. In addition, we detected 7 potentially novel ARGs in upstream sediment, including thymidylate synthases, dihydrofolate reductases, and class D beta-lactamase. Taken together, in addition to finding known gene types, we report the discovery of novel and diverse ARGs in antibiotic-polluted industrial effluents and sediments, providing a qualitative basis for monitoring the dispersal of ARGs

  1. Drug resistance associated genetic polymorphisms in Plasmodium falciparum and Plasmodium vivax collected in Honduras, Central America.

    Science.gov (United States)

    Jovel, Irina T; Mejía, Rosa E; Banegas, Engels; Piedade, Rita; Alger, Jackeline; Fontecha, Gustavo; Ferreira, Pedro E; Veiga, Maria I; Enamorado, Irma G; Bjorkman, Anders; Ursing, Johan

    2011-12-19

    In Honduras, chloroquine and primaquine are recommended and still appear to be effective for treatment of Plasmodium falciparum and Plasmodium vivax malaria. The aim of this study was to determine the proportion of resistance associated genetic polymorphisms in P. falciparum and P. vivax collected in Honduras. Blood samples were collected from patients seeking medical attention at the Hospital Escuela in Tegucigalpa from 2004 to 2006 as well as three regional hospitals, two health centres and one regional laboratory during 2009. Single nucleotide polymorphisms in P. falciparum chloroquine resistance transporter (pfcrt), multidrug resistance 1 (pfmdr1), dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes and in P. vivax multidrug resistance 1 (pvmdr1) and dihydrofolate reductase (pvdhfr) genes were detected using PCR based methods. Thirty seven P. falciparum and 64 P. vivax samples were collected. All P. falciparum infections acquired in Honduras carried pfcrt, pfmdr1, pfdhps and pfdhfr alleles associated with chloroquine, amodiaquine and sulphadoxine-pyrimethamine sensitivity only. One patient with parasites acquired on a Pacific Island had pfcrt 76 T and pfmdr1 86Y alleles. That patient and a patient infected in West Africa had pfdhfr 51I, 59 R and 108 N alleles. Pvmdr1 976 F was found in 7/37 and two copies of pvmdr1 were found in 1/37 samples. Pvdhfr 57 L + 58 R was observed in 2/57 samples. The results indicate that P. falciparum from Honduras remain sensitive to chloroquine and sulphadoxine-pyrimethamine. This suggests that chloroquine and sulphadoxine-pyrimethamine should be efficacious for treatment of uncomplicated P. falciparum malaria, supporting current national treatment guidelines. However, genetic polymorphisms associated with chloroquine and sulphadoxine-pyrimethamine tolerance were detected in local P. vivax and imported P. falciparum infections. Continuous monitoring of the prevalence of drug resistant/tolerant P

  2. X-Ray crystal structure of GarR—tartronate semialdehyde reductase from Salmonella typhimurium

    OpenAIRE

    Osipiuk, J.; Zhou, M.; Moy, S.; Collart, F.; Joachimiak, A.

    2009-01-01

    Tartronate semialdehyde reductases (TSRs), also known as 2-hydroxy-3-oxopropionate reductases, catalyze the reduction of tartronate semialdehyde using NAD as cofactor in the final stage of D-glycerate biosynthesis. These enzymes belong to family of structurally and mechanically related β-hydroxyacid dehydrogenases which differ in substrate specificity and catalyze reactions in specific metabolic pathways. Here, we present the crystal structure of GarR a TSR from Salmonella typhimurium determi...

  3. MRP4 knockdown enhances migration, suppresses apoptosis, and produces aggregated morphology in human retinal vascular endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Tagami, Mizuki [Department of Surgery Related, Division of Ophthalmology, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Kusuhara, Sentaro, E-mail: kusu@med.kobe-u.ac.jp [Department of Surgery Related, Division of Ophthalmology, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Imai, Hisanori [Department of Surgery Related, Division of Ophthalmology, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Uemura, Akiyoshi [Department of Surgery Related, Division of Ophthalmology, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Department of Vascular Biology, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Honda, Shigeru; Tsukahara, Yasutomo; Negi, Akira [Department of Surgery Related, Division of Ophthalmology, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan)

    2010-10-01

    Research highlights: {yields} Exogenous VEGF decreases MRP4 expression in a dose-dependent manner. {yields} MRP4 knockdown leads to enhanced cell migration. {yields} MRP4 knockdown suppresses caspase-3-mediated cell apoptosis. {yields} MRP4 knockdown produces cell assembly and cell aggregation. -- Abstract: The multidrug resistance protein (MRP) MRP4/ABCC4 is an ATP-binding cassette transporter that actively effluxes endogenous and xenobiotic substrates out of cells. In the rodent retina, Mrp4 mRNA and protein are exclusively expressed in vascular endothelial cells, but the angiogenic properties of Mrp4 are poorly understood so far. This study aims to explore the angiogenic properties of MRP4 in human retinal microvascular endothelial cells (HRECs) utilizing the RNA interference (RNAi) technique. MRP4 expression was decreased at the mRNA and protein levels after stimulation with exogenous vascular endothelial growth factor in a dose-dependent manner. RNAi-mediated MRP4 knockdown in HRECs do not affect cell proliferation but enhances cell migration. Moreover, cell apoptosis induced by serum starvation was less prominent in MRP4 siRNA-treated HRECs as compared to control siRNA-treated HRECs. In a Matrigel-based tube-formation assay, although MRP4 knockdown did not lead to a significant change in the total tube length, MRP4 siRNA-treated HRECs assembled and aggregated into a massive tube-like structure, which was not observed in control siRNA-treated HRECs. These results suggest that MRP4 is uniquely involved in retinal angiogenesis.

  4. Reduced bone mass and muscle strength in male 5α-reductase type 1 inactivated mice.

    Directory of Open Access Journals (Sweden)

    Sara H Windahl

    Full Text Available Androgens are important regulators of bone mass but the relative importance of testosterone (T versus dihydrotestosterone (DHT for the activation of the androgen receptor (AR in bone is unknown. 5α-reductase is responsible for the irreversible conversion of T to the more potent AR activator DHT. There are two well established isoenzymes of 5α-reductase (type 1 and type 2, encoded by separate genes (Srd5a1 and Srd5a2. 5α-reductase type 2 is predominantly expressed in male reproductive tissues whereas 5α-reductase type 1 is highly expressed in liver and moderately expressed in several other tissues including bone. The aim of the present study was to investigate the role of 5α-reductase type 1 for bone mass using Srd5a1⁻/⁻ mice. Four-month-old male Srd5a1⁻/⁻ mice had reduced trabecular bone mineral density (-36%, p<0.05 and cortical bone mineral content (-15%, p<0.05 but unchanged serum androgen levels compared with wild type (WT mice. The cortical bone dimensions were reduced in the male Srd5a1⁻/⁻ mice as a result of a reduced cortical periosteal circumference compared with WT mice. T treatment increased the cortical periosteal circumference (p<0.05 in orchidectomized WT mice but not in orchidectomized Srd5a1⁻/⁻ mice. Male Srd5a1⁻/⁻ mice demonstrated a reduced forelimb muscle grip strength compared with WT mice (p<0.05. Female Srd5a1⁻/⁻ mice had slightly increased cortical bone mass associated with elevated circulating levels of androgens. In conclusion, 5α-reductase type 1 inactivated male mice have reduced bone mass and forelimb muscle grip strength and we propose that these effects are due to lack of 5α-reductase type 1 expression in bone and muscle. In contrast, the increased cortical bone mass in female Srd5a1⁻/⁻ mice, is an indirect effect mediated by elevated circulating androgen levels.

  5. Lifespan and reproduction in brain-specific miR-29-knockdown mouse.

    Science.gov (United States)

    Takeda, Toru; Tanabe, Hiroyuki

    2016-03-18

    The microRNA miR-29 is widely distributed and highly expressed in adult mouse brain during the mouse's lifetime. We recently created conditional mutant mice whose miR-29 was brain-specifically knocked down through overexpression of an antisense RNA transgene against miR-29. To explore a role for brain miR-29 in maximizing organismal fitness, we assessed somatic growth, reproduction, and lifespan in the miR-29-knockdown (KD) mice and their wild-type (WT) littermates. The KD mice were developmentally indistinguishable from WT mice with respect to gross morphology and physical activity. Fertility testing revealed that KD males were subfertile, whereas KD females were hyperfertile, only in terms of reproductive success, when compared to their gender-matched WT correspondents. Another phenotypic difference between KD and WT animals appeared in their lifespan data; KD males displayed an overall increasing tendency in post-reproductive survival relative to WT males. In contrast, KD females were prone to shorter lifespans than WT females. These results clarify that brain-targeted miR-29 knockdown affects both lifespan and reproduction in a gender-dependent manner, and moreover that the reciprocal responsiveness to the miR-29 knockdown between these two phenotypes in both genders closely follow life-course models based on the classical trade-off prediction wherein elaborate early-life energetic investment in reproduction entails accelerated late-life declines in survival, and vice versa. Thus, this study identified miR-29 as the first mammalian miRNA that is directly implicated in the lifetime trade-off between the two major fitness components, lifespan and reproduction. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Effect of cystamine on rat tissue GSH level and glutathione reductase activity

    International Nuclear Information System (INIS)

    Kovarova, H.; Pulpanova, J.

    1979-01-01

    Reduced glutathione (GSH) level and glutathione reductase activity were determined by means of the spectrophotometric method in various rat tissues after i.p. administration of cystamine (50 mg/kg and 20 mg/kg). GSH amount dropped in the spleen and kidney at 10 and 20 min; following this interval, an increase of GSH level was observed in the liver at 20-30 min, in the spleen and kidney at 60 min after the treatment with a radioprotective cystamine dose (50 mg/kg). The changes in GSH level induced by a non-radioprotective cystamine dose (20 mg/kg) had an opposite tendency. The activity of glutathione reductase was decreased in all tissues studied. As to the mechanism of the radioprotective action, both the inactivation of glutathione reductase activity and the changes in GSH level seem to be the factors contributing to the radioprotective effect of cystamine by strengthening the cellular radioresistance. (orig.) 891 MG/orig. 892 RKD [de

  7. Stable Toll-Like Receptor 10 Knockdown in THP-1 Cells Reduces TLR-Ligand-Induced Proinflammatory Cytokine Expression

    Directory of Open Access Journals (Sweden)

    Hai Van Le

    2016-06-01

    Full Text Available Toll-like receptor 10 (TLR10 is the only orphan receptor whose natural ligand and function are unknown among the 10 human TLRs. In this study, to test whether TLR10 recognizes some known TLR ligands, we established a stable TLR10 knockdown human monocytic cell line THP-1 using TLR10 short hairpin RNA lentiviral particle and puromycin selection. Among 60 TLR10 knockdown clones that were derived from each single transduced cell, six clones were randomly selected, and then one of those clones, named E7, was chosen for the functional study. E7 exhibited approximately 50% inhibition of TLR10 mRNA and protein expression. Of all the TLRs, only the expression of TLR10 changed significantly in this cell line. Additionally, phorbol 12-myristate 13-acetate-induced macrophage differentiation of TLR10 knockdown cells was not affected in the knockdown cells. When exposed to TLR ligands, such as synthetic diacylated lipoprotein (FSL-1, lipopolysaccharide (LPS, and flagellin, significant induction of proinflammatory cytokine gene expression including Interleukin-8 (IL-8, Interleukin-1 beta (IL-1β, Tumor necrosis factor-alpha (TNF-α and Chemokine (C–C Motif Ligand 20 (CCL20 expression, was found in the control THP-1 cells, whereas the TLR10 knockdown cells exhibited a significant reduction in the expression of IL-8, IL-1β, and CCL20. TNF-α was the only cytokine for which the expression did not decrease in the TLR10 knockdown cells from that measured in the control cells. Analysis of putative binding sites for transcription factors using a binding-site-prediction program revealed that the TNF-α promoter does not have putative binding sites for AP-1 or c-Jun, comprising a major transcription factor along with NF-κB for TLR signaling. Our results suggest that TLR10 is involved in the recognition of FSL-1, LPS, and flagellin and TLR-ligand-induced expression of TNF-α does not depend on TLR10.

  8. Stable Toll-Like Receptor 10 Knockdown in THP-1 Cells Reduces TLR-Ligand-Induced Proinflammatory Cytokine Expression.

    Science.gov (United States)

    Le, Hai Van; Kim, Jae Young

    2016-06-01

    Toll-like receptor 10 (TLR10) is the only orphan receptor whose natural ligand and function are unknown among the 10 human TLRs. In this study, to test whether TLR10 recognizes some known TLR ligands, we established a stable TLR10 knockdown human monocytic cell line THP-1 using TLR10 short hairpin RNA lentiviral particle and puromycin selection. Among 60 TLR10 knockdown clones that were derived from each single transduced cell, six clones were randomly selected, and then one of those clones, named E7, was chosen for the functional study. E7 exhibited approximately 50% inhibition of TLR10 mRNA and protein expression. Of all the TLRs, only the expression of TLR10 changed significantly in this cell line. Additionally, phorbol 12-myristate 13-acetate-induced macrophage differentiation of TLR10 knockdown cells was not affected in the knockdown cells. When exposed to TLR ligands, such as synthetic diacylated lipoprotein (FSL-1), lipopolysaccharide (LPS), and flagellin, significant induction of proinflammatory cytokine gene expression including Interleukin-8 (IL-8), Interleukin-1 beta (IL-1β), Tumor necrosis factor-alpha (TNF-α) and Chemokine (C-C Motif) Ligand 20 (CCL20) expression, was found in the control THP-1 cells, whereas the TLR10 knockdown cells exhibited a significant reduction in the expression of IL-8, IL-1β, and CCL20. TNF-α was the only cytokine for which the expression did not decrease in the TLR10 knockdown cells from that measured in the control cells. Analysis of putative binding sites for transcription factors using a binding-site-prediction program revealed that the TNF-α promoter does not have putative binding sites for AP-1 or c-Jun, comprising a major transcription factor along with NF-κB for TLR signaling. Our results suggest that TLR10 is involved in the recognition of FSL-1, LPS, and flagellin and TLR-ligand-induced expression of TNF-α does not depend on TLR10.

  9. Expression, purification, crystallization and preliminary X-ray diffraction analysis of carbonyl reductase from Candida parapsilosis ATCC 7330

    International Nuclear Information System (INIS)

    Aggarwal, Nidhi; Mandal, P. K.; Gautham, Namasivayam; Chadha, Anju

    2013-01-01

    The expression, purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies on C. parapsilosis carbonyl reductase are reported. The NAD(P)H-dependent carbonyl reductase from Candida parapsilosis ATCC 7330 catalyses the asymmetric reduction of ethyl 4-phenyl-2-oxobutanoate to ethyl (R)-4-phenyl-2-hydroxybutanoate, a precursor of angiotensin-converting enzyme inhibitors such as Cilazapril and Benazepril. The carbonyl reductase was expressed in Escherichia coli and purified by GST-affinity and size-exclusion chromatography. Crystals were obtained by the hanging-drop vapour-diffusion method and diffracted to 1.86 Å resolution. The asymmetric unit contained two molecules of carbonyl reductase, with a solvent content of 48%. The structure was solved by molecular replacement using cinnamyl alcohol dehydrogenase from Saccharomyces cerevisiae as a search model

  10. Proanthocyanidin synthesis in Theobroma cacao: genes encoding anthocyanidin synthase, anthocyanidin reductase, and leucoanthocyanidin reductase.

    Science.gov (United States)

    Liu, Yi; Shi, Zi; Maximova, Siela; Payne, Mark J; Guiltinan, Mark J

    2013-12-05

    The proanthocyanidins (PAs), a subgroup of flavonoids, accumulate to levels of approximately 10% total dry weight of cacao seeds. PAs have been associated with human health benefits and also play important roles in pest and disease defense throughout the plant. To dissect the genetic basis of PA biosynthetic pathway in cacao (Theobroma cacao), we have isolated three genes encoding key PA synthesis enzymes, anthocyanidin synthase (ANS), anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR). We measured the expression levels of TcANR, TcANS and TcLAR and PA content in cacao leaves, flowers, pod exocarp and seeds. In all tissues examined, all three genes were abundantly expressed and well correlated with PA accumulation levels, suggesting their active roles in PA synthesis. Overexpression of TcANR in an Arabidopsis ban mutant complemented the PA deficient phenotype in seeds and resulted in reduced anthocyanidin levels in hypocotyls. Overexpression of TcANS in tobacco resulted in increased content of both anthocyanidins and PAs in flower petals. Overexpression of TcANS in an Arabidopsis ldox mutant complemented its PA deficient phenotype in seeds. Recombinant TcLAR protein converted leucoanthocyanidin to catechin in vitro. Transgenic tobacco overexpressing TcLAR had decreased amounts of anthocyanidins and increased PAs. Overexpressing TcLAR in Arabidopsis ldox mutant also resulted in elevated synthesis of not only catechin but also epicatechin. Our results confirm the in vivo function of cacao ANS and ANR predicted based on sequence homology to previously characterized enzymes from other species. In addition, our results provide a clear functional analysis of a LAR gene in vivo.

  11. Photoaffinity labeling of steroid 5 alpha-reductase of rat liver and prostate microsomes

    International Nuclear Information System (INIS)

    Liang, T.; Cheung, A.H.; Reynolds, G.F.; Rasmusson, G.H.

    1985-01-01

    21-Diazo-4-methyl-4-aza-5 alpha-pregnane-3,20-dione (Diazo-MAPD) inhibits steroid 5 alpha-reductase in liver microsomes of female rats with a K/sub i/ value of 8.7 +/- 1.7 nM, and the inhibition is competitive with testosterone. It also inhibits the binding of a 5 alpha-reductase inhibitor, [ 3 H] 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one ([ 3 H]4-MA), to the enzyme in liver microsomes. The inhibition of 5 alpha-reductase activity and of inhibitor binding activity by diazo-MAPD becomes irreversible upon UV irradiation. [1,2- 3 H]Diazo-MAPD binds to a single high affinity site in liver microsomes of female rats, and this binding requires NADPH. Without UV irradiation, this binding is reversible, and it becomes irreversible upon UV irradiation. Both the initial reversible binding and the subsequent irreversible conjugation after UV irradiation are inhibited by inhibitors (diazo-MAPD and 4-MA) and substrates (progesterone and testosterone) of 5 alpha-reductase, but they are not inhibited by 5 alpha-reduced steroids. Photoaffinity labeled liver microsomes of female rats were solubilized and fractionated by high performance gel filtration. The radioactive conjugate eluted in one major peak at Mr 50,000

  12. Hydroxyurea-Mediated Cytotoxicity Without Inhibition of Ribonucleotide Reductase.

    Science.gov (United States)

    Liew, Li Phing; Lim, Zun Yi; Cohen, Matan; Kong, Ziqing; Marjavaara, Lisette; Chabes, Andrei; Bell, Stephen D

    2016-11-01

    In many organisms, hydroxyurea (HU) inhibits class I ribonucleotide reductase, leading to lowered cellular pools of deoxyribonucleoside triphosphates. The reduced levels for DNA precursors is believed to cause replication fork stalling. Upon treatment of the hyperthermophilic archaeon Sulfolobus solfataricus with HU, we observe dose-dependent cell cycle arrest, accumulation of DNA double-strand breaks, stalled replication forks, and elevated levels of recombination structures. However, Sulfolobus has a HU-insensitive class II ribonucleotide reductase, and we reveal that HU treatment does not significantly impact cellular DNA precursor pools. Profiling of protein and transcript levels reveals modulation of a specific subset of replication initiation and cell division genes. Notably, the selective loss of the regulatory subunit of the primase correlates with cessation of replication initiation and stalling of replication forks. Furthermore, we find evidence for a detoxification response induced by HU treatment. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  13. Nitrate reductase gene involvement in hexachlorobiphenyl dechlorination by Phanerochaete chrysosporium

    International Nuclear Information System (INIS)

    De, Supriyo; Perkins, Michael; Dutta, Sisir K.

    2006-01-01

    Polychlorobiphenyl (PCB) degradation usually occurs through reductive dechlorination under anaerobic conditions and phenolic ring cleavage under aerobic conditions. In this paper, we provide evidence of nitrate reductase (NaR) mediated dechlorination of hexachlorobiphenyl (PCB-153) in Phanerochaete chrysosporium under non-ligninolytic condition and the gene involved. The NaR enzyme and its cofactor, molybdenum (Mo), were found to mediate reductive dechlorination of PCBs even in aerobic condition. Tungsten (W), a competitive inhibitor of this enzyme, was found to suppress this dechlorination. Chlorine release assay provided further evidence of this nitrate reductase mediated dechlorination. Commercially available pure NaR enzyme from Aspergillus was used to confirm these results. Through homology search using TBLASTN program, NaR gene was identified, primers were designed and the RT-PCR product was sequenced. The NaR gene was then annotated in the P. chrysosporium genome (GenBank accession no. AY700576). This is the first report regarding the presence of nitrate reductase gene in this fungus with the explanation why this fungus can dechlorinate PCBs even in aerobic condition. These fungal inoculums are used commercially as pellets in sawdust for enhanced bioremediation of PCBs at the risk of depleting soil nitrates. Hence, the addition of nitrates to the pellets will reduce this risk as well as enhance its activity

  14. Isolation and characterization of cDNAs encoding leucoanthocyanidin reductase and anthocyanidin reductase from Populus trichocarpa.

    Directory of Open Access Journals (Sweden)

    Lijun Wang

    Full Text Available Proanthocyanidins (PAs contribute to poplar defense mechanisms against biotic and abiotic stresses. Transcripts of PA biosynthetic genes accumulated rapidly in response to infection by the fungus Marssonina brunnea f.sp. multigermtubi, treatments of salicylic acid (SA and wounding, resulting in PA accumulation in poplar leaves. Anthocyanidin reductase (ANR and leucoanthocyanidin reductase (LAR are two key enzymes of the PA biosynthesis that produce the main subunits: (+-catechin and (--epicatechin required for formation of PA polymers. In Populus, ANR and LAR are encoded by at least two and three highly related genes, respectively. In this study, we isolated and functionally characterized genes PtrANR1 and PtrLAR1 from P. trichocarpa. Phylogenetic analysis shows that Populus ANR1 and LAR1 occurr in two distinct phylogenetic lineages, but both genes have little difference in their tissue distribution, preferentially expressed in roots. Overexpression of PtrANR1 in poplar resulted in a significant increase in PA levels but no impact on catechin levels. Antisense down-regulation of PtrANR1 showed reduced PA accumulation in transgenic lines, but increased levels of anthocyanin content. Ectopic expression of PtrLAR1 in poplar positively regulated the biosynthesis of PAs, whereas the accumulation of anthocyanin and flavonol was significantly reduced (P<0.05 in all transgenic plants compared to the control plants. These results suggest that both PtrANR1 and PtrLAR1 contribute to PA biosynthesis in Populus.

  15. Targeted Knock-Down of miR21 Primary Transcripts Using snoMEN Vectors Induces Apoptosis in Human Cancer Cell Lines.

    Directory of Open Access Journals (Sweden)

    Motoharu Ono

    Full Text Available We have previously reported an antisense technology, 'snoMEN vectors', for targeted knock-down of protein coding mRNAs using human snoRNAs manipulated to contain short regions of sequence complementarity with the mRNA target. Here we characterise the use of snoMEN vectors to target the knock-down of micro RNA primary transcripts. We document the specific knock-down of miR21 in HeLa cells using plasmid vectors expressing miR21-targeted snoMEN RNAs and show this induces apoptosis. Knock-down is dependent on the presence of complementary sequences in the snoMEN vector and the induction of apoptosis can be suppressed by over-expression of miR21. Furthermore, we have also developed lentiviral vectors for delivery of snoMEN RNAs and show this increases the efficiency of vector transduction in many human cell lines that are difficult to transfect with plasmid vectors. Transduction of lentiviral vectors expressing snoMEN targeted to pri-miR21 induces apoptosis in human lung adenocarcinoma cells, which express high levels of miR21, but not in human primary cells. We show that snoMEN-mediated suppression of miRNA expression is prevented by siRNA knock-down of Ago2, but not by knock-down of Ago1 or Upf1. snoMEN RNAs colocalise with Ago2 in cell nuclei and nucleoli and can be co-immunoprecipitated from nuclear extracts by antibodies specific for Ago2.

  16. Targeted Knock-Down of miR21 Primary Transcripts Using snoMEN Vectors Induces Apoptosis in Human Cancer Cell Lines.

    Science.gov (United States)

    Ono, Motoharu; Yamada, Kayo; Avolio, Fabio; Afzal, Vackar; Bensaddek, Dalila; Lamond, Angus I

    2015-01-01

    We have previously reported an antisense technology, 'snoMEN vectors', for targeted knock-down of protein coding mRNAs using human snoRNAs manipulated to contain short regions of sequence complementarity with the mRNA target. Here we characterise the use of snoMEN vectors to target the knock-down of micro RNA primary transcripts. We document the specific knock-down of miR21 in HeLa cells using plasmid vectors expressing miR21-targeted snoMEN RNAs and show this induces apoptosis. Knock-down is dependent on the presence of complementary sequences in the snoMEN vector and the induction of apoptosis can be suppressed by over-expression of miR21. Furthermore, we have also developed lentiviral vectors for delivery of snoMEN RNAs and show this increases the efficiency of vector transduction in many human cell lines that are difficult to transfect with plasmid vectors. Transduction of lentiviral vectors expressing snoMEN targeted to pri-miR21 induces apoptosis in human lung adenocarcinoma cells, which express high levels of miR21, but not in human primary cells. We show that snoMEN-mediated suppression of miRNA expression is prevented by siRNA knock-down of Ago2, but not by knock-down of Ago1 or Upf1. snoMEN RNAs colocalise with Ago2 in cell nuclei and nucleoli and can be co-immunoprecipitated from nuclear extracts by antibodies specific for Ago2.

  17. Identification of Pneumocystis carinii chromosomes and mapping of five genes

    DEFF Research Database (Denmark)

    Lundgren, B; Cotton, R; Lundgren, J D

    1990-01-01

    Pulsed field gel electrophoresis was used to identify the chromosome-size DNA of Pneumocystis carinii, a major pathogen of immunocompromised patients. Thirteen chromosomes of rodent Pneumocystis carinii, ranging in size from 300 to 700 kilobases (kb), were identified. The minimum genome size for P....... carinii, estimated on the basis of the sizes of chromosomes, is 7,000 kb. Genetic heterogeneity among different P. carinii isolates was documented by demonstration of chromosomal size variability. By hybridization studies, the genes for topoisomerase I, dihydrofolate reductase, rRNA, actin......, and thymidylate synthase were mapped to single chromosomes of approximately 650, 590, 550, 460, and 350 kb, respectively. Hybridization studies further confirmed the genetic heterogeneity of P. carinii....

  18. Isolation and primary structural analysis of two conjugated polyketone reductases from Candida parapsilosis.

    Science.gov (United States)

    Hidalgo, A R; Akond, M A; Kita, K; Kataoka, M; Shimizu, S

    2001-12-01

    Two conjugated polyketone reductases (CPRs) were isolated from Candida parapsilosis IFO 0708. The primary structures of CPRs (C1 and C2) were analyzed by amino acid sequencing. The amino acid sequences of both enzymes had high similarity to those of several proteins of the aldo-keto-reductase (AKR) superfamily. However, several amino acid residues in the putative active sites of AKRs were not conserved in CPRs-C1 and -C2.

  19. Molecular Cloning and Expression of Bacterial Mercuric Reductase ...

    African Journals Online (AJOL)

    In order to characterize the bacterial mercuric reductase (merA) gene, mercury resistant (Hgr) Escherichia coli strains have been isolated from various mercury contaminated sites of India. Their minimum inhibitory concentration (MIC) for Hg and zone of inhibition for different antibiotics were measured, and finally mer operon ...

  20. Molecular Cloning and Expression of Bacterial Mercuric Reductase ...

    African Journals Online (AJOL)

    USER

    2010-06-21

    Jun 21, 2010 ... In order to characterize the bacterial mercuric reductase (merA) gene, mercury resistant (Hgr). Escherichia coli strains have been isolated from various mercury contaminated sites of India. Their minimum inhibitory concentration (MIC) for Hg and zone of inhibition for different antibiotics were measured, and ...

  1. Potency of a novel saw palmetto ethanol extract, SPET-085, for inhibition of 5alpha-reductase II.

    Science.gov (United States)

    Pais, Pilar

    2010-08-01

    The nicotinamide adenine dinucleotide phosphate (NADPH)-dependent membrane protein 5alpha-reductase irreversibly catalyses the conversion of testosterone to the most potent androgen, 5alpha-dihydrotestosterone (DHT). In humans, two 5alpha-reductase isoenyzmes are expressed: type I and type II. Type II is found primarily in prostate tissue. Saw palmetto extract (SPE) has been widely used for the treatment of lower urinary tract symptoms secondary to benign prostatic hyperplasia (BPH). The mechanisms of the pharmacological effects of SPE include the inhibition of 5alpha-reductase, among other actions. Clinical studies of SPE have been equivocal, with some showing significant results and others not. These inconsistent results may be due, in part, to varying bioactivities of the SPE used in the studies. The aim of the present study was to determine the in vitro potency of a novel saw palmetto ethanol extract (SPET-085), an inhibitor of the 5alpha-reductase isoenzyme type II, in a cell-free test system. On the basis of the enzymatic conversion of the substrate androstenedione to the 5alpha-reduced product 5alpha-androstanedione, the inhibitory potency was measured and compared to those of finasteride, an approved 5alpha-reductase inhibitor. SPET-085 concentration-dependently inhibited 5alpha-reductase type II in vitro (IC(50)=2.88+/-0.45 microg/mL). The approved 5alpha-reductase inhibitor, finasteride, tested as positive control, led to 61% inhibition of 5alpha-reductase type II. SPET-085 effectively inhibits the enzyme that has been linked to BPH, and the amount of extract required for activity is very low compared to data reported for other extracts. It can be concluded from data in the literature that SPET-085 is as effective as a hexane extract of saw palmetto that exhibited the highest levels of bioactivity, and is more effective than other SPEs tested. This study confirmed that SPET-085 has prostate health-promoting bioactivity that also corresponds favorably to

  2. Intraethnic variation in steroid-5-alpha-reductase polymorphisms in ...

    Indian Academy of Sciences (India)

    2015-06-01

    Jun 1, 2015 ... in prostate cancer patients: a potential factor implicated ... reductase alpha polypeptides 1 and 2 in a set of 601 prostate cancer patients from four ..... tion in the key androgen-regulating genes androgen receptor, cytochrome ...

  3. N-Myc knockdown and apigenin treatment controlled growth of malignant neuroblastoma cells having N-Myc amplification.

    Science.gov (United States)

    Hossain, Md Motarab; Banik, Naren L; Ray, Swapan K

    2013-10-15

    Malignant neuroblastomas mostly occur in children and are frequently associated with N-Myc amplification. Oncogene amplification, which is selective increase in copy number of the oncogene, provides survival advantages in solid tumors including malignant neuroblastoma. We have decreased expression of N-Myc oncogene using short hairpin RNA (shRNA) plasmid to increase anti-tumor efficacy of the isoflavonoid apigenin (APG) in human malignant neuroblastoma SK-N-DZ and SK-N-BE2 cell lines that harbor N-Myc amplification. N-Myc knockdown induced morphological and biochemical features of neuronal differentiation. Combination of N-Myc knockdown and APG most effectively induced morphological and biochemical features of apoptotic death. This combination therapy also prevented cell migration and decreased N-Myc driven survival, angiogenic, and invasive factors. Collectively, N-Myc knockdown and APG treatment is a promising strategy for controlling the growth of human malignant neuroblastoma cell lines that harbor N-Myc amplification. © 2013 Elsevier B.V. All rights reserved.

  4. Molecular and phenotypic characterization of transgenic soybean expressing the Arabidopsis ferric chelate reductase gene, FRO2.

    Science.gov (United States)

    Vasconcelos, Marta; Eckert, Helene; Arahana, Venancio; Graef, George; Grusak, Michael A; Clemente, Tom

    2006-10-01

    Soybean (Glycine max Merr.) production is reduced under iron-limiting calcareous soils throughout the upper Midwest regions of the US. Like other dicotyledonous plants, soybean responds to iron-limiting environments by induction of an active proton pump, a ferric iron reductase and an iron transporter. Here we demonstrate that heterologous expression of the Arabidopsis thaliana ferric chelate reductase gene, FRO2, in transgenic soybean significantly enhances Fe(+3) reduction in roots and leaves. Root ferric reductase activity was up to tenfold higher in transgenic plants and was not subjected to post-transcriptional regulation. In leaves, reductase activity was threefold higher in the transgenic plants when compared to control. The enhanced ferric reductase activity led to reduced chlorosis, increased chlorophyll concentration and a lessening in biomass loss in the transgenic events between Fe treatments as compared to control plants grown under hydroponics that mimicked Fe-sufficient and Fe-deficient soil environments. However, the data indicate that constitutive FRO2 expression under non-iron stress conditions may lead to a decrease in plant productivity as reflected by reduced biomass accumulation in the transgenic events under non-iron stress conditions. When grown at Fe(III)-EDDHA levels greater than 10 microM, iron concentration in the shoots of transgenic plants was significantly higher than control. The same observation was found in the roots in plants grown at iron levels higher than 32 microM Fe(III)-EDDHA. These results suggest that heterologous expression of an iron chelate reductase in soybean can provide a route to alleviate iron deficiency chlorosis.

  5. Keratin23 (KRT23) knockdown decreases proliferation and affects the DNA damage response of colon cancer cells

    DEFF Research Database (Denmark)

    Birkenkamp-Demtröder, Karin; Hahn, Stephan; Mansilla, Francisco

    2013-01-01

    correlated with absent expression, while increased KRT23 expression in tumor samples correlated with promoter hypomethylation, as confirmed by bisulfite sequencing. Demethylation induced KRT23 expression in vitro. Expression profiling of shRNA mediated stable KRT23 knockdown in colon cancer cell lines showed...... response, mainly molecules of the double strand break repair homologous recombination pathway. KRT23 knockdown decreased the transcript and protein expression of key molecules as e.g. MRE11A, E2F1, RAD51 and BRCA1. Knockdown of KRT23 rendered colon cancer cells more sensitive to irradiation and reduced......Keratin 23 (KRT23) is strongly expressed in colon adenocarcinomas but absent in normal colon mucosa. Array based methylation profiling of 40 colon samples showed that the promoter of KRT23 was methylated in normal colon mucosa, while hypomethylated in most adenocarcinomas. Promoter methylation...

  6. Research progress on the roles of aldose reductase in diabetic retinopathy

    Directory of Open Access Journals (Sweden)

    Hong-Zhe Li

    2015-07-01

    Full Text Available Aldose reductase(ARbelonging to nicotinamide-adenine dinucleotide phosphate(NADPH-dependent aldehyde-keto reductase superfamily, is the key rate-limiting enzyme in the polyol pathway which plays an important role in the body's high-sugar metabolism. AR is widely present in the kidneys, blood vessels, lens, retina, heart, skeletal muscle and other tissues and organs, converts glucose to sorbitol which easy permeability of cell membranes, cause cell swelling, degeneration, necrosis, and have a close relationship with the development of chronic complications of diabetes mellitus. Diabetic retinopathy(DRis a multifactorial disease, the exact cause is currently unknown, but polyol pathway has been demonstrated to play an important role in the pathogenesis of DR. Clinical risk factors such as blood sugar control, blood pressure and other treatments for DR only play a part effect of remission or invalid, if we can find out DR genes associated with the disease, this will contribute to a better understanding of the pathological mechanisms and contribute to the development of new treatments and drugs. The current research progress of AR, AR gene polymorphism, Aldose reductase inhibitors to DR was reviewed in this article.

  7. Inhibition of steroid 5 alpha-reductase by specific aliphatic unsaturated fatty acids.

    Science.gov (United States)

    Liang, T; Liao, S

    1992-01-01

    Human or rat microsomal 5 alpha-reductase activity, as measured by enzymic conversion of testosterone into 5 alpha-dihydrotestosterone or by binding of a competitive inhibitor, [3H]17 beta-NN-diethulcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one ([3H]4-MA) to the reductase, is inhibited by low concentrations (less than 10 microM) of certain polyunsaturated fatty acids. The relative inhibitory potencies of unsaturated fatty acids are, in decreasing order: gamma-linolenic acid greater than cis-4,7,10,13,16,19-docosahexaenoic acid = cis-6,9,12,15-octatetraenoic acid = arachidonic acid = alpha-linolenic acid greater than linoleic acid greater than palmitoleic acid greater than oleic acid greater than myristoleic acid. Other unsaturated fatty acids such as undecylenic acid, erucic acid and nervonic acid, are inactive. The methyl esters and alcohol analogues of these compounds, glycerols, phospholipids, saturated fatty acids, retinoids and carotenes were inactive even at 0.2 mM. The results of the binding assay and the enzymic assay correlated well except for elaidic acid and linolelaidic acid, the trans isomers of oleic acid and linoleic acid respectively, which were much less active than their cis isomers in the binding assay but were as potent in the enzymic assay. gamma-Linolenic acid had no effect on the activities of two other rat liver microsomal enzymes: NADH:menadione reductase and glucuronosyl transferase. gamma-Linolenic acid, the most potent inhibitor tested, decreased the Vmax. and increased Km values of substrates, NADPH and testosterone, and promoted dissociation of [3H]4-MA from the microsomal reductase. gamma-Linolenic acid, but not the corresponding saturated fatty acid (stearic acid), inhibited the 5 alpha-reductase activity, but not the 17 beta-dehydrogenase activity, of human prostate cancer cells in culture. These results suggest that unsaturated fatty acids may play an important role in regulating androgen action in target cells. PMID:1637346

  8. Knockdown of RMI1 impairs DNA repair under DNA replication stress.

    Science.gov (United States)

    Xu, Chang; Fang, Lianying; Kong, Yangyang; Xiao, Changyan; Yang, Mengmeng; Du, Li-Qing; Liu, Qiang

    2017-12-09

    RMI1 (RecQ-mediated genome instability protein 1) forms a conserved BTR complex with BLM, Topo IIIα, and RMI2, and its absence causes genome instability. It has been revealed that RMI1 localizes to nuclear foci with BLM and Topo IIIα in response to replication stress, and that RMI1 functions downstream of BLM in promoting replication elongation. However, the precise functions of RMI1 during replication stress are not completely understood. Here we report that RMI1 knockdown cells are hypersensitive to hydroxyurea (HU). Using comet assay, we show that RMI1 knockdown cells exhibit accumulation of broken DNAs after being released from HU treatment. Moreover, we demonstrate that RMI1 facilitates the recovery from activated checkpoint and resuming the cell cycle after replicative stress. Surprisingly, loss of RMI1 results in a failure of RAD51 loading onto DNA damage sites. These findings reveal the importance of RMI1 in response to replication stress, which could explain the molecular basis for its function in maintaining genome integrity. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Expression, purification, crystallization and preliminary X-ray analysis of perakine reductase, a new member of the aldo-keto reductase enzyme superfamily from higher plants

    Science.gov (United States)

    Rosenthal, Cindy; Mueller, Uwe; Panjikar, Santosh; Sun, Lianli; Ruppert, Martin; Zhao, Yu; Stöckigt, Joachim

    2006-01-01

    Perakine reductase (PR) is a novel member of the aldo-keto reductase enzyme superfamily from higher plants. PR from the plant Rauvolfia serpentina is involved in the biosynthesis of monoterpenoid indole alkaloids by performing NADPH-dependent reduction of perakine, yielding raucaffrinoline. However, PR can also reduce cinnamic aldehyde and some of its derivatives. After heterologous expression of a triple mutant of PR in Escherichia coli, crystals of the purified and methylated enzyme were obtained by the hanging-drop vapour-diffusion technique at 293 K with 100 mM sodium citrate pH 5.6 and 27% PEG 4000 as precipitant. Crystals belong to space group C2221 and diffract to 2.0 Å, with unit-cell parameters a = 58.9, b = 93.0, c = 143.4 Å. PMID:17142919

  10. The Nox/Ferric reductase/Ferric reductase-like families of Eumycetes.

    Science.gov (United States)

    Grissa, Ibtissem; Bidard, Frédérique; Grognet, Pierre; Grossetete, Sandrine; Silar, Philippe

    2010-09-01

    Reactive Oxygen Species (ROS) are involved in plant biomass degradation by fungi and development of fungal structures. While the ROS-generating NADPH oxidases from filamentous fungi are under strong scrutiny, much less is known about the related integral Membrane (or Ferric) Reductases (IMRs). Here, we present a survey of these enzymes in 29 fungal genomes covering the entire available range of fungal diversity. IMRs are present in all fungal genomes. They can be classified into at least 24 families, underscoring the high diversity of these enzymes. Some are differentially regulated during colony or fruiting body development, as well as by the nature of the carbon source of the growth medium. Importantly, functional characterization of IMRs has been made on proteins belonging to only two families, while nothing or very little is known about the proteins of the other 22 families. Copyright © 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  11. Crystallization and preliminary X-ray diffraction studies of ferredoxin reductase from Leptospira interrogans

    International Nuclear Information System (INIS)

    Nascimento, Alessandro S.; Ferrarezi, Thiago; Catalano-Dupuy, Daniela L.; Ceccarelli, Eduardo A.; Polikarpov, Igor

    2006-01-01

    Crystals adequate for X-ray diffraction analysis have been prepared from L. interrogans ferredoxin-NADP + reductase. Ferredoxin-NADP + reductase (FNR) is an FAD-containing enzyme that catalyzes electron transfer between NADP(H) and ferredoxin. Here, results are reported of the recombinant expression, purification and crystallization of FNR from Leptospira interrogans, a parasitic bacterium of animals and humans. The L. interrogans FNR crystals belong to a primitive monoclinic space group and diffract to 2.4 Å resolution at a synchrotron source

  12. Crystallization and preliminary X-ray diffraction studies of ferredoxin reductase from Leptospira interrogans

    Energy Technology Data Exchange (ETDEWEB)

    Nascimento, Alessandro S.; Ferrarezi, Thiago [Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Saocarlense 400, São Carlos, SP, 13560-970 (Brazil); Catalano-Dupuy, Daniela L.; Ceccarelli, Eduardo A. [Facultad de Ciencias Bioquímicas y Farmacéuticas, Molecular Biology Division, Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET, Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario (Argentina); Polikarpov, Igor, E-mail: ipolikarpov@if.sc.usp.br [Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Saocarlense 400, São Carlos, SP, 13560-970 (Brazil)

    2006-07-01

    Crystals adequate for X-ray diffraction analysis have been prepared from L. interrogans ferredoxin-NADP{sup +} reductase. Ferredoxin-NADP{sup +} reductase (FNR) is an FAD-containing enzyme that catalyzes electron transfer between NADP(H) and ferredoxin. Here, results are reported of the recombinant expression, purification and crystallization of FNR from Leptospira interrogans, a parasitic bacterium of animals and humans. The L. interrogans FNR crystals belong to a primitive monoclinic space group and diffract to 2.4 Å resolution at a synchrotron source.

  13. The Drosophila carbonyl reductase sniffer is an efficient 4-oxonon-2-enal (4ONE) reductase.

    Science.gov (United States)

    Martin, Hans-Jörg; Ziemba, Marta; Kisiela, Michael; Botella, José A; Schneuwly, Stephan; Maser, Edmund

    2011-05-30

    Studies with the fruit-fly Drosophila melanogaster demonstrated that the enzyme sniffer prevented oxidative stress-induced neurodegeneration. Mutant flies overexpressing sniffer had significantly extended life spans in a 99.5% oxygen atmosphere compared to wild-type flies. However, the molecular mechanism of this protection remained unclear. Sequence analysis and database searches identified sniffer as a member of the short-chain dehydrogenase/reductase superfamily with a 27.4% identity to the human enzyme carbonyl reductase type I (CBR1). As CBR1 catalyzes the reduction of the lipid peroxidation products 4HNE and 4ONE, we tested whether sniffer is able to metabolize these lipid derived aldehydes by carbonyl reduction. To produce recombinant enzyme, the coding sequence of sniffer was amplified from a cDNA-library, cloned into a bacterial expression vector and the His-tagged protein was purified by Ni-chelate chromatography. We found that sniffer catalyzed the NADPH-dependent carbonyl reduction of 4ONE (K(m)=24±2 μM, k(cat)=500±10 min(-1), k(cat)/K(m)=350 s(-1) mM(-1)) but not that of 4HNE. The reaction product of 4ONE reduction by sniffer was mainly 4HNE as shown by HPLC- and GC/MS analysis. Since 4HNE, though still a potent electrophile, is less neurotoxic and protein reactive than 4ONE, one mechanism by which sniffer exerts its neuroprotective effects in Drosophila after oxidative stress may be enzymatic reduction of 4ONE. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  14. Crystallization of purple nitrous oxide reductase from Pseudomonas stutzeri

    International Nuclear Information System (INIS)

    Pomowski, Anja; Zumft, Walter G.; Kroneck, Peter M. H.; Einsle, Oliver

    2010-01-01

    The physiologically active form of nitrous oxide reductase was isolated and crystallized under strict exclusion of dioxygen and diffraction data were collected from crystals belonging to two different space groups. Nitrous oxide reductase (N 2 OR) from Pseudomonas stutzeri catalyzes the final step in denitrification: the two-electron reduction of nitrous oxide to molecular dinitrogen. Crystals of the enzyme were grown under strict exclusion of dioxygen by sitting-drop vapour diffusion using 2R,3R-butanediol as a cryoprotectant. N 2 OR crystallized in either space group P1 or P6 5 . Interestingly, the key determinant for the resulting space group was the crystallization temperature. Crystals belonging to space group P1 contained four 130 kDa dimers in the asymmetric unit, while crystals belonging to space group P6 5 contained a single dimer in the asymmetric unit. Diffraction data were collected to resolutions better than 2 Å

  15. Purification and kinetic analysis of cytosolic and mitochondrial thioredoxin glutathione reductase extracted from Taenia solium cysticerci.

    Science.gov (United States)

    Plancarte, Agustin; Nava, Gabriela

    2015-02-01

    Thioredoxin glutathione reductases (TGRs) (EC 1.8.1.9) were purified to homogeneity from the cytosolic (cTsTGR) and mitochondrial (mTsTGR) fractions of Taenia solium, the agent responsible for neurocysticercosis, one of the major central nervous system parasitic diseases in humans. TsTGRs had a relative molecular weight of 132,000, while the corresponding value per subunit obtained under denaturing conditions, was of 62,000. Specific activities for thioredoxin reductase and glutathione reductase substrates for both TGRs explored were in the range or lower than values obtained for other platyhelminths and mammalian TGRs. cTsTGR and mTsTGR also showed hydroperoxide reductase activity using hydroperoxide as substrate. Km(DTNB) and Kcat(DTNB) values for cTsTGR and mTsTGR (88 µM and 1.9 s(-1); 45 µM and 12.6 s(-1), respectively) and Km(GSSG) and Kcat(GSSG) values for cTsTGR and mTsTGR (6.3 µM and 0.96 s(-1); 4 µM and 1.62 s(-1), respectively) were similar to or lower than those reported for mammalian TGRs. Mass spectrometry analysis showed that 12 peptides from cTsTGR and seven from mTsTGR were a match for gi|29825896 thioredoxin glutathione reductase [Echinococcus granulosus], confirming that both enzymes are TGRs. Both T. solium TGRs were inhibited by the gold compound auranofin, a selective inhibitor of thiol-dependent flavoreductases (I₅₀ = 3.25, 2.29 nM for DTNB and GSSG substrates, respectively for cTsTGR; I₅₀ = 5.6, 25.4 nM for mTsTGR toward the same substrates in the described order). Glutathione reductase activity of cTsTGR and mTsTGR exhibited hysteretic behavior with moderate to high concentrations of GSSG; this result was not observed either with thioredoxin, DTNB or NADPH. However, the observed hysteretic kinetics was suppressed with increasing amounts of both parasitic TGRs. These data suggest the existence of an effective substitute which may account for the lack of the detoxification enzymes glutathione reductase

  16. Role of aldose reductase C-106T polymorphism among diabetic Egyptian patients with different microvascular complications

    Directory of Open Access Journals (Sweden)

    Nermine Hossam Zakaria

    2014-04-01

    Full Text Available The aldose reductase pathway proves that elevated blood glucose promotes cellular dysfunction. The polyol pathway converts excess intracellular glucose into alcohols via activity of the aldose reductase. This enzyme catalyzes the conversion of glucose to sorbitol which triggers variety of intracellular changes in the tissues. Among diabetes, activity is drastically increased in association with three main consequences inside the cells. The aim of this study was to detect the association of the C-106 T polymorphism of the aldose reductase gene and its frequency among a sample of 150 Egyptian adults with type 2 diabetic patients having diabetic microvascular. The detection of the aldose reductase C-106 T polymorphism gene was done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP. The genotype distribution of the C-106 T polymorphism showed that CC genotype was statistically significantly higher among patients with retinopathy compared to nephropathy. Patients with nephropathy had significant association with the TT genotype when compared with diabetic retinopathy patients. Follow up study after the genotype detection among recently diagnosed diabetic patients in order to give a prophylactic aldose reductase inhibitors; studying the microvascular complications and its relation to the genotype polymorphisms. The study may include multiple gene polymorphisms to make the relation between the gene and the occurrence of these complications more evident.

  17. Nitrate reductase and nitrous oxide production by Fusarium oxysporum 11dn1 under aerobic and anaerobic conditions.

    Science.gov (United States)

    Kurakov, A V; Nosikov, A N; Skrynnikova, E V; L'vov, N P

    2000-08-01

    The fungus Fusarium oxysporum 11dn1 was found to be able to grow and produce nitrous oxide on nitrate-containing medium in anaerobic conditions. The rate of nitrous oxide formation was three to six orders of magnitude lower than the rates of molecular nitrogen production by common denitrifying bacteria. Acetylene and ammonia did not affect the release of nitrous oxide release. It was shown that under anaerobic conditions fast increase of nitrate reductase activity occurred, caused by the synthesis of enzyme de novo and protein dephosphorylation. Reverse transfer of the mycelium to aerobic conditions led to a decline in nitrate reductase activity and stopped nitrous oxide production. The presence of two nitrate reductases was shown, which differed in molecular mass, location, temperature optima, and activity in nitrate- and ammonium-containing media. Two enzymes represent assimilatory and dissimilatory nitrate reductases, which are active in aerobic and anaerobic conditions, respectively.

  18. A multicolor panel of TALE-KRAB based transcriptional repressor vectors enabling knockdown of multiple gene targets.

    Science.gov (United States)

    Zhang, Zhonghui; Wu, Elise; Qian, Zhijian; Wu, Wen-Shu

    2014-12-05

    Stable and efficient knockdown of multiple gene targets is highly desirable for dissection of molecular pathways. Because it allows sequence-specific DNA binding, transcription activator-like effector (TALE) offers a new genetic perturbation technique that allows for gene-specific repression. Here, we constructed a multicolor lentiviral TALE-Kruppel-associated box (KRAB) expression vector platform that enables knockdown of multiple gene targets. This platform is fully compatible with the Golden Gate TALEN and TAL Effector Kit 2.0, a widely used and efficient method for TALE assembly. We showed that this multicolor TALE-KRAB vector system when combined together with bone marrow transplantation could quickly knock down c-kit and PU.1 genes in hematopoietic stem and progenitor cells of recipient mice. Furthermore, our data demonstrated that this platform simultaneously knocked down both c-Kit and PU.1 genes in the same primary cell populations. Together, our results suggest that this multicolor TALE-KRAB vector platform is a promising and versatile tool for knockdown of multiple gene targets and could greatly facilitate dissection of molecular pathways.

  19. Expression profile of CREB knockdown in myeloid leukemia cells

    International Nuclear Information System (INIS)

    Pellegrini, Matteo; Cheng, Jerry C; Voutila, Jon; Judelson, Dejah; Taylor, Julie; Nelson, Stanley F; Sakamoto, Kathleen M

    2008-01-01

    The cAMP Response Element Binding Protein, CREB, is a transcription factor that regulates cell proliferation, differentiation, and survival in several model systems, including neuronal and hematopoietic cells. We demonstrated that CREB is overexpressed in acute myeloid and leukemia cells compared to normal hematopoietic stem cells. CREB knockdown inhibits leukemic cell proliferation in vitro and in vivo, but does not affect long-term hematopoietic reconstitution. To understand downstream pathways regulating CREB, we performed expression profiling with RNA from the K562 myeloid leukemia cell line transduced with CREB shRNA. By combining our expression data from CREB knockdown cells with prior ChIP data on CREB binding we were able to identify a list of putative CREB regulated genes. We performed extensive analyses on the top genes in this list as high confidence CREB targets. We found that this list is enriched for genes involved in cancer, and unexpectedly, highly enriched for histone genes. Furthermore, histone genes regulated by CREB were more likely to be specifically expressed in hematopoietic lineages. Decreased expression of specific histone genes was validated in K562, TF-1, and primary AML cells transduced with CREB shRNA. We have identified a high confidence list of CREB targets in K562 cells. These genes allow us to begin to understand the mechanisms by which CREB contributes to acute leukemia. We speculate that regulation of histone genes may play an important role by possibly altering the regulation of DNA replication during the cell cycle

  20. Transcriptional modulation of genes encoding nitrate reductase in ...

    African Journals Online (AJOL)

    The free aluminum (Al) content in soil can reach levels that are toxic to plants, and this has frequently limited increased productivity of cultures. Four genes encoding nitrate reductase (NR) were identified, named ZmNR1–4. With the aim of evaluating NR activity and the transcriptional modulation of the ZmNR1, ZmNR2, ...

  1. DNA damage induction of ribonucleotide reductase.

    OpenAIRE

    Elledge, S J; Davis, R W

    1989-01-01

    RNR2 encodes the small subunit of ribonucleotide reductase, the enzyme that catalyzes the first step in the pathway for the production of deoxyribonucleotides needed for DNA synthesis. RNR2 is a member of a group of genes whose activities are cell cycle regulated and that are transcriptionally induced in response to the stress of DNA damage. An RNR2-lacZ fusion was used to further characterize the regulation of RNR2 and the pathway responsible for its response to DNA damage. beta-Galactosidas...

  2. Cell death by SecTRAPs: thioredoxin reductase as a prooxidant killer of cells.

    Directory of Open Access Journals (Sweden)

    Karin Anestål

    Full Text Available BACKGROUND: SecTRAPs (selenium compromised thioredoxin reductase-derived apoptotic proteins can be formed from the selenoprotein thioredoxin reductase (TrxR by targeting of its selenocysteine (Sec residue with electrophiles, or by its removal through C-terminal truncation. SecTRAPs are devoid of thioredoxin reductase activity but can induce rapid cell death in cultured cancer cell lines by a gain of function. PRINCIPAL FINDINGS: Both human and rat SecTRAPs killed human A549 and HeLa cells. The cell death displayed both apoptotic and necrotic features. It did not require novel protein synthesis nor did it show extensive nuclear fragmentation, but it was attenuated by use of caspase inhibitors. The redox active disulfide/dithiol motif in the N-terminal domain of TrxR had to be maintained for manifestation of SecTRAP cytotoxicity. Stopped-flow kinetics showed that NADPH can reduce the FAD moiety in SecTRAPs at similar rates as in native TrxR and purified SecTRAPs could maintain NADPH oxidase activity, which was accelerated by low molecular weight substrates such as juglone. In a cellular context, SecTRAPs triggered extensive formation of reactive oxygen species (ROS and consequently antioxidants could protect against the cell killing by SecTRAPs. CONCLUSIONS: We conclude that formation of SecTRAPs could contribute to the cytotoxicity seen upon exposure of cells to electrophilic agents targeting TrxR. SecTRAPs are prooxidant killers of cells, triggering mechanisms beyond those of a mere loss of thioredoxin reductase activity.

  3. Crystallization and preliminary X-ray analysis of the NADPH-dependent 3-quinuclidinone reductase from Rhodotorula rubra

    International Nuclear Information System (INIS)

    Takeshita, Daijiro; Kataoka, Michihiko; Miyakawa, Takuya; Miyazono, Ken-ichi; Uzura, Atsuko; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2009-01-01

    The NADPH-dependent 3-quinuclidinone reductase from Rhodotorula rubra was expressed, purified, and crystallized and X-ray diffraction data of this crystal were collected to 2.2 Å resolution. (R)-3-Quinuclidinol is a useful compound that is applicable to the synthesis of various pharmaceuticals. The NADPH-dependent carbonyl reductase 3-quinuclidinone reductase from Rhodotorula rubra catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol and is expected to be utilized in industrial production of this alcohol. 3-Quinuclidinone reductase from R. rubra was expressed in Escherichia coli and purified using Ni-affinity and ion-exchange column chromatography. Crystals of the protein were obtained by the sitting-drop vapour-diffusion method using PEG 8000 as the precipitant. The crystals belonged to space group P4 1 2 1 2, with unit-cell parameters a = b = 91.3, c = 265.4 Å, and diffracted X-rays to 2.2 Å resolution. The asymmetric unit contained four molecules of the protein and the solvent content was 48.4%

  4. Regulation of ribonucleotide reductase by Spd1 involves multiple mechanisms

    DEFF Research Database (Denmark)

    Nestoras, Konstantinos; Mohammed, Asma Hadi; Schreurs, Ann-Sofie

    2010-01-01

    The correct levels of deoxyribonucleotide triphosphates and their relative abundance are important to maintain genomic integrity. Ribonucleotide reductase (RNR) regulation is complex and multifaceted. RNR is regulated allosterically by two nucleotide-binding sites, by transcriptional control, and...

  5. Relationship between nitrate reductase and nitrate uptake in phytoplankton in the Peru upwelling region

    International Nuclear Information System (INIS)

    Blasco, D.; MacIsaac, J.J.; Packard, T.T.; Dugdale, R.C.

    1984-01-01

    Nitrate reductase (NR) activity and 15 NO 3 - uptake in phytoplankton were compared under different environmental conditions on two cruises in the upwelling region off Peru. The NR activity and NO 3 - uptake rates responded differently to light and nutrients and the differences led to variations in the uptake: reductase ratio. Analysis of these variations suggests that the re-equilibration time of the two processes in response to environmental perturbation is an important source of variability. The nitrate uptake system responds faster than the nitrate reductase system. Considering these differences in response time the basic differences in the two processes, and the differences in their measurement, the authors conclude that the Nr activity measures the current nitrate-reducing potential, which reflects NO 3 - assimilation before the sampling time, while 15 NO 3 - uptake measures NO 3 - assimilation in the 6-h period following sampling

  6. Deiodinase knockdown during early zebrafish development affects growth, development, energy metabolism, motility and phototransduction.

    Directory of Open Access Journals (Sweden)

    Enise Bagci

    Full Text Available Thyroid hormone (TH balance is essential for vertebrate development. Deiodinase type 1 (D1 and type 2 (D2 increase and deiodinase type 3 (D3 decreases local intracellular levels of T3, the most important active TH. The role of deiodinase-mediated TH effects in early vertebrate development is only partially understood. Therefore, we investigated the role of deiodinases during early development of zebrafish until 96 hours post fertilization at the level of the transcriptome (microarray, biochemistry, morphology and physiology using morpholino (MO knockdown. Knockdown of D1+D2 (D1D2MO and knockdown of D3 (D3MO both resulted in transcriptional regulation of energy metabolism and (muscle development in abdomen and tail, together with reduced growth, impaired swim bladder inflation, reduced protein content and reduced motility. The reduced growth and impaired swim bladder inflation in D1D2MO could be due to lower levels of T3 which is known to drive growth and development. The pronounced upregulation of a large number of transcripts coding for key proteins in ATP-producing pathways in D1D2MO could reflect a compensatory response to a decreased metabolic rate, also typically linked to hypothyroidism. Compared to D1D2MO, the effects were more pronounced or more frequent in D3MO, in which hyperthyroidism is expected. More specifically, increased heart rate, delayed hatching and increased carbohydrate content were observed only in D3MO. An increase of the metabolic rate, a decrease of the metabolic efficiency and a stimulation of gluconeogenesis using amino acids as substrates may have been involved in the observed reduced protein content, growth and motility in D3MO larvae. Furthermore, expression of transcripts involved in purine metabolism coupled to vision was decreased in both knockdown conditions, suggesting that both may impair vision. This study provides new insights, not only into the role of deiodinases, but also into the importance of a correct

  7. Alpha2,3-sialyltransferase III knockdown sensitized ovarian cancer cells to cisplatin-induced apoptosis.

    Science.gov (United States)

    Wang, Xiaoyu; Zhang, Yiting; Lin, Haiyingjie; Liu, Yan; Tan, Yi; Lin, Jie; Gao, Fenze; Lin, Shaoqiang

    2017-01-22

    Emerging evidence indicates that β-galactoside-α2,3-sialyltransferase III (ST3Gal3) involves in development, inflammation, neoplastic transformation, and metastasis. However, the role of ST3Gal3 in regulating cancer chemoresistance remains elusive. Herein, we investigated the functional effects of ST3Gal3 in cisplatin-resistant ovarian cancer cells. We found that the levels of ST3Gal3 mRNA differed significantly among ovarian cancer cell lines. HO8910PM cells that have high invasive and metastatic capacity express elevated ST3Gal3 mRNA and are resistant to cisplatin, comparing to SKOV3 cells that have a lower level of ST3Gal3 expression and are more chemosensitive to cisplatin. We found that the expression of ST3Gal3 has reverse correlation with the dosage of cisplatin used in both SKOV3 and HO8910PM cells, and high dose of cisplatin could down-regulate ST3Gal3 expression. We then examined the functional effects of ST3Gal3 knockdown in cancer cell lines using FACS analysis. The number of apoptotic cells was much higher in cells if ST3Gal3 expression was knocked down by siRNA and/or by treating cells with higher dosage of cisplatin in comparison to control cells. Interestingly, in HO8910PM cells with ST3Gal3 knockdown, the levels of caspase 8 and caspase 3 proteins increased, which was more obvious in cells treated with both ST3Gal3 knockdown and cisplatin, suggesting that ST3Gal3 knockdown synergistically enhanced cisplatin-induced apoptosis in ovarian cancer cells. Taken together, these results uncover an alternative mechanism of cisplatin-resistance through ST3Gal3 and open a window for effective prevention of chemoresistance and relapse of ovarian cancer by targeting ST3Gal3. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Cloning and characterization of a nitrite reductase gene related to ...

    African Journals Online (AJOL)

    STORAGESEVER

    2010-03-01

    Mar 1, 2010 ... Alexander et al., 2005) and heme-type nitrite reductase gene (Smith and ... owing to a genotype-dependent response (Zhang et al.,. 1991; Sakhanokho et al., ..... Improvement of cell culture conditions for rice. Jpn. Agric. Res.

  9. Effect of ammonium and nitrate on ferric chelate reductase and nitrate reductase in Vaccinium species.

    Science.gov (United States)

    Poonnachit, U; Darnell, R

    2004-04-01

    Most Vaccinium species have strict soil requirements for optimal growth, requiring low pH, high iron availability and nitrogen primarily in the ammonium form. These soils are limited and are often located near wetlands. Vaccinium arboreum is a wild species adapted to a wide range of soils, including high pH, low iron, and nitrate-containing soils. This broader soil adaptation in V. arboreum may be related to increased efficiency of iron or nitrate uptake compared with the cultivated Vaccinium species. Nitrate, ammonium and iron uptake, and nitrate reductase (NR) and ferric chelate reductase (FCR) activities were compared in two Vaccinium species grown hydroponically in either nitrate or ammonia, with or without iron. The species studied were the wild V. arboreum and the cultivated V. corymbosum interspecific hybrid, which exhibits the strict soil requirements of most Vaccinium species. Ammonium uptake was significantly greater than nitrate uptake in both species, while nitrate uptake was greater in the wild species, V. arboreum, compared with the cultivated species, V. corymbosum. The increased nitrate uptake in V. arboreum was correlated with increased root NR activity compared with V. corymbosum. The lower nitrate uptake in V. corymbosum was reflected in decreased plant dry weight in this species compared with V. arboreum. Root FCR activity increased significantly in V. corymbosum grown under iron-deficient conditions, compared with the same species grown under iron-sufficient conditions or with V. arboreum grown under either iron condition. V. arboreum appears to be more efficient in acquiring nitrate compared with V. corymbosum, possibly due to increased NR activity and this may partially explain the wider soil adaptation of V. arboreum.

  10. Towards New Antifolates Targeting Eukaryotic Opportunistic Infections

    Energy Technology Data Exchange (ETDEWEB)

    Liu, J.; Bolstad, D; Bolstad, E; Wright, D; Anderson, A

    2009-01-01

    Trimethoprim, an antifolate commonly prescribed in combination with sulfamethoxazole, potently inhibits several prokaryotic species of dihydrofolate reductase (DHFR). However, several eukaryotic pathogenic organisms are resistant to trimethoprim, preventing its effective use as a therapeutic for those infections. We have been building a program to reengineer trimethoprim to more potently and selectively inhibit eukaryotic species of DHFR as a viable strategy for new drug discovery targeting several opportunistic pathogens. We have developed a series of compounds that exhibit potent and selective inhibition of DHFR from the parasitic protozoa Cryptosporidium and Toxoplasma as well as the fungus Candida glabrata. A comparison of the structures of DHFR from the fungal species Candida glabrata and Pneumocystis suggests that the compounds may also potently inhibit Pneumocystis DHFR.

  11. Shp2 knockdown and Noonan/LEOPARD mutant Shp2-induced gastrulation defects.

    Directory of Open Access Journals (Sweden)

    Chris Jopling

    2007-12-01

    Full Text Available Shp2 is a cytoplasmic protein-tyrosine phosphatase that is essential for normal development. Activating and inactivating mutations have been identified in humans to cause the related Noonan and LEOPARD syndromes, respectively. The cell biological cause of these syndromes remains to be determined. We have used the zebrafish to assess the role of Shp2 in early development. Here, we report that morpholino-mediated knockdown of Shp2 in zebrafish resulted in defects during gastrulation. Cell tracing experiments demonstrated that Shp2 knockdown induced defects in convergence and extension cell movements. In situ hybridization using a panel of markers indicated that cell fate was not affected by Shp2 knock down. The Shp2 knockdown-induced defects were rescued by active Fyn and Yes and by active RhoA. We generated mutants of Shp2 with mutations that were identified in human patients with Noonan or LEOPARD Syndrome and established that Noonan Shp2 was activated and LEOPARD Shp2 lacked catalytic protein-tyrosine phosphatase activity. Expression of Noonan or LEOPARD mutant Shp2 in zebrafish embryos induced convergence and extension cell movement defects without affecting cell fate. Moreover, these embryos displayed craniofacial and cardiac defects, reminiscent of human symptoms. Noonan and LEOPARD mutant Shp2s were not additive nor synergistic, consistent with the mutant Shp2s having activating and inactivating roles in the same signaling pathway. Our results demonstrate that Shp2 is required for normal convergence and extension cell movements during gastrulation and that Src family kinases and RhoA were downstream of Shp2. Expression of Noonan or LEOPARD Shp2 phenocopied the craniofacial and cardiac defects of human patients. The finding that defective Shp2 signaling induced cell movement defects as early as gastrulation may have implications for the monitoring and diagnosis of Noonan and LEOPARD syndrome.

  12. Expression, purification and molecular structure modeling of thioredoxin (Trx) and thioredoxin reductase (TrxR) from Acidithiobacillus ferrooxidans.

    Science.gov (United States)

    Wang, Yiping; Zhang, Xiaojian; Liu, Qing; Ai, Chenbing; Mo, Hongyu; Zeng, Jia

    2009-07-01

    The thioredoxin system consists of thioredoxin (Trx), thioredoxin reductase (TrxR) and NADPH, which plays several key roles in maintaining the redox environment of the cell. In Acidithiobacillus ferrooxidans, thioredoxin system may play important functions in the activity regulation of periplasmic proteins and energy metabolism. Here, we cloned thioredoxin (trx) and thioredoxin reductase (trxR) genes from Acidithiobacillus ferrooxidans, and expressed the genes in Escherichia coli. His-Trx and His-TrxR were purified to homogeneity with one-step Ni-NTA affinity column chromatography. Site-directed mutagenesis results confirmed that Cys33, Cys36 of thioredoxin, and Cys142, Cys145 of thioredoxin reductase were active-site residues.

  13. Knockdown of Zebrafish Blood Vessel Epicardial Substance Results in Incomplete Retinal Lamination

    Directory of Open Access Journals (Sweden)

    Yu-Ching Wu

    2014-01-01

    Full Text Available Cell polarity during eye development determines the normal retinal lamination and differentiation of photoreceptor cells in the retina. In vertebrates, blood vessel epicardial substance (Bves is known to play an important role in the formation and maintenance of the tight junctions essential for epithelial cell polarity. In the current study, we generated a transgenic zebrafish Bves (zbves promoter-EGFP zebrafish line to investigate the expression pattern of Bves in the retina and to study the role of zbves in retinal lamination. Immunostaining with different specific antibodies from retinal cells and transmission electron microscopy were used to identify the morphological defects in normal and Bves knockdown zebrafish. In normal zebrafish, Bves is located at the apical junctions of embryonic retinal neuroepithelia during retinogenesis; later, it is strongly expressed around inner plexiform layer (IPL and retinal pigment epithelium (RPE. In contrast, a loss of normal retinal lamination and cellular polarity was found with undifferentiated photoreceptor cells in Bves knockdown zebrafish. Herein, our results indicated that disruption of Bves will result in a loss of normal retinal lamination.

  14. The NADPH thioredoxin reductase C functions as an electron donor to 2-Cys peroxiredoxin in a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1

    International Nuclear Information System (INIS)

    Sueoka, Keigo; Yamazaki, Teruaki; Hiyama, Tetsuo; Nakamoto, Hitoshi

    2009-01-01

    An NADPH thioredoxin reductase C was co-purified with a 2-Cys peroxiredoxin by the combination of anion exchange chromatography and electroelution from gel slices after native PAGE from a thermophilic cyanobacterium Thermosynechococcus elongatus as an NAD(P)H oxidase complex induced by oxidative stress. The result provided a strong evidence that the NADPH thioredoxin reductase C interacts with the 2-Cys peroxiredoxin in vivo. An in vitro reconstitution assay with purified recombinant proteins revealed that both proteins were essential for an NADPH-dependent reduction of H 2 O 2 . These results suggest that the reductase transfers the reducing power from NADPH to the peroxiredoxin, which reduces peroxides in the cyanobacterium under oxidative stress. In contrast with other NADPH thioredoxin reductases, the NADPH thioredoxin reductase C contains a thioredoxin-like domain in addition to an NADPH thioredoxin reductase domain in the same polypeptide. Each domain contains a conserved CXYC motif. A point mutation at the CXYC motif in the NADPH thioredoxin reductase domain resulted in loss of the NADPH oxidation activity, while a mutation at the CXYC motif in the thioredoxin-like domain did not affect the electron transfer, indicating that this motif is not essential in the electron transport from NADPH to the 2-Cys peroxiredoxin.

  15. Alpha 1-blockers vs 5 alpha-reductase inhibitors in benign prostatic hyperplasia. A comparative review

    DEFF Research Database (Denmark)

    Andersen, J T

    1995-01-01

    During recent years, pharmacological treatment of symptomatic benign prostatic hyperplasia (BPH) has become the primary treatment choice for an increasing number of patients. The 2 principal drug classes employed are alpha 1-blockers and 5 alpha-reductase inhibitors. Current information from...... of patients who will respond well to alpha 1-blockers have yet to be identified, and data concerning the long term effects of these drugs are not yet available. 5 alpha-Reductase inhibitors have a slow onset of effect, but treatment leads to improvement in symptoms, reduction of the size of the prostate gland...... and improvement in objective parameters for bladder outflow obstruction. Approximately 30 to 50% of patients will respond to treatment with 5 alpha-reductase inhibitors. The definitive role of pharmacological treatment in symptomatic BPH remains to be established, although it seems that patients unfit...

  16. Reconstitution of FMN-free NADPH-cytochrome P-450 reductase with a phosphorothioate analog of FMN: 31P NMR studies of the reconstituted protein

    International Nuclear Information System (INIS)

    Krum, D.P.; Otvos, J.D.; Calhoun, J.P.; Miziorko, H.M.; Masters, B.S.S.

    1987-01-01

    A phosphorothioate analog of FMN (FMNS) has been synthesized and shown to be completely competent in reconstituting the FMN-free form of NADPH-cytochrome P-450 reductase as evidenced by flavin determinations and cytochrome c reductase activity assays. The FMNS-reconstituted FMN-free reductase gives rise to an air-stable semiquinone, and the fluorescence of FMNS is quenched upon addition of FMN-free reductase. 31 P NMR spectra of the FMN-free reductase reveal only two resonances (-7.3 and -11.3 ppm), which are attributable to FAD. This result confirms the assignments of Otvos et al, and demonstrates unequivocally that there are no phosphate residues other than those of FMN and FAD attached to the steapsin-solubilized reductase. The addition of FMN to the FMN-free reductase resulted in the appearance of one additional resonance at 3.9 ppm. Addition of FMNS to the FMN-free reductase caused no change, surprisingly, in the 31 P NMR spectrum until Mn(II) was added, after which a peak centered at ∼ 45 ppm was observed. This unexpected result may be explained if the T 1 for the phosphate of FMNS is significantly longer than that of FMN, and suggests that the sulfur atom of FMNS may perturb the interaction of the phosphate with its protein environment. These results demonstrate the utility of phosphorothioate analogs as mechanistic probes for proteins containing nucleotide cofactors

  17. Evaluation of the conserve flavin reductase gene from three ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-12-15

    Dec 15, 2009 ... means of PCR technique. The nucleic acid sequences of the PCR primers were designed using conserved nucleic acid sequences of the flavin reductase enzyme from. Rhodococcus sp. strain IGTS8. The oligonucleotide primers were as follows: 5'-GAA TTC ATG TCT GAC. AAG CCG AAT GCC-3' (forward) ...

  18. Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains

    Directory of Open Access Journals (Sweden)

    Hahn-Hägerdal Bärbel

    2008-10-01

    Full Text Available Abstract Background Ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. This process would greatly benefit from recombinant Saccharomyces cerevisiae strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and xylose. Different pathways can be introduced in S. cerevisiae to provide arabinose and xylose utilisation. In this study, the bacterial arabinose isomerase pathway was combined with two different xylose utilisation pathways: the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways, respectively, in genetically identical strains. The strains were compared with respect to aerobic growth in arabinose and xylose batch culture and in anaerobic batch fermentation of a mixture of glucose, arabinose and xylose. Results The specific aerobic arabinose growth rate was identical, 0.03 h-1, for the xylose reductase/xylitol dehydrogenase and xylose isomerase strain. The xylose reductase/xylitol dehydrogenase strain displayed higher aerobic growth rate on xylose, 0.14 h-1, and higher specific xylose consumption rate in anaerobic batch fermentation, 0.09 g (g cells-1 h-1 than the xylose isomerase strain, which only reached 0.03 h-1 and 0.02 g (g cells-1h-1, respectively. Whereas the xylose reductase/xylitol dehydrogenase strain produced higher ethanol yield on total sugars, 0.23 g g-1 compared with 0.18 g g-1 for the xylose isomerase strain, the xylose isomerase strain achieved higher ethanol yield on consumed sugars, 0.41 g g-1 compared with 0.32 g g-1 for the xylose reductase/xylitol dehydrogenase strain. Anaerobic fermentation of a mixture of glucose, arabinose and xylose resulted in higher final ethanol concentration, 14.7 g l-1 for the xylose reductase/xylitol dehydrogenase strain compared with 11.8 g l-1 for the xylose isomerase strain, and in higher specific ethanol productivity, 0.024 g (g cells-1 h-1 compared with 0.01 g (g cells-1 h-1

  19. Attenuated food anticipatory activity and abnormal circadian locomotor rhythms in Rgs16 knockdown mice.

    Directory of Open Access Journals (Sweden)

    Naoto Hayasaka

    Full Text Available Regulators of G protein signaling (RGS are a multi-functional protein family, which functions in part as GTPase-activating proteins (GAPs of G protein α-subunits to terminate G protein signaling. Previous studies have demonstrated that the Rgs16 transcripts exhibit robust circadian rhythms both in the suprachiasmatic nucleus (SCN, the master circadian light-entrainable oscillator (LEO of the hypothalamus, and in the liver. To investigate the role of RGS16 in the circadian clock in vivo, we generated two independent transgenic mouse lines using lentiviral vectors expressing short hairpin RNA (shRNA targeting the Rgs16 mRNA. The knockdown mice demonstrated significantly shorter free-running period of locomotor activity rhythms and reduced total activity as compared to the wild-type siblings. In addition, when feeding was restricted during the daytime, food-entrainable oscillator (FEO-driven elevated food-anticipatory activity (FAA observed prior to the scheduled feeding time was significantly attenuated in the knockdown mice. Whereas the restricted feeding phase-advanced the rhythmic expression of the Per2 clock gene in liver and thalamus in the wild-type animals, the above phase shift was not observed in the knockdown mice. This is the first in vivo demonstration that a common regulator of G protein signaling is involved in the two separate, but interactive circadian timing systems, LEO and FEO. The present study also suggests that liver and/or thalamus regulate the food-entrained circadian behavior through G protein-mediated signal transduction pathway(s.

  20. Targeted siRNA Delivery and mRNA Knockdown Mediated by Bispecific Digoxigenin-binding Antibodies

    Directory of Open Access Journals (Sweden)

    Britta Schneider

    2012-01-01

    Full Text Available Bispecific antibodies (bsAbs that bind to cell surface antigens and to digoxigenin (Dig were used for targeted small interfering RNA (siRNA delivery. They are derivatives of immunoglobulins G (IgGs that bind tumor antigens, such as Her2, IGF1-R, CD22, and LeY, with stabilized Dig-binding variable domains fused to the C-terminal ends of the heavy chains. siRNA that was digoxigeninylated at its 3′end was bound in a 2:1 ratio to the bsAbs. These bsAb–siRNA complexes delivered siRNAs specifically to cells that express the corresponding antigen as demonstrated by flow cytometry and confocal microscopy. The complexes internalized into endosomes and Dig-siRNAs separated from bsAbs, but Dig-siRNA was not released into the cytoplasm; bsAb-targeting alone was thus not sufficient for effective mRNA knockdown. This limitation was overcome by formulating the Dig-siRNA into nanoparticles consisting of dynamic polyconjugates (DPCs or into lipid-based nanoparticles (LNPs. The resulting complexes enabled bsAb-targeted siRNA-specific messenger RNA (mRNA knockdown with IC50 siRNA values in the low nanomolar range for a variety of bsAbs, siRNAs, and target cells. Furthermore, pilot studies in mice bearing tumor xenografts indicated mRNA knockdown in endothelial cells following systemic co-administration of bsAbs and siRNA formulated in LNPs that were targeted to the tumor vasculature.

  1. Identification and functional evaluation of the reductases and dehydrogenases from Saccharomyces cerevisiae involved in vanillin resistance.

    Science.gov (United States)

    Wang, Xinning; Liang, Zhenzhen; Hou, Jin; Bao, Xiaoming; Shen, Yu

    2016-04-01

    Vanillin, a type of phenolic released during the pre-treatment of lignocellulosic materials, is toxic to microorganisms and therefore its presence inhibits the fermentation. The vanillin can be reduced to vanillyl alcohol, which is much less toxic, by the ethanol producer Saccharomyces cerevisiae. The reducing capacity of S. cerevisiae and its vanillin resistance are strongly correlated. However, the specific enzymes and their contribution to the vanillin reduction are not extensively studied. In our previous work, an evolved vanillin-resistant strain showed an increased vanillin reduction capacity compared with its parent strain. The transcriptome analysis suggested the reductases and dehydrogenases of this vanillin resistant strain were up-regulated. Using this as a starting point, 11 significantly regulated reductases and dehydrogenases were selected in the present work for further study. The roles of these reductases and dehydrogenases in the vanillin tolerance and detoxification abilities of S. cerevisiae are described. Among the candidate genes, the overexpression of the alcohol dehydrogenase gene ADH6, acetaldehyde dehydrogenase gene ALD6, glucose-6-phosphate 1-dehydrogenase gene ZWF1, NADH-dependent aldehyde reductase gene YNL134C, and aldo-keto reductase gene YJR096W increased 177, 25, 6, 15, and 18 % of the strain μmax in the medium containing 1 g L(-1) vanillin. The in vitro detected vanillin reductase activities of strain overexpressing ADH6, YNL134C and YJR096W were notably higher than control. The vanillin specific reduction rate increased by 8 times in ADH6 overexpressed strain but not in YNL134C and YJR096W overexpressed strain. This suggested that the enzymes encoded by YNL134C and YJR096W might prefer other substrate and/or could not show their effects on vanillin on the high background of Adh6p in vivo. Overexpressing ALD6 and ZWF1 mainly increased the [NADPH]/[NADP(+)] and [GSH]/[GSSG] ratios but not the vanillin reductase activities. Their

  2. Structural insights into the neuroprotective-acting carbonyl reductase Sniffer of Drosophila melanogaster.

    Science.gov (United States)

    Sgraja, Tanja; Ulschmid, Julia; Becker, Katja; Schneuwly, Stephan; Klebe, Gerhard; Reuter, Klaus; Heine, Andreas

    2004-10-01

    In vivo studies with the fruit-fly Drosophila melanogaster have shown that the Sniffer protein prevents age-dependent and oxidative stress-induced neurodegenerative processes. Sniffer is a NADPH-dependent carbonyl reductase belonging to the enzyme family of short-chain dehydrogenases/reductases (SDRs). The crystal structure of the homodimeric Sniffer protein from Drosophila melanogaster in complex with NADP+ has been determined by multiple-wavelength anomalous dispersion and refined to a resolution of 1.75 A. The observed fold represents a typical dinucleotide-binding domain as detected for other SDRs. With respect to the cofactor-binding site and the region referred to as substrate-binding loop, the Sniffer protein shows a striking similarity to the porcine carbonyl reductase (PTCR). This loop, in both Sniffer and PTCR, is substantially shortened compared to other SDRs. In most enzymes of the SDR family this loop adopts a well-defined conformation only after substrate binding and remains disordered in the absence of any bound ligands or even if only the dinucleotide cofactor is bound. In the structure of the Sniffer protein, however, the conformation of this loop is well defined, although no substrate is present. Molecular modeling studies provide an idea of how binding of substrate molecules to Sniffer could possibly occur.

  3. Direct enzyme assay evidence confirms aldehyde reductase function of Ydr541cp and Ygl039wp from Saccharomyces cerevisiae

    Science.gov (United States)

    Aldehyde reductase gene ARI1 is a recently characterized member of intermediate subfamily under SDR (short-chain dehydrogenase/reductase) superfamily that revealed mechanisms of in situ detoxification of furfural and HMF for tolerance of Saccharomyces cerevisiae. Uncharacterized open reading frames ...

  4. Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and cholesterol biosynthesis by oxylanosterols

    Energy Technology Data Exchange (ETDEWEB)

    Panini, S.R.; Sexton, R.C.; Gupta, A.K.; Parish, E.J.; Chitrakorn, S.; Rudney, H.

    1986-11-01

    Treatment of rat intestinal epithelial cell cultures with the oxidosqualene cyclase inhibitor, 3 beta-(2-(diethylamino)-ethoxy)androst-5-en-17-one (U18666A), resulted in an accumulation of squalene 2,3:22,23-dioxide (SDO). When U18666A was withdrawn and the cells were treated with the sterol 14 alpha-demethylase inhibitor, ketoconazole, SDO was metabolized to a product identified as 24(S),25-epoxylanosterol. To test the biological effects and cellular metabolism of this compound, we prepared 24(RS),25-epoxylanosterol by chemical synthesis. The epimeric mixture of 24,25-epoxylanosterols could be resolved by high performance liquid chromatography on a wide-pore, non-endcapped, reverse phase column. Both epimers were effective suppressors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity of IEC-6 cells. The suppressive action of the natural epimer, 24(S),25-epoxylanosterol, but not that of 24(R),25-epoxylanosterol could be completely prevented by ketoconazole. IEC-6 cells could efficiently metabolize biosynthetic 24(S),25-epoxy(/sup 3/H)anosterol mainly to the known reductase-suppressor 24(S),25-epoxycholesterol. This metabolism was substantially reduced by ketoconazole. These data support the conclusion that 24(S),25-epoxylanosterol per se is not a suppressor of HMG-CoA reductase activity but is a precursor to a regulatory oxysterol(s). It has recently been reported that 25-hydroxycholesterol can occur naturally in cultured cells in amounts sufficient to effect regulation of HMG-CoA reductase. In order to investigate the biological effects of possible precursors of 25-hydroxycholesterol, we chemically synthesized 25-hydroxylanosterol and 25-hydroxylanostene-3-one. Both oxylanosterol derivatives suppressed cellular sterol synthesis at the level of HMG-CoA reductase. (Abstract Truncated)

  5. Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and cholesterol biosynthesis by oxylanosterols

    International Nuclear Information System (INIS)

    Panini, S.R.; Sexton, R.C.; Gupta, A.K.; Parish, E.J.; Chitrakorn, S.; Rudney, H.

    1986-01-01

    Treatment of rat intestinal epithelial cell cultures with the oxidosqualene cyclase inhibitor, 3 beta-[2-(diethylamino)-ethoxy]androst-5-en-17-one (U18666A), resulted in an accumulation of squalene 2,3:22,23-dioxide (SDO). When U18666A was withdrawn and the cells were treated with the sterol 14 alpha-demethylase inhibitor, ketoconazole, SDO was metabolized to a product identified as 24(S),25-epoxylanosterol. To test the biological effects and cellular metabolism of this compound, we prepared 24(RS),25-epoxylanosterol by chemical synthesis. The epimeric mixture of 24,25-epoxylanosterols could be resolved by high performance liquid chromatography on a wide-pore, non-endcapped, reverse phase column. Both epimers were effective suppressors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity of IEC-6 cells. The suppressive action of the natural epimer, 24(S),25-epoxylanosterol, but not that of 24(R),25-epoxylanosterol could be completely prevented by ketoconazole. IEC-6 cells could efficiently metabolize biosynthetic 24(S),25-epoxy[ 3 H]anosterol mainly to the known reductase-suppressor 24(S),25-epoxycholesterol. This metabolism was substantially reduced by ketoconazole. These data support the conclusion that 24(S),25-epoxylanosterol per se is not a suppressor of HMG-CoA reductase activity but is a precursor to a regulatory oxysterol(s). It has recently been reported that 25-hydroxycholesterol can occur naturally in cultured cells in amounts sufficient to effect regulation of HMG-CoA reductase. In order to investigate the biological effects of possible precursors of 25-hydroxycholesterol, we chemically synthesized 25-hydroxylanosterol and 25-hydroxylanostene-3-one. Both oxylanosterol derivatives suppressed cellular sterol synthesis at the level of HMG-CoA reductase. (Abstract Truncated)

  6. Gene-knockdown in the honey bee mite Varroa destructor by a non-invasive approach: studies on a glutathione S-transferase

    Directory of Open Access Journals (Sweden)

    Campbell Ewan M

    2010-08-01

    Full Text Available Abstract Background The parasitic mite Varroa destructor is considered the major pest of the European honey bee (Apis mellifera and responsible for declines in honey bee populations worldwide. Exploiting the full potential of gene sequences becoming available for V. destructor requires adaptation of modern molecular biology approaches to this non-model organism. Using a mu-class glutathione S-transferase (VdGST-mu1 as a candidate gene we investigated the feasibility of gene knockdown in V. destructor by double-stranded RNA-interference (dsRNAi. Results Intra-haemocoelic injection of dsRNA-VdGST-mu1 resulted in 97% reduction in VdGST-mu1 transcript levels 48 h post-injection compared to mites injected with a bolus of irrelevant dsRNA (LacZ. This gene suppression was maintained to, at least, 72 h. Total GST catalytic activity was reduced by 54% in VdGST-mu1 gene knockdown mites demonstrating the knockdown was effective at the translation step as well as the transcription steps. Although near total gene knockdown was achieved by intra-haemocoelic injection, only half of such treated mites survived this traumatic method of dsRNA administration and less invasive methods were assessed. V. destructor immersed overnight in 0.9% NaCl solution containing dsRNA exhibited excellent reduction in VdGST-mu1 transcript levels (87% compared to mites immersed in dsRNA-LacZ. Importantly, mites undergoing the immersion approach had greatly improved survival (75-80% over 72 h, approaching that of mites not undergoing any treatment. Conclusions Our findings on V. destructor are the first report of gene knockdown in any mite species and demonstrate that the small size of such organisms is not a major impediment to applying gene knockdown approaches to the study of such parasitic pests. The immersion in dsRNA solution method provides an easy, inexpensive, relatively high throughput method of gene silencing suitable for studies in V. destructor, other small mites and

  7. Ubiquinol-cytochrome c reductase (Complex III) electrochemistry at multi-walled carbon nanotubes/Nafion modified glassy carbon electrodes

    International Nuclear Information System (INIS)

    Pelster, Lindsey N.; Minteer, Shelley D.

    2012-01-01

    Highlights: ► The electron transport chain is important to the understanding of metabolism in the living cell. ► Ubiquinol-cytochrome c reductase is a membrane bound complex of the electron transport chain (Complex III). ► The paper details the first bioelectrochemical characterization of ubiquinol-cytochrome c reductase at an electrode. - Abstract: Electron transport chain complexes are critical to metabolism in living cells. Ubiquinol-cytochrome c reductase (Complex III) is responsible for carrying electrons from ubiquinol to cytochrome c, but the complex has not been evaluated electrochemically. This work details the bioelectrochemistry of ubiquinol-cytochrome c reductase of the electron transport chain of tuber mitochondria. The characterization of the electrochemistry of this enzyme is investigated in carboxylated multi-walled carbon nanotube/tetrabutyl ammonium bromide-modified Nafion ® modified glassy carbon electrodes by cyclic voltammetry. Increasing concentrations of cytochrome c result in a catalytic response from the active enzyme in the nanotube sandwich. The experiments show that the enzyme followed Michaelis–Menten kinetics with a K m for the immobilized enzyme of 2.97 (±0.11) × 10 −6 M and a V max of 6.31 (±0.82) × 10 −3 μmol min −1 at the electrode, but the K m and V max values decreased compared to the free enzyme in solution, which is expected for immobilized redox proteins. This is the first evidence of ubiquinol-cytochrome c reductase bioelectrocatalysis.

  8. Investigating knockdown resistance (kdr) mechanism against pyrethroids/DDT in the malaria vector Anopheles funestus across Africa.

    Science.gov (United States)

    Irving, Helen; Wondji, Charles S

    2017-08-09

    Understanding the molecular basis of insecticide resistance is key to improve the surveillance and monitoring of malaria vector populations under control. In the major malaria vector Anopheles funestus, little is currently known about the role of the knockdown resistance (kdr) mechanism. Here, we investigated the presence and contribution of knockdown resistance (kdr) to pyrethroids/DDT resistance observed in Anopheles funestus across Africa. Pyrosequencing genotyping and sequencing of the voltage gated sodium channel (VGSC) gene did not detect the common L1014F mutation in field collected An. funestus across Africa. Amplification and cloning of the full-length of the sodium channel gene in pyrethroid resistant mosquitoes revealed evidences of alternative splicing events with three transcripts of 2092, 2061 and 2117 amino acids (93% average similarity to An. gambiae). Several amino acid changes were detected close to the domain II of the protein such as L928R, F938 W, I939S, L802S and T1008 M. However, all these mutations are found at low frequency and their role in pyrethroid resistance could not be established. The presence of the exclusive alternative splicing at exon 19 was not associated with resistance phenotype. Analysis of patterns of genetic diversity of the VGSC gene revealed a high polymorphism level of this gene across Africa with no evidence of directional selection suggesting a limited role for knockdown resistance in pyrethroid resistance in An. funestus. Patterns of genetic differentiation correlate with previous observations of the existence of barriers to gene flow Africa-wide with southern population significantly differentiated from other regions. Despite an apparent limited role of knockdown resistance in An. funestus, it is necessary to continue to monitor the contribution of the mutations detected here as increasing selection from insecticide-based interventions may change the dynamic in field populations as previously observed in other

  9. Prevalence of Dihydrofolate reductase gene mutations in Plasmodium falciparum isolate from pregnant women in Nigeria

    Directory of Open Access Journals (Sweden)

    Olusola Ojurongbe

    2011-12-01

    Full Text Available We assessed the prevalence of Plasmodium falciparum and the frequency of the dhfr triple mutation that is associated with antifolate drug resistance among P. falciparumisolates obtained from pregnant women in Ilorin, Nigeria. The study included 179 women in the second and third trimester of pregnancy who have been exposed to intermittent preventive treatment in pregnancy (IPTp with sulfadoxinepyrimethamine. Thick and thin blood films and PCR were used for malaria parasite detection. Blood group and hemoglobin concentration were also determined. Mutations in P. falciparum dhfr were analyzed by sequencing DNA obtained from blood spots on filter paper. Prevalence of P. falciparum in the population (PCR corrected was 44.1% (79/179 with 66.7% and 33.3% in the second and third trimester, respectively. Primigravide (51.3% were more infected than multigravide (48.7% but the difference was not statistically significant. Women in blood group A had the highest P. falciparum malaria infection (30.8%. The mean hemoglobin concentration was lower among those infected with malaria parasite. Also, more women with the malaria parasite (38.4% had anemia compare to those without (21.4%. The prevalence of the P. falciparum dhfr mutant alleles was 64.1%, 61.5%, 38.5%, and 12.8% for I51, R59, N108 and T108, respectively. None of the samples had the L164 mutation. The combined triple dhfr mutation (51 + 59 + 108 in the population was 17.9% (7 of 39. Also, the prevalence of the triple mutant alleles was not significantly associated to the number of doses of SP taken by the women. These findings highlight the need for a regular assessment of IPTp/SP efficacy, and evaluation of possible alternative drugs.

  10. Kinetics and thermodynamics of the thermal inactivation and chaperone assisted folding of zebrafish dihydrofolate reductase.

    Science.gov (United States)

    Thapliyal, Charu; Jain, Neha; Rashid, Naira; Chaudhuri Chattopadhyay, Pratima

    2018-01-01

    The maintenance of thermal stability is a major issue in protein engineering as many proteins tend to form inactive aggregates at higher temperatures. Zebrafish DHFR, an essential protein for the survival of cells, shows irreversible thermal unfolding transition. The protein exhibits complete unfolding and loss of activity at 50 °C as monitored by UV-Visible, fluorescence and far UV-CD spectroscopy. The heat induced inactivation of zDHFR follows first-order kinetics and Arrhenius law. The variation in the value of inactivation rate constant, k with increasing temperatures depicts faster inactivation at elevated temperatures. We have attempted to study the chaperoning ability of a shorter variant of GroEL (minichaperone) and compared it with that of conventional GroEL-GroES chaperone system. Both the chaperone system prevented the aggregation and assisted in refolding of zDHFR. The rate of thermal inactivation was significantly retarded in the presence of chaperones which indicate that it enhances the thermal stability of the enzyme. As minichaperone is less complex, and does not require high energy co-factors like ATP, for its function as compared to conventional GroEL-GroES system, it can act as a very good in vitro as well as in vivo chaperone model for monitoring assisted protein folding phenomenon. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. The 1-hydroxy-2-methyl-butenyl 4-diphosphate reductase gene from ...

    African Journals Online (AJOL)

    The 1-hydroxy-2-methyl-butenyl 4-diphosphate reductase gene from Taxus media: Cloning, characterization and functional identification. Y Sun, M Chen, J Tang, W Liu, C Yang, Y Yang, X Lan, M Hsieh, Z Liao ...

  12. Knockdown of the placental growth factor gene inhibits laser induced choroidal neovascularization in a murine model.

    Science.gov (United States)

    Nourinia, Ramin; Soheili, Zahra-Soheila; Ahmadieh, Hamid; Akrami, Hassan; Rezaei Kanavi, Mozhgan; Samiei, Shahram

    2013-01-01

    To evaluate the effect of placental growth factor (PlGF) gene knockdown in a murine model of laser-induced choroidal neovascularization. Choroidal neovascularization was induced in the left eyes of 11 mice by infrared laser. Small interfering RNA (siRNA, 20 picomoles/10 μl) corresponding to PlGF mRNA was administered intravitreally by Hamilton syringe in all subjects. One month later, fluorescein angiography and histolologic examination were performed. No leakage was apparent in the 11 eyes treated with siRNA cognate to PlGF. The results of histological evaluation were consistent with angiographic findings showing absence of choroidal neovascularization. Knockdown of the PlGF gene can inhibit the growth of laser-induced choroidal neovascularization in mice.

  13. Atomic Structure of Salutaridine Reductase from the Opium Poppy (Papaver somniferum)

    Energy Technology Data Exchange (ETDEWEB)

    Higashi, Yasuhiro; Kutchan, Toni M.; Smith, Thomas J. (Danforth)

    2011-11-18

    The opium poppy (Papaver somniferum L.) is one of the oldest known medicinal plants. In the biosynthetic pathway for morphine and codeine, salutaridine is reduced to salutaridinol by salutaridine reductase (SalR; EC 1.1.1.248) using NADPH as coenzyme. Here, we report the atomic structure of SalR to a resolution of {approx}1.9 {angstrom} in the presence of NADPH. The core structure is highly homologous to other members of the short chain dehydrogenase/reductase family. The major difference is that the nicotinamide moiety and the substrate-binding pocket are covered by a loop (residues 265-279), on top of which lies a large 'flap'-like domain (residues 105-140). This configuration appears to be a combination of the two common structural themes found in other members of the short chain dehydrogenase/reductase family. Previous modeling studies suggested that substrate inhibition is due to mutually exclusive productive and nonproductive modes of substrate binding in the active site. This model was tested via site-directed mutagenesis, and a number of these mutations abrogated substrate inhibition. However, the atomic structure of SalR shows that these mutated residues are instead distributed over a wide area of the enzyme, and many are not in the active site. To explain how residues distal to the active site might affect catalysis, a model is presented whereby SalR may undergo significant conformational changes during catalytic turnover.

  14. Relationship between nitrate reductase and nitrate uptake in phytoplankton in the Peru upwelling region

    International Nuclear Information System (INIS)

    Blasco, D.; MacIsaac, J.J.; Packard, T.T.; Dugdale, R.C.

    1984-01-01

    Nitrate reductase (NR) activity and 15 NO 3 - uptake in phytoplankton were compared under different environmental conditions on two cruises in the upwelling region off Peru. The NR activity and NO 3 - uptake rates responded differently to light and nutrients and the differences led to variations in the uptake:reductase ratio. Analysis of these variations suggests that the re-equilibration time of the two processes in response to environmental perturbation is an important source of variability. The nitrate uptake system responds faster than the nitrate reductase system. Considering these differences in response time, the basic differences in the two processes, and the differences in their measurement, the authors conclude that the NR activity measures the current nitrate-reducing potential, which relfects NO 3 - assimilation before the sampling time, while 15 NO 3 - uptake measures NO 3 - assimilation in the 6-h period following sampling. Thus, considering the sampling time as a point of reference, the former is a measure of the past and the latter is a measure of the future

  15. Lactococcus lactis Thioredoxin Reductase Is Sensitive to Light Inactivation

    DEFF Research Database (Denmark)

    Björnberg, Olof; Viennet, Thibault; Skjoldager, Nicklas

    2015-01-01

    Thioredoxin, involved in numerous redox pathways, is maintained in the dithiol state by the nicotinamide adenine dinucleotide phosphate-dependent flavoprotein thioredoxin reductase (TrxR). Here, TrxR from Lactococcus lactis is compared with the well-characterized TrxR from Escherichia coli. The two...... enzymes belong to the same class of low-molecular weight thioredoxin reductases and display similar kcat values (∼25 s-1) with their cognate thioredoxin. Remarkably, however, the L. lactis enzyme is inactivated by visible light and furthermore reduces molecular oxygen 10 times faster than E. coli Trx......-resolution mass spectrometric analysis of heat-extracted FAD from light-damaged TrxR revealed a mass increment of 13.979 Da, relative to that of unmodified FAD, corresponding to the addition of one oxygen atom and the loss of two hydrogen atoms. Tandem mass spectrometry confined the increase in mass...

  16. Novel bacterial sulfur oxygenase reductases from bioreactors treating gold-bearing concentrates

    DEFF Research Database (Denmark)

    Chen, Z-W; Liu, Y-Y; Wu, J-F

    2007-01-01

    The microbial community and sulfur oxygenase reductases of metagenomic DNA from bioreactors treating gold-bearing concentrates were studied by 16S rRNA library, real-time polymerase chain reaction (RT-PCR), conventional cultivation, and molecular cloning. Results indicated that major bacterial......) of bacteria and archaea were 4.59 x 10(9) and 6.68 x 10(5), respectively. Bacterial strains representing Acidithiobacillus, Leptospirillum, and Sulfobacillus were isolated from the bioreactors. To study sulfur oxidation in the reactors, pairs of new PCR primers were designed for the detection of sulfur...... oxygenase reductase (SOR) genes. Three sor-like genes, namely, sor (Fx), sor (SA), and sor (SB) were identified from metagenomic DNAs of the bioreactors. The sor (Fx) is an inactivated SOR gene and is identical to the pseudo-SOR gene of Ferroplasma acidarmanus. The sor (SA) and sor (SB) showed...

  17. TRAF1 knockdown alleviates palmitate-induced insulin resistance in HepG2 cells through NF-κB pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Wanlu [Department of Pathogen Biology, Medical College, Nantong University, 19 Qixiu Road, Nantong 226001, Jiangsu Province (China); Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, 19 Qixiu Road, Nantong 226001, Jiangsu Province (China); Tang, Zhuqi; Zhu, Xiaohui [Department of Endocrinology, Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong 226001, Jiangsu Province (China); Xia, Nana; Zhao, Yun; Wang, Suxin [Department of Pathogen Biology, Medical College, Nantong University, 19 Qixiu Road, Nantong 226001, Jiangsu Province (China); Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, 19 Qixiu Road, Nantong 226001, Jiangsu Province (China); Cui, Shiwei, E-mail: neifenmicui@163.com [Department of Endocrinology, Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong 226001, Jiangsu Province (China); Wang, Cuifang, E-mail: binghuodinghuo@163.com [Department of Endocrinology, Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong 226001, Jiangsu Province (China)

    2015-11-20

    High-fat diet (HFD) and inflammation are key contributors to insulin resistance (IR) and Type 2 diabetes mellitus (T2DM). With HFD, plasma free fatty acids (FFAs) can activate the nuclear factor-κB (NF-κB) in target tissues, then initiate negative crosstalk between FFAs and insulin signaling. However, the molecular link between IR and inflammation remains to be identified. We here reported that tumor necrosis factor receptor-associated factor 1 (TRAF1), an adapter in signal transduction, was involved in the onset of IR in hepatocytes. TRAF1 was significantly up-regulated in insulin-resistant liver tissues and palmitate (PA)-treated HepG2 cells. In addition, we showed that depletion of TRAF1 led to inhibition of the activity of NF-κB. Given the fact that the activation of NF-κB played a facilitating role in IR, the phosphorylation of Akt and GSK3β was also analyzed. We found that depletion of TRAF1 markedly reversed PA-induced attenuation of the phosphorylation of Akt and GSK3β in the cells. The accumulation of lipid droplets in hepatocyte and expression of two key gluconeogenic enzymes, PEPCK and G6Pase, were also determined and found to display a similar tendency with the phosphorylation of Akt and GSK3β. Glucose uptake assay indicated that knocking down TRAF1 blocked the effect of PA on the suppression of glucose uptake. These data implicated that TRAF1 knockdown might alleviate PA-induced IR in HepG2 cells through NF-κB pathway. - Highlights: • TRAF1 accelerated PA-induced IR in HepG2 cells mediated through NF-κB signaling. • Knockdown of TRAF1 alleviated PA-induced IR in HepG2 cells. • Knockdown of TRAF1 alleviated PA-induced lipid accumulation in HepG2 cells. • Knockdown of TRAF1 reversed PA-induced suppression of glucose uptake in HepG2 cells. • Knockdown of TRAF1 reversed PA-induced gluconeogenesis in HepG2 cells.

  18. TRAF1 knockdown alleviates palmitate-induced insulin resistance in HepG2 cells through NF-κB pathway

    International Nuclear Information System (INIS)

    Zhang, Wanlu; Tang, Zhuqi; Zhu, Xiaohui; Xia, Nana; Zhao, Yun; Wang, Suxin; Cui, Shiwei; Wang, Cuifang

    2015-01-01

    High-fat diet (HFD) and inflammation are key contributors to insulin resistance (IR) and Type 2 diabetes mellitus (T2DM). With HFD, plasma free fatty acids (FFAs) can activate the nuclear factor-κB (NF-κB) in target tissues, then initiate negative crosstalk between FFAs and insulin signaling. However, the molecular link between IR and inflammation remains to be identified. We here reported that tumor necrosis factor receptor-associated factor 1 (TRAF1), an adapter in signal transduction, was involved in the onset of IR in hepatocytes. TRAF1 was significantly up-regulated in insulin-resistant liver tissues and palmitate (PA)-treated HepG2 cells. In addition, we showed that depletion of TRAF1 led to inhibition of the activity of NF-κB. Given the fact that the activation of NF-κB played a facilitating role in IR, the phosphorylation of Akt and GSK3β was also analyzed. We found that depletion of TRAF1 markedly reversed PA-induced attenuation of the phosphorylation of Akt and GSK3β in the cells. The accumulation of lipid droplets in hepatocyte and expression of two key gluconeogenic enzymes, PEPCK and G6Pase, were also determined and found to display a similar tendency with the phosphorylation of Akt and GSK3β. Glucose uptake assay indicated that knocking down TRAF1 blocked the effect of PA on the suppression of glucose uptake. These data implicated that TRAF1 knockdown might alleviate PA-induced IR in HepG2 cells through NF-κB pathway. - Highlights: • TRAF1 accelerated PA-induced IR in HepG2 cells mediated through NF-κB signaling. • Knockdown of TRAF1 alleviated PA-induced IR in HepG2 cells. • Knockdown of TRAF1 alleviated PA-induced lipid accumulation in HepG2 cells. • Knockdown of TRAF1 reversed PA-induced suppression of glucose uptake in HepG2 cells. • Knockdown of TRAF1 reversed PA-induced gluconeogenesis in HepG2 cells.

  19. Synthesis and Activity of a New Series of(Z-3-Phenyl-2-benzoylpropenoic Acid Derivatives as Aldose Reductase Inhibitors

    Directory of Open Access Journals (Sweden)

    Shao-Jie Wang

    2007-04-01

    Full Text Available During the course of studies directed towards the discovery of novel aldose reductase inhibitors for the treatment of diabetic complications, we synthesized a series of new (Z-3-phenyl-2-benzoylpropenoic acid derivatives and tested their in vitro inhibitory activities on rat lens aldose reductase. Of these compounds, (Z-3-(3,4-dihydroxyphenyl-2-(4-methylbenzoylpropenoicacid(3k was identified as the most potent inhibitor, with an IC50 of 0.49μM. The theoretical binding mode of 3k was obtained by simulation of its docking into the active site of the human aldose reductase crystal structure.

  20. Influence of rete testis fluid deprivation on the kinetic parameters of goat epididymal 5 alpha-reductase.

    Science.gov (United States)

    Kelce, W R; Lubis, A M; Braun, W F; Youngquist, R S; Ganjam, V K

    1990-01-01

    A surgical technique to cannulate the rete testis of the goat was utilized to examine the effects of rete testis fluid (RTF) deprivation on the enzymatic activity of epididymal 5 alpha-reductase. Kinetic techniques were used to determine whether the regional enzymatic effect of RTF deprivation is to decrease the apparent number of 5 alpha-reductase active sites or the catalytic activity of each active site within the epididymal epithelium. Paired comparisons of (Vmax)app and (Km)app values between control and RTF-deprived epididymides indicated that RTF deprivation affected the value of (Vmax)app with no apparent change in the values of (Km)app in caput, corpus, and cauda epididymal regions. We conclude that RTF deprivation in the goat epididymis for 7 days results in a decreased number of apparent 5 alpha-reductase active sites within the epididymal epithelium.

  1. Crystallization and preliminary X-ray analysis of the NADPH-dependent 3-quinuclidinone reductase from Rhodotorula rubra

    Science.gov (United States)

    Takeshita, Daijiro; Kataoka, Michihiko; Miyakawa, Takuya; Miyazono, Ken-ichi; Uzura, Atsuko; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2009-01-01

    (R)-3-Quinuclidinol is a useful compound that is applicable to the synthesis of various pharmaceuticals. The NADPH-dependent carbonyl reductase 3-­quinuclidinone reductase from Rhodotorula rubra catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol and is expected to be utilized in industrial production of this alcohol. 3-Quinuclidinone reductase from R. rubra was expressed in Escherichia coli and purified using Ni-affinity and ion-exchange column chromatography. Crystals of the protein were obtained by the sitting-drop vapour-diffusion method using PEG 8000 as the precipitant. The crystals belonged to space group P41212, with unit-cell parameters a = b = 91.3, c = 265.4 Å, and diffracted X-rays to 2.2 Å resolution. The asymmetric unit contained four molecules of the protein and the solvent content was 48.4%. PMID:19478454

  2. Ferric reductase genes involved in high-affinity iron uptake are differentially regulated in yeast and hyphae of Candida albicans.

    Science.gov (United States)

    Jeeves, Rose E; Mason, Robert P; Woodacre, Alexandra; Cashmore, Annette M

    2011-09-01

    The pathogenic yeast Candida albicans possesses a reductive iron uptake system which is active in iron-restricted conditions. The sequestration of iron by this mechanism initially requires the reduction of free iron to the soluble ferrous form, which is catalysed by ferric reductase proteins. Reduced iron is then taken up into the cell by a complex of a multicopper oxidase protein and an iron transport protein. Multicopper oxidase proteins require copper to function and so reductive iron and copper uptake are inextricably linked. It has previously been established that Fre10 is the major cell surface ferric reductase in C. albicans and that transcription of FRE10 is regulated in response to iron levels. We demonstrate here that Fre10 is also a cupric reductase and that Fre7 also makes a significant contribution to cell surface ferric and cupric reductase activity. It is also shown, for the first time, that transcription of FRE10 and FRE7 is lower in hyphae compared to yeast and that this leads to a corresponding decrease in cell surface ferric, but not cupric, reductase activity. This demonstrates that the regulation of two virulence determinants, the reductive iron uptake system and the morphological form of C. albicans, are linked. Copyright © 2011 John Wiley & Sons, Ltd.

  3. Mosquito knock-down and adulticidal activities of essential oils by vaporizer, impregnated filter paper and aerosol methods

    Directory of Open Access Journals (Sweden)

    M. Ramar

    2014-09-01

    Full Text Available Essential oils from 12 medicinal plants were evaluated by three different bioassay methods (Vaporizer, Filter paper and Aerosol for Knock-down and adulticidal efficacy on the filarial vector mosquito, Culex quinquefasciatus. Based on screening results the effective plants were selected for investigating Knock-down and adulticidal potential against adult female of the laboratory-reared mosquito species, Cx. quinquefasciatus. In vaporizer bioassay method four different doses (1.25, 2.5, 5 and 10% were used. Four different doses (0.625, 1.25, 2.5 and 10% were used both filter paper (cm2 and aerosol (cm3 bioassay methods. Five essential oils (calamus, camphor, citronella, clove and eucalyptus were identified as potential treatments in vaporizer bioassay. The result showed that the knock down time decreased with increased concentration in clove oil treatment; the Knock-down time (KT 50 = 46.1 ± 0.1, 38.5 ± 0.1, 30.7 ± 0.2, and 20.1 ± 0.1 minutes was recorded at 1.25, 2.5, 5 and 10% /cm3 respectively. In filter paper method nine essential oils were identified as potential treatments. After 1 hr exposure period clove oil recorded the lowest median Knock-down time (KT50 which was calculated as 9.15 ± 0.1min/cm2. Followed by citronella (KT50 =11.4 ± 0.1 min and eucalyptus (KT50 =11.4 ±0.1min oils since they recorded lower median Knock-down time. All the twelve essential oils were identified as potential treatments in aerosol activity. The lethal time decreased when the concentration increased. At 5 % concentration the median lethal time (LT50 for clove oil was calculated as (LT50=3.80 ± 0.1minutes. The Cinnamon oil was effective which recorded (LT50 = 1.99 mins as median lethal time. Camphor (LT50 =19.6± 0.1 min oil were found to be less toxic by aerosol method. These results suggest that clove oil and cinnamon oil have the potential to be used as a eco-friendly approach for the control of the major important filaria vector Cx. quinquefasciatus

  4. Intramolecular electron transfer in Pseudomonas aeruginosa cd(1) nitrite reductase

    DEFF Research Database (Denmark)

    Farver, Ole; Brunori, Maurizio; Cutruzzolà, Francesca

    2009-01-01

    ) as the level of reduction increased in both the WT and the His mutant. Equilibrium standard enthalpy and entropy changes and activation parameters of this ET process were determined. We concluded that negative cooperativity is a common feature among the cd(1) nitrite reductases, and we discuss this control...

  5. Identification of a Novel Epoxyqueuosine Reductase Family by Comparative Genomics.

    Science.gov (United States)

    Zallot, Rémi; Ross, Robert; Chen, Wei-Hung; Bruner, Steven D; Limbach, Patrick A; de Crécy-Lagard, Valérie

    2017-03-17

    The reduction of epoxyqueuosine (oQ) is the last step in the synthesis of the tRNA modification queuosine (Q). While the epoxyqueuosine reductase (EC 1.17.99.6) enzymatic activity was first described 30 years ago, the encoding gene queG was only identified in Escherichia coli in 2011. Interestingly, queG is absent from a large number of sequenced genomes that harbor Q synthesis or salvage genes, suggesting the existence of an alternative epoxyqueuosine reductase in these organisms. By analyzing phylogenetic distributions, physical gene clustering, and fusions, members of the Domain of Unknown Function 208 (DUF208) family were predicted to encode for an alternative epoxyqueuosine reductase. This prediction was validated with genetic methods. The Q modification is present in Lactobacillus salivarius, an organism missing queG but harboring the duf208 gene. Acinetobacter baylyi ADP1 is one of the few organisms that harbor both QueG and DUF208, and deletion of both corresponding genes was required to observe the absence of Q and the accumulation of oQ in tRNA. Finally, the conversion oQ to Q was restored in an E. coli queG mutant by complementation with plasmids harboring duf208 genes from different bacteria. Members of the DUF208 family are not homologous to QueG enzymes, and thus, duf208 is a non-orthologous replacement of queG. We propose to name DUF208 encoding genes as queH. While QueH contains conserved cysteines that could be involved in the coordination of a Fe/S center in a similar fashion to what has been identified in QueG, no cobalamin was identified associated with recombinant QueH protein.

  6. Nitrate reductase activity of Staphylococcus carnosus affecting the color formation in cured raw ham.

    Science.gov (United States)

    Bosse Née Danz, Ramona; Gibis, Monika; Schmidt, Herbert; Weiss, Jochen

    2016-07-01

    The influence of the nitrate reductase activity of two Staphylococcus carnosus strains used as starter cultures on the formation of nitrate, nitrite and color pigments in cured raw ham was investigated. In this context, microbiological, chemical and multivariate image analyses were carried out on cured raw hams, which were injected with different brines containing either nitrite or nitrate, with or without the S. carnosus starter cultures. During processing and storage, the viable counts of staphylococci remained constant at 6.5logcfu/g in the hams inoculated with starter cultures, while the background microbiota of the hams processed without the starter cultures developed after 14days. Those cured hams inoculated with S. carnosus LTH 7036 (high nitrate reductase activity) showed the highest decrease in nitrate and high nitrite concentrations in the end product, but were still in the range of the legal European level. The hams cured with nitrate and without starter culture or with the other strain, S. carnosus LTH 3838 (low nitrate reductase activity) showed higher residual nitrate levels and a lower nitrite content in the end product. The multivariate image analysis identified spatial and temporal differences in the meat pigment profiles of the differently cured hams. The cured hams inoculated with S. carnosus LTH 3838 showed an uncured core due to a delay in pigment formation. Therefore, the selection of starter cultures based on their nitrate reductase activity is a key point in the formation of curing compounds and color pigments in cured raw ham manufacture. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Insecticidal potency of RNAi-based catalase knockdown in Rhynchophorus ferrugineus (Oliver) (Coleoptera: Curculionidae).

    Science.gov (United States)

    Al-Ayedh, Hassan; Rizwan-Ul-Haq, Muhammad; Hussain, Abid; Aljabr, Ahmed M

    2016-11-01

    Palm trees around the world are prone to notorious Rhynchophorus ferrugineus, which causes heavy losses of palm plantations. In Middle Eastern countries, this pest is a major threat to date palm orchards. Conventional pest control measures with the major share of synthetic insecticides have resulted in insect resistance and environmental issues. Therefore, in order to explore better alternatives, the RNAi approach was employed to knock down the catalase gene in fifth and tenth larval instars with different dsRNA application methods, and their insecticidal potency was studied. dsRNA of 444 bp was prepared to knock down catalase in R. ferrugineus. Out of the three dsRNA application methods, dsRNA injection into larvae was the most effective, followed by dsRNA application by artificial feeding. Both methods resulted in significant catalase knockdown in various tissues, especially the midgut. As a result, the highest growth inhibition of 123.49 and 103.47% and larval mortality of 80 and 40% were observed in fifth-instar larvae, whereas larval growth inhibition remained at 86.83 and 69.08% with larval mortality at 30 and 10% in tenth-instar larvae after dsRNA injection and artificial diet treatment. The topical application method was the least efficient, with the lowest larval growth inhibition of 57.23 and 45.61% and 0% mortality in fifth- and tenth-instar larvae. Generally, better results were noted at the high dsRNA dose of 5 µL. Catalase enzyme is found in most insect body tissues, and thus its dsRNA can cause broad-scale gene knockdown within the insect body, depending upon the application method. Significant larval mortality and growth inhibition after catalase knockdown in R. ferrugineus confirms its insecticidal potency and suggests a bright future for RNAi-based bioinsecticides in pest control. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  8. NADPH-Thioredoxin Reductase C Mediates the Response to Oxidative Stress and Thermotolerance in the Cyanobacterium Anabaena sp PCC7120

    NARCIS (Netherlands)

    Sanchez-Riego, Ana M.; Mata-Cabana, Alejandro; Galmozzi, CarlaV.; Florencio, Francisco J.

    2016-01-01

    NADPH-thioredoxin reductase C (NTRC) is a bimodular enzyme composed of an NADPH-thioredoxin reductase and a thiioredoxin domain extension in the same protein. In plants, NTRC has been described to be involved in the protection of the chloroplast against oxidative stress damage through reduction of

  9. Sterol-induced Dislocation of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase from Endoplasmic Reticulum Membranes into the Cytosol through a Subcellular Compartment Resembling Lipid Droplets*

    Science.gov (United States)

    Hartman, Isamu Z.; Liu, Pingsheng; Zehmer, John K.; Luby-Phelps, Katherine; Jo, Youngah; Anderson, Richard G. W.; DeBose-Boyd, Russell A.

    2010-01-01

    Sterol-induced binding to Insigs in the endoplasmic reticulum (ER) allows for ubiquitination of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme in cholesterol synthesis. This ubiquitination marks reductase for recognition by the ATPase VCP/p97, which mediates extraction and delivery of reductase from ER membranes to cytosolic 26 S proteasomes for degradation. Here, we report that reductase becomes dislocated from ER membranes into the cytosol of sterol-treated cells. This dislocation exhibits an absolute requirement for the actions of Insigs and VCP/p97. Reductase also appears in a buoyant fraction of sterol-treated cells that co-purifies with lipid droplets, cytosolic organelles traditionally regarded as storage depots for neutral lipids such as triglycerides and cholesteryl esters. Genetic, biochemical, and localization studies suggest a model in which reductase is dislodged into the cytosol from an ER subdomain closely associated with lipid droplets. PMID:20406816

  10. Knockdown of autophagy enhances innate immune response in hepatitis C virus infected hepatocytes

    Science.gov (United States)

    Shrivastava, Shubham; Raychoudhuri, Amit; Steele, Robert; Ray, Ranjit; Ray, Ratna B.

    2010-01-01

    The role of autophagy in disease pathogenesis following viral infection is beginning to be elucidated. We have previously reported that hepatitis C virus (HCV) infection in hepatocytes induces autophagy. However, the biological significance of HCV induced autophagy has not been clarified. Autophagy has recently been identified as a novel component of innate immune system against viral infection. In the present study, we have shown that knockdown of autophagy related protein Beclin1 or ATG7 in immortalized human hepatocytes (IHH) inhibited HCV growth. Beclin1 or ATG7 knockdown IHH when infected with HCV exhibited an increased expression of IFN-β, OAS-1, IFN-α and IFI27 mRNAs of the interferon signaling pathways as compared to infection of control IHH. Subsequent study demonstrated that HCV infection in autophagy impaired IHH displayed caspase activation, PARP cleavage and apoptotic cell death. Conclusion The disruption of autophagy machinery in HCV infected hepatocytes activated IFN signaling pathway, and induced apoptosis. Together, these results suggest that HCV induced autophagy impairs innate immune response. PMID:21274862

  11. RNAi-mediated knockdown of MTNR1B without disrupting the effects of melatonin on apoptosis and cell cycle in bovine granulose cells

    Directory of Open Access Journals (Sweden)

    Wenju Liu

    2018-04-01

    Full Text Available Melatonin is well known as a powerful free radical scavenger and exhibits the ability to prevent cell apoptosis. In the present study, we investigated the role of melatonin and its receptor MTNR1B in regulating the function of bovine granulosa cells (GCs and hypothesized the involvement of MTNR1B in mediating the effect of melatonin on GCs. Our results showed that MTNR1B knockdown significantly promoted GCs apoptosis but did not affect the cell cycle. These results were further verified by increasing the expression of pro-apoptosis genes (BAX and CASP3, decreasing expression of the anti-apoptosis genes (BCL2 and BCL-XL and anti-oxidant genes (SOD1 and GPX4 without affecting cell cycle factors (CCND1, CCNE1 and CDKN1A and TP53. In addition, MTNR1B knockdown did not disrupt the effects of melatonin in suppressing the GCs apoptosis or blocking the cell cycle. Moreover, MTNR1B knockdown did not affect the role of melatonin in increasing BCL2, BCL-XL, and CDKN1A expression, or decreasing BAX, CASP3, TP53, CCND1 and CCNE1 expression. The expression of MTNR1A was upregulated after MTNR1B knockdown, and melatonin promoted MTNR1A expression with or without MTNR1B knockdown. However, despite melatonin supplementation, the expression of SOD1 and GPX4 was still suppressed after MTNR1B knockdown. In conclusion, these findings indicate that melatonin and MTNR1B are involved in BCL2 family and CASP3-dependent apoptotic pathways in bovine GCs. MTNR1A and MTNR1B may coordinate the work of medicating the appropriate melatonin responses to GCs.

  12. Characterization of the methotrexate transport pathway in murine L1210 leukemia cells: Involvement of a membrane receptor and a cytosolic protein

    International Nuclear Information System (INIS)

    Price, E.M.; Ratnam, M.; Rodeman, K.M.; Freisheim, J.H.

    1988-01-01

    A radioiodinated photoaffinity analogue of methotrexate, N α -(4-amino-4-deoxy-10-methyl-pteroyl)-N ε -(4-azidosalicylyl)-L-lysine (APA-ASA-Lys), was recently used to identify the plasma membrane derived binding protein involved in the transport of this folate antagonist into murine L1210 cells. The labeled protein has an apparent molecular weight of 46K-48K when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but no such labeling occurs in a methotrexate transport-defective cell line (L1210/R81). Labeling of the total cytosolic protein from disrupted cells, followed by electrophoresis and autoradiography, showed, among other proteins, a 21K band, corresponding to dihydrofolate reductase (DHFR), in both the parent and R81 cells and a 38K band only in the parent cells. However, when whole cells were UV irradiated at various times at 37 degree C following addition of radiolabeled APA-ASA-Lys, the 38K protein and DHFR were the only cytosolic proteins labeled in the parent cells, while the intact R81 cells showed no labeled cytosolic protein, since the photoprobe is not transported. Further, when the parent cells were treated with a pulse of radiolabeled photoprobe, followed by UV irradiation at different times at 37 degree C, the probe appeared sequentially on the 48K membrane protein and both the 38K cytosolic protein and dihydrofolate reductase. A 48K protein could be detected in both parent L1210 cells and the R81 cells on Western blots using antisera to a membrane folate binding protein from human placenta. These results suggest a vectorial transport of APA-ASA-Lys or methotrexate and reduced folate coenzymes into murine L1210 cells mediated by a 48K integral membrane protein and a 38K cytosolic or peripheral membrane protein. The 38K protein may help in the trafficking of reduced folate coenzymes, shuttling them to various cytosolic targets

  13. Knockdown of long non-coding RNA Taurine Up-Regulated 1 inhibited doxorubicin resistance of bladder urothelial carcinoma via Wnt/β-catenin pathway.

    Science.gov (United States)

    Xie, Dalong; Zhang, Hui; Hu, Xuanhao; Shang, Chao

    2017-10-24

    In genitourinary system, bladder cancer (BC) is the most common and lethal malignant tumor, which most common type is bladder urothelial carcinoma (BUC). Long non-coding RNA (lncRNA) Taurine Up-Regulated 1 (TUG1) gene is high-expressed in several malignant tumors, including BC. In this study, over-expression of TUG1 was found in BUC tissues and cell line resistant to doxorubicin (Dox). Knockdown of TUG1 inhibited the Dox resistance and promoted the cytotoxicity induced by Dox in T24/Dox cells. TUG1 knockdown also depressed the Wnt/β-catenin pathway, and the activation the Wnt/β-catenin pathway partly reversed the inhibitory effects of TUG1 knockdown on Dox resistance in T24/Dox cells. In conclusion, up-regulation of lncRNA TUG1 was related with the poor response of BUC patients to Dox chemotherapy, knockdown of TUG1 inhibited the Dox resistance of BUC cells via Wnt/β-catenin pathway. These findings might assist in the discovery of novel potential diagnostic and therapeutic target for BUC, thereby improve the effects of clinical treatment in patients.

  14. Gene knockdown of CENPA reduces sphere forming ability and stemness of glioblastoma initiating cells

    Directory of Open Access Journals (Sweden)

    Jinan Behnan

    2016-09-01

    Knockdown of CENPA reduced sphere forming ability, proliferation and cell viability of GICs. We also detected significant reduction in the expression of stemness marker SOX2 and the proliferation marker Ki67. These results indicate that CENPA might represent a promising therapeutic target for GBM treatment.

  15. A Simple Retroelement Based Knock-Down System in Dictyostelium: Further Insights into RNA Interference Mechanisms.

    Science.gov (United States)

    Friedrich, Michael; Meier, Doreen; Schuster, Isabelle; Nellen, Wolfgang

    2015-01-01

    We have previously shown that the most abundant Dictyostelium discoideum retroelement DIRS-1 is suppressed by RNAi mechanisms. Here we provide evidence that both inverted terminal repeats have strong promoter activity and that bidirectional expression apparently generates a substrate for Dicer. A cassette containing the inverted terminal repeats and a fragment of a gene of interest was sufficient to activate the RNAi response, resulting in the generation of ~21 nt siRNAs, a reduction of mRNA and protein expression of the respective endogene. Surprisingly, no transitivity was observed on the endogene. This was in contrast to previous observations, where endogenous siRNAs caused spreading on an artificial transgene. Knock-down was successful on seven target genes that we examined. In three cases a phenotypic analysis proved the efficiency of the approach. One of the target genes was apparently essential because no knock-out could be obtained; the RNAi mediated knock-down, however, resulted in a very slow growing culture indicating a still viable reduction of gene expression. ADVANTAGES OF THE DIRS-1–RNAI SYSTEM: The knock-down system required a short DNA fragment (~400 bp) of the target gene as an initial trigger. Further siRNAs were generated by RdRPs since we have shown some siRNAs with a 5'-triphosphate group. Extrachromosomal vectors facilitate the procedure and allowed for molecular and phenotypic analysis within one week. The system provides an efficient and rapid method to reduce protein levels including those of essential genes.

  16. Knockdown of the Placental Growth Factor Gene Inhibits Laser Induced Choroidal Neovascularization in a Murine Model

    Directory of Open Access Journals (Sweden)

    Ramin Nourinia

    2013-01-01

    Full Text Available Purpose: To evaluate the effect of placental growth factor (PlGF gene knockdown in a murine model of laser-induced choroidal neovascularization. Methods: Choroidal neovascularization was induced in the left eyes of 11 mice by infrared laser. Small interfering RNA (siRNA, 20 picomoles/10 μl corresponding to PlGF mRNA was administered intravitreally by Hamilton syringe in all subjects. One month later, fluorescein angiography and histolologic examination were performed. Results: No leakage was apparent in the 11 eyes treated with siRNA cognate to PlGF. The results of histological evaluation were consistent with angiographic findings showing absence of choroidal neovascularization. Conclusion: Knockdown of the PlGF gene can inhibit the growth of laser-induced choroidal neovascularization in mice.

  17. Acute Podocyte Vascular Endothelial Growth Factor (VEGF-A) Knockdown Disrupts alphaVbeta3 Integrin Signaling in the Glomerulus

    Science.gov (United States)

    Veron, Delma; Villegas, Guillermo; Aggarwal, Pardeep Kumar; Bertuccio, Claudia; Jimenez, Juan; Velazquez, Heino; Reidy, Kimberly; Abrahamson, Dale R.; Moeckel, Gilbert; Kashgarian, Michael; Tufro, Alda

    2012-01-01

    Podocyte or endothelial cell VEGF-A knockout causes thrombotic microangiopathy in adult mice. To study the mechanism involved in acute and local injury caused by low podocyte VEGF-A we developed an inducible, podocyte-specific VEGF-A knockdown mouse, and we generated an immortalized podocyte cell line (VEGFKD) that downregulates VEGF-A upon doxycycline exposure. Tet-O-siVEGF:podocin-rtTA mice express VEGF shRNA in podocytes in a doxycycline-regulated manner, decreasing VEGF-A mRNA and VEGF-A protein levels in isolated glomeruli to ∼20% of non-induced controls and urine VEGF-A to ∼30% of control values a week after doxycycline induction. Induced tet-O-siVEGF:podocin-rtTA mice developed acute renal failure and proteinuria, associated with mesangiolysis and microaneurisms. Glomerular ultrastructure revealed endothelial cell swelling, GBM lamination and podocyte effacement. VEGF knockdown decreased podocyte fibronectin and glomerular endothelial alphaVbeta3 integrin in vivo. VEGF receptor-2 (VEGFR2) interacts with beta3 integrin and neuropilin-1 in the kidney in vivo and in VEGFKD podocytes. Podocyte VEGF knockdown disrupts alphaVbeta3 integrin activation in glomeruli, detected by WOW1-Fab. VEGF silencing in cultured VEGFKD podocytes downregulates fibronectin and disrupts alphaVbeta3 integrin activation cell-autonomously. Collectively, these studies indicate that podocyte VEGF-A regulates alphaVbeta3 integrin signaling in the glomerulus, and that podocyte VEGF knockdown disrupts alphaVbeta3 integrin activity via decreased VEGFR2 signaling, thereby damaging the three layers of the glomerular filtration barrier, causing proteinuria and acute renal failure. PMID:22808199

  18. Prevention of hemodynamic and vascular albumin filtration changes in diabetic rats by aldose reductase inhibitors

    International Nuclear Information System (INIS)

    Tilton, R.G.; Chang, K.; Pugliese, G.; Eades, D.M.; Province, M.A.; Sherman, W.R.; Kilo, C.; Williamson, J.R.

    1989-01-01

    This study investigated hemodynamic changes in diabetic rats and their relationship to changes in vascular albumin permeation and increased metabolism of glucose to sorbitol. The effects of 6 wk of streptozocin-induced diabetes and three structurally different inhibitors of aldose reductase were examined on (1) regional blood flow (assessed with 15-microns 85Sr-labeled microspheres) and vascular permeation by 125I-labeled bovine serum albumin (BSA) and (2) glomerular filtration rate (assessed by plasma clearance of 57Co-labeled EDTA) and urinary albumin excretion (determined by radial immunodiffusion assay). In diabetic rats, blood flow was significantly increased in ocular tissues (anterior uvea, posterior uvea, retina, and optic nerve), sciatic nerve, kidney, new granulation tissue, cecum, and brain. 125I-BSA permeation was increased in all of these tissues except brain. Glomerular filtration rate and 24-h urinary albumin excretion were increased 2- and 29-fold, respectively, in diabetic rats. All three aldose reductase inhibitors completely prevented or markedly reduced these hemodynamic and vascular filtration changes and increases in tissue sorbitol levels in the anterior uvea, posterior uvea, retina, sciatic nerve, and granulation tissue. These observations indicate that early diabetes-induced hemodynamic changes and increased vascular albumin permeation and urinary albumin excretion are aldose reductase-linked phenomena. Discordant effects of aldose reductase inhibitors on blood flow and vascular albumin permeation in some tissues suggest that increased vascular albumin permeation is not entirely attributable to hemodynamic change

  19. Cloning, expression and antigenicity of the L. donovani reductase

    DEFF Research Database (Denmark)

    Jensen, A T; Kemp, K; Theander, T G

    2001-01-01

    (K). Only 2 of 22 plasma samples from patients with visceral leishmaniasis were found to have detectable anti-reductase antibodies and peripheral blood mononuclear cells (PBMC) from one of three individuals previously infected with visceral leishmaniasis proliferated in the presence of recombinant...

  20. A New Type of YumC-Like Ferredoxin (Flavodoxin) Reductase Is Involved in Ribonucleotide Reduction

    DEFF Research Database (Denmark)

    Chen, Jun; Shen, Jing; Solem, Christian

    2015-01-01

    . subtilis but that the addition of deoxynucleosides cannot compensate for the lethal phenotype displayed by the B. subtilis yumC knockout mutant. Ferredoxin (flavodoxin) reductase (FdR) is involved in many important reactions in both eukaryotes and prokaryotes, such as photosynthesis, nitrate reduction, etc. The recently...... ribonucleotide reductase, which represents the workhorse for the bioconversion of nucleotides to deoxynucleotides in many prokaryotes and eukaryotic pathogens under aerobic conditions. As the partner of the flavodoxin (NrdI), the key FdR is missing in the current model describing the class Ib system...

  1. Development of a Rapid Real-Time PCR Assay for Quantitation of Pneumocystis carinii f. sp. Carinii

    DEFF Research Database (Denmark)

    Larsen, Hans Henrik; Kovacs, Joseph A; Stock, Frida

    2002-01-01

    A method for reliable quantification of Pneumocystis carinii in research models of P. carinii pneumonia (PCP) that is more convenient and reproducible than microscopic enumeration of organisms would greatly facilitate investigations of this organism. We developed a rapid quantitative touchdown (QTD......) PCR assay for detecting P. carinii f. sp. carinii, the subspecies of P. carinii commonly used in research models of PCP. The assay was based on the single-copy dihydrofolate reductase gene and was able to detect ... 6 log values for standards containing > or =5 copies/tube. Application of the assay to a series of 10-fold dilutions of P. carinii organisms isolated from rat lung demonstrated that it was reproducibly quantitative over 5 log values (r = 0.99). The assay was applied to a recently reported in vitro...

  2. Knockdown of HIF-1α and IL-8 induced apoptosis of hepatocellular carcinoma triggers apoptosis of vascular endothelial cells.

    Science.gov (United States)

    Choi, Sung Hoon; Park, Jun Yong; Kang, Wonseok; Kim, Seung Up; Kim, Do Young; Ahn, Sang Hoon; Ro, Simon Wonsang; Han, Kwang-Hyub

    2016-01-01

    A local hypoxic microenvironment is one of the most important characteristics of solid tumors. Hypoxia inducible factor-1α (HIF-1α) and Interleukin-8 (IL-8) activate tumor survival from hypoxic-induced apoptosis in each pathway. This study aimed to evaluate whether knockdown of HIF-1α and IL-8 induced apoptosis of the hepatocellular carcinoma (HCC) and endothelial cell lines. HCC cell lines were infected with adenovirus-expressing shRNA for HIF-1α and IL-8 and maintained under hypoxic conditions (1% O2, 24 h). The expression levels of HIF-1α and both apoptotic and growth factors were examined by real-time quantitative PCR and western blot. We also investigated apoptosis by TUNEL assay (FACS and Immunofluorescence) and measured the concentration of cytochrome C. Inhibition of HIF-1α and IL-8 up-regulated the expression of apoptotic factors while downregulating anti-apoptotic factors simultaneously. Knockdown of HIF-1α and IL-8 increased the concentration of cytochrome C and enhanced DNA fragmentation in HCC cell lines. Moreover, culture supernatant collected from the knockdown of HIF-1α and IL-8 in HCC cell lines induced apoptosis in human umbilical vein endothelial cells under hypoxia, and the expression of variable apoptotic ligand increased from HCC cell lines, time-dependently. These data suggest that adenovirus-mediated knockdown of HIF-1α and IL-8 induced apoptosis in HCC cells and triggered apoptosis of vascular endothelial cells.

  3. Can RNAi-mediated hsp90α knockdown in combination with 17-AAG be a therapy for glioma?

    Science.gov (United States)

    Mehta, Adi; Shervington, Amal; Howl, John; Jones, Sarah; Shervington, Leroy

    2013-01-01

    Heat shock protein 90 promotes tumor progression and survival and has emerged as a vital therapeutic target. Previously we reported that the combinatorial treatment of 17AAG/sihsp90α significantly downregulated Hsp90α mRNA and protein levels in Glioblastoma Multiforme (GBM). Here we investigated the ability of cell penetrating peptide (Tat48-60 CPP)-mediated siRNA-induced hsp90α knockdown as a single agent and in combination with 17-allylamino-17-demethoxygeldanamycin (17-AAG) to induce tumor growth inhibition in GBM and whether it possessed therapeutic implications. GBM and non-tumorigenic cells exposed to siRNA and/or 17-AAG were subsequently assessed by qRT-PCR, immunofluorescence, FACS analysis, quantitative Akt, LDH leakage and cell viability assays. PAGE was performed for serum stability assessment. A combination of siRNA/17-AAG treatment significantly induced Hsp90α gene and protein knockdown by 95% and 98%, respectively, concomitant to 84% Akt kinase activity attenuation, induced cell cycle arrest and tumor-specific cytotoxicity by 88%. Efficient complex formation between CPP and siRNA exhibited improved serum stability of the siRNA with minimal intrinsic toxicity in vitro. The preliminary in vivo results showed that combination therapy induced hsp90α knockdown and attenuated Akt kinase activity in intracranial glioblastoma mouse models. The results imply that RNAi-mediated hsp90α knockdown increases 17-AAG treatment efficacy in GBM. In addition, the cytotoxic response observed was the consequence of downregulation of hsp90α gene expression, reduced Akt kinase activity and S-G2/M cell cycle arrest. These results are novel and highlight the ability of Tat to efficiently deliver siRNA in GBM and suggest that the dual inhibition of Hsp90 has therapeutic potentials.

  4. Signal Transducer and Activator of Transcription 1 (STAT1) Knock-down Induces Apoptosis in Malignant Pleural Mesothelioma.

    Science.gov (United States)

    Arzt, Lisa; Halbwedl, Iris; Gogg-Kamerer, Margit; Popper, Helmut H

    2017-07-01

    Malignant pleural mesothelioma (MPM) is the most common primary tumor of the pleura. Its incidence is still increasing in Europe and the prognosis remains poor. We investigated the oncogenic function of signal transducer and activator of transcription 1 (STAT1) in MPM in more detail. A miRNA profiling was performed on 52 MPM tissue samples. Upregulated miRNAs (targeting SOCS1/3) were knocked-down using miRNA inhibitors. mRNA expression levels of STAT1/3, SOCS1/3 were detected in MPM cell lines. STAT1 has been knocked-down using siRNA and qPCR was used to detect mRNA expression levels of all JAK/STAT family members and genes that regulate them. An immunohistochemical staining was performed to detect the expression of caspases. STAT1 was upregulated and STAT3 was downregulated, SOCS1/3 protein was not detected but it was possible to detect SOCS1/3 mRNA in MPM cell lines. The upregulated miRNAs were successfully knocked-down, however the expected effect on SOCS1 expression was not detected. STAT1 knock-down had different effects on STAT3/5 expression. Caspase 3a and 8 expression was found to be increased after STAT1 knock-down. The physiologic regulation of STAT1 via SOCS1 is completely lost in MPM and it does not seem that the miRNAs identified by now, do inhibit the expression of SOCS1. MPM cell lines compensate STAT1 knock-down by increasing the expression of STAT3 or STAT5a, two genes which are generally considered to be oncogenes. And much more important, STAT1 knock-down induces apoptosis in MPM cell lines and STAT1 might therefore be a target for therapeutic intervention.

  5. CD147 knockdown improves the antitumor efficacy of trastuzumab in HER2-positive breast cancer cells.

    Science.gov (United States)

    Xiong, Lijuan; Ding, Li; Ning, Haoyong; Wu, Chenglin; Fu, Kaifei; Wang, Yuxiao; Zhang, Yan; Liu, Yan; Zhou, Lijun

    2016-09-06

    Trastuzumab is widely used in the clinical treatment of human epidermal growth factor receptor-2 (HER2)-positive breast cancer, but the patient response rate is low. CD147 stimulates cancer cell proliferation, migration, metastasis and differentiation and is involved in chemoresistance in many types of cancer cells. Whether CD147 alters the effect of trastuzumab on HER2-positive breast cancer cells has not been previously reported. Our study confirmed that CD147 suppression enhances the effects of trastuzumab both in vitro and in vivo. CD147 suppression increased the inhibitory rate of trastuzumab and cell apoptosis in SKBR3, BT474, HCC1954 and MDA-MB453 cells compared with the controls. Furthermore, CD147 knockdown increased expression of cleaved Caspase-3/9 and poly (ADP-ribose) polymerase (PARP) and decreased both mitogen-activated protein kinase (MAPK) and Akt phosphorylation in the four cell lines. In an HCC1954 xenograft model, trastuzumab achieved greater suppression of tumor growth in the CD147-knockdown group than in the shRNA negative control (NC) group. These data indicated that enhancement of the effect of trastuzumab on HER2-positive cells following CD147 knockdown might be attributed to increased apoptosis and decreased phosphorylation of signaling proteins. CD147 may be a key protein for enhancing the clinical efficacy of trastuzumab.

  6. Manipulating the in vivo immune response by targeted gene knockdown.

    Science.gov (United States)

    Lieberman, Judy

    2015-08-01

    Aptamers, nucleic acids selected for high affinity binding to proteins, can be used to activate or antagonize immune mediators or receptors in a location and cell-type specific manner and to enhance antigen presentation. They can also be linked to other molecules (other aptamers, siRNAs or miRNAs, proteins, toxins) to produce multifunctional compounds for targeted immune modulation in vivo. Aptamer-siRNA chimeras (AsiCs) that induce efficient cell-specific knockdown in immune cells in vitro and in vivo can be used as an immunological research tool or potentially as an immunomodulating therapeutic. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Microbial production of branched-chain dicarboxylate 2-methylsuccinic acid via enoate reductase-mediated bioreduction.

    Science.gov (United States)

    Wang, Jian; Yang, Yaping; Zhang, Ruihua; Shen, Xiaolin; Chen, Zhenya; Wang, Jia; Yuan, Qipeng; Yan, Yajun

    2018-01-01

    2-Methylsuccinic acid (2-MSA) is a C5 branched-chain dicarboxylate that serves as an attractive synthon for the synthesis of polymers with extensive applications in coatings, cosmetic solvents and bioplastics. However, the lack of natural pathways for 2-MSA biosynthesis has limited its application as a promising bio-replacement. Herein, we conceived a non-natural three-step biosynthetic route for 2-MSA, via employing the citramalate pathway in combination with enoate reductase-mediated bioreduction of the pathway intermediate citraconate. First, over-expression of codon-optimized citramalate synthase variant CimA* from Methanococcus jannaschii, endogenous isopropylmalate isomerase EcLeuCD and enoate reductase YqjM from Bacillus subtilis allowed the production of 2-MSA in Escherichia coli for the first time, with a titer of 0.35g/L in shake flask experiments. Subsequent screening of YqjM-like enoate reductases of different bacterial origins enabled identification and characterization of a new NAD(P)H-dependent enoate reductase KpnER from Klebsiella pneumoniae, which exhibited higher activity towards citraconate than YqjM. Incorporation of KpnER into the 2-MSA biosynthetic pathway led to 2-MSA production improvement to a titer of 0.96g/L in aerobic condition. Subsequent optimizations including cofactor regeneration, microaerobic cultivation and host strain engineering, boosted 2-MSA titer to 3.61g/L with a molar yield of 0.36 in shake flask experiments. This work established a promising platform for 2-MSA bioproduction, which enabled the highest titer of 2-MSA production in microbial hosts so far. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  8. Functional Characterization of Four Putative δ1-Pyrroline-5-Carboxylate Reductases from Bacillus subtilis

    Energy Technology Data Exchange (ETDEWEB)

    Forlani, Giuseppe; Nocek, Boguslaw; Chakravarthy, Srinivas; Joachimiak, Andrzej

    2017-08-02

    In most living organisms, the amino acid proline is synthesized starting from both glutamate and ornithine. In prokaryotes, in the absence of an ornithine cyclodeaminase that has been identified to date only in a small number of soil and plant bacteria, these pathways share the last step, the reduction of δ1-pyrroline-5-carboxylate (P5C) catalyzed by P5C reductase (EC 1.5.1.2). In several species, multiple forms of P5C reductase have been reported, possibly reflecting the dual function of proline. Aside from its common role as a building block of proteins, proline is indeed also involved in the cellular response to osmotic and oxidative stress conditions. Genome analysis of Bacillus subtilis identifies the presence of four genes (ProH, ProI, ProG, and ComER) that, based on bioinformatic and phylogenic studies, were defined as respectively coding a putative P5C reductase. Here we describe the cloning, heterologous expression, functional analysis and small-angle X-ray scattering studies of the four affinity-purified proteins. Results showed that two of them, namely ProI and ComER, lost their catalytic efficiency or underwent subfunctionalization. In the case of ComER, this could be likely explained by the loss of the ability to form a dimer, which has been previously shown to be an essential structural feature of the catalytically active P5C reductase. The properties of the two active enzymes are consistent with a constitutive role for ProG, and suggest that ProH expression may be beneficial to satisfy an increased need for proline.

  9. Functional Characterization of Four Putative δ1-Pyrroline-5-Carboxylate Reductases from Bacillus subtilis

    Energy Technology Data Exchange (ETDEWEB)

    Forlani, Giuseppe; Nocek, Boguslaw; Chakravarthy, Srinivas; Joachimiak, Andrzej

    2017-08-02

    In most living organisms, the amino acid proline is synthesized starting from both glutamate and ornithine. In prokaryotes, in the absence of an ornithine cyclodeaminase that has been identified to date only in a small number of soil and plant bacteria, these pathways share the last step, the reduction of delta(1)-pyrroline-5-carboxylate (P5C) catalyzed by P5C reductase (EC 1.5.1.2). In several species, multiple forms of P5C reductase have been reported, possibly reflecting the dual function of proline. Aside from its common role as a building block of proteins, proline is indeed also involved in the cellular response to osmotic and oxidative stress conditions. Genome analysis of Bacillus subtilis identifies the presence of four genes (ProH, ProI, ProG, and ComER) that, based on bioinformatic and phylogenic studies, were defined as respectively coding a putative P5C reductase. Here we describe the cloning, heterologous expression, functional analysis and small-angle X-ray scattering studies of the four affinity-purified proteins. Results showed that two of them, namely ProI and ComER, lost their catalytic efficiency or underwent subfunctionalization. In the case of ComER, this could be likely explained by the loss of the ability to form a dimer, which has been previously shown to be an essential structural feature of the catalytically active P5C reductase. The properties of the two active enzymes are consistent with a constitutive role for ProG, and suggest that ProH expression may be beneficial to satisfy an increased need for proline.

  10. Functional Characterization of Four Putative δ1-Pyrroline-5-Carboxylate Reductases from Bacillus subtilis

    Directory of Open Access Journals (Sweden)

    Giuseppe Forlani

    2017-08-01

    Full Text Available In most living organisms, the amino acid proline is synthesized starting from both glutamate and ornithine. In prokaryotes, in the absence of an ornithine cyclodeaminase that has been identified to date only in a small number of soil and plant bacteria, these pathways share the last step, the reduction of δ1-pyrroline-5-carboxylate (P5C catalyzed by P5C reductase (EC 1.5.1.2. In several species, multiple forms of P5C reductase have been reported, possibly reflecting the dual function of proline. Aside from its common role as a building block of proteins, proline is indeed also involved in the cellular response to osmotic and oxidative stress conditions. Genome analysis of Bacillus subtilis identifies the presence of four genes (ProH, ProI, ProG, and ComER that, based on bioinformatic and phylogenic studies, were defined as respectively coding a putative P5C reductase. Here we describe the cloning, heterologous expression, functional analysis and small-angle X-ray scattering studies of the four affinity-purified proteins. Results showed that two of them, namely ProI and ComER, lost their catalytic efficiency or underwent subfunctionalization. In the case of ComER, this could be likely explained by the loss of the ability to form a dimer, which has been previously shown to be an essential structural feature of the catalytically active P5C reductase. The properties of the two active enzymes are consistent with a constitutive role for ProG, and suggest that ProH expression may be beneficial to satisfy an increased need for proline.

  11. Overexpression of Soybean Isoflavone Reductase (GmIFR) Enhances Resistance to Phytophthora sojae in Soybean.

    Science.gov (United States)

    Cheng, Qun; Li, Ninghui; Dong, Lidong; Zhang, Dayong; Fan, Sujie; Jiang, Liangyu; Wang, Xin; Xu, Pengfei; Zhang, Shuzhen

    2015-01-01

    Isoflavone reductase (IFR) is an enzyme involved in the biosynthetic pathway of isoflavonoid phytoalexin in plants. IFRs are unique to the plant kingdom and are considered to have crucial roles in plant response to various biotic and abiotic environmental stresses. Here, we report the characterization of a novel member of the soybean isoflavone reductase gene family GmIFR. Overexpression of GmIFR transgenic soybean exhibited enhanced resistance to Phytophthora sojae. Following stress treatments, GmIFR was significantly induced by P. sojae, ethephon (ET), abscisic acid (placeCityABA), salicylic acid (SA). It is located in the cytoplasm when transiently expressed in soybean protoplasts. The daidzein levels reduced greatly for the seeds of transgenic plants, while the relative content of glyceollins in transgenic plants was significantly higher than that of non-transgenic plants. Furthermore, we found that the relative expression levels of reactive oxygen species (ROS) of transgenic soybean plants were significantly lower than those of non-transgenic plants after incubation with P. sojae, suggesting an important role of GmIFR might function as an antioxidant to reduce ROS in soybean. The enzyme activity assay suggested that GmIFR has isoflavone reductase activity.

  12. Association study of methylenetetrahydrofolate reductase C677T mutation with cerebral venous thrombosis in an Iranian population.

    Science.gov (United States)

    Ghaznavi, Habib; Soheili, Zahra; Samiei, Shahram; Soltanpour, Mohammad S

    2015-12-01

    There are limited data on the role of methylenetetrahydrofolate reductase C677T polymorphism and hyperhomocysteinemia as risk factors for cerebral venous thrombosis in Iranian population. We examined a possible association between fasting plasma homocysteine levels, methylenetetrahydrofolate reductase C677T polymorphism, and cerebral venous thrombosis in 50 patients with a diagnosis of cerebral venous thrombosis (20-63 years old) and 75 healthy controls (18-65 years old). Genotyping of the methylenetetrahydrofolate reductase C677T gene polymorphism was performed by PCR-restriction fragment length polymorphism analysis, and homocysteine levels were measured by enzyme immunoassay. Fasting plasma homocysteine levels were significantly higher in cerebral venous thrombosis patients than in controls (P = 0.015). Moreover, plasma homocysteine levels were significantly higher in methylenetetrahydrofolate reductase 677TT genotype compared to 677CT and 677CC genotypes in both cerebral venous thrombosis patients (P = 0.01) and controls (P = 0.03). Neither 677CT heterozygote genotype [odds ratio (OR) 1.35, 95% confidence interval (CI) 0.64-2.84, P = 0.556] nor 677TT homozygote genotype (OR 1.73, 95% CI 0.32-9.21, P = 0.833) was significantly associated with cerebral venous thrombosis. Additionally, no significant differences in the frequency of 677T allele between cerebral venous thrombosis patients and controls were identified (OR 1.31, 95% CI 0.69-2.50, P = 0.512). In conclusion, our study demonstrated that elevated plasma homocysteine levels are significant risk factors for cerebral venous thrombosis. Also, methylenetetrahydrofolate reductase 677TT genotype is not linked with cerebral venous thrombosis, but is a determinant of elevated plasma homocysteine levels.

  13. Novel siRNA formulation to effectively knockdown mutant p53 in osteosarcoma.

    Science.gov (United States)

    Kundu, Anup K; Iyer, Swathi V; Chandra, Sruti; Adhikari, Amit S; Iwakuma, Tomoo; Mandal, Tarun K

    2017-01-01

    The tumor suppressor p53 plays a crucial role in the development of osteosarcoma. The primary objective of this study is to develop and optimize lipid based nanoparticle formulations that can carry siRNA and effectively silence mutant p53 in 318-1, a murine osteosarcoma cell line. The nanoparticles were composed of a mixture of two lipids (cholesterol and DOTAP) and either PLGA or PLGA-PEG and prepared by using an EmulsiFlex-B3 high pressure homogenizer. A series of studies that include using different nanoparticles, different amount of siRNAs, cell numbers, incubation time, transfection media volume, and storage temperature was performed to optimize the gene silencing efficiency. Replacement of lipids by PLGA or PLGA-PEG decreased the particle size and overall cytotoxicity. Among all lipid-polymer nanoformulations, nanoparticles with 10% PLGA showed highest mutant p53 knockdown efficiency while maintaining higher cell viability when a nanoparticle to siRNA ratio equal to 6.8:0.66 and 75 nM siRNA was used. With long term storage the mutant p53 knockdown efficiency decreased to a greater extent. This study warrants a future evaluation of this formulation for gene silencing efficiency of mutant p53 in tissue culture and animal models for the treatment of osteosarcoma.

  14. Novel siRNA formulation to effectively knockdown mutant p53 in osteosarcoma.

    Directory of Open Access Journals (Sweden)

    Anup K Kundu

    Full Text Available The tumor suppressor p53 plays a crucial role in the development of osteosarcoma. The primary objective of this study is to develop and optimize lipid based nanoparticle formulations that can carry siRNA and effectively silence mutant p53 in 318-1, a murine osteosarcoma cell line.The nanoparticles were composed of a mixture of two lipids (cholesterol and DOTAP and either PLGA or PLGA-PEG and prepared by using an EmulsiFlex-B3 high pressure homogenizer. A series of studies that include using different nanoparticles, different amount of siRNAs, cell numbers, incubation time, transfection media volume, and storage temperature was performed to optimize the gene silencing efficiency.Replacement of lipids by PLGA or PLGA-PEG decreased the particle size and overall cytotoxicity. Among all lipid-polymer nanoformulations, nanoparticles with 10% PLGA showed highest mutant p53 knockdown efficiency while maintaining higher cell viability when a nanoparticle to siRNA ratio equal to 6.8:0.66 and 75 nM siRNA was used. With long term storage the mutant p53 knockdown efficiency decreased to a greater extent.This study warrants a future evaluation of this formulation for gene silencing efficiency of mutant p53 in tissue culture and animal models for the treatment of osteosarcoma.

  15. The Drosophila carbonyl reductase sniffer prevents oxidative stress-induced neurodegeneration.

    Science.gov (United States)

    Botella, Jose A; Ulschmid, Julia K; Gruenewald, Christoph; Moehle, Christoph; Kretzschmar, Doris; Becker, Katja; Schneuwly, Stephan

    2004-05-04

    A growing body of evidence suggests that oxidative stress is a common underlying mechanism in the pathogenesis of neurodegenerative disorders such as Alzheimer's, Huntington's, Creutzfeld-Jakob and Parkinson's diseases. Despite the increasing number of reports finding a causal relation between oxidative stress and neurodegeneration, little is known about the genetic elements that confer protection against the deleterious effects of oxidation in neurons. We have isolated and characterized the Drosophila melanogaster gene sniffer, whose function is essential for preventing age-related neurodegeneration. In addition, we demonstrate that oxidative stress is a direct cause of neurodegeneration in the Drosophila central nervous system and that reduction of sniffer activity leads to neuronal cell death. The overexpression of the gene confers neuronal protection against oxygen-induced apoptosis, increases resistance of flies to experimental normobaric hyperoxia, and improves general locomotor fitness. Sniffer belongs to the family of short-chain dehydrogenase/reductase (SDR) enzymes and exhibits carbonyl reductase activity. This is the first in vivo evidence of the direct and important implication of this enzyme as a neuroprotective agent in the cellular defense mechanisms against oxidative stress.

  16. Cytochrome b5 reductase is the component from neuronal synaptic plasma membrane vesicles that generates superoxide anion upon stimulation by cytochrome c

    Directory of Open Access Journals (Sweden)

    Alejandro K. Samhan-Arias

    2018-05-01

    Full Text Available In this work, we measured the effect of cytochrome c on the NADH-dependent superoxide anion production by synaptic plasma membrane vesicles from rat brain. In these membranes, the cytochrome c stimulated NADH-dependent superoxide anion production was inhibited by antibodies against cytochrome b5 reductase linking the production to this enzyme. Measurement of the superoxide anion radical generated by purified recombinant soluble and membrane cytochrome b5 reductase corroborates the production of the radical by different enzyme isoforms. In the presence of cytochrome c, a burst of superoxide anion as well as the reduction of cytochrome c by cytochrome b5 reductase was measured. Complex formation between both proteins suggests that cytochrome b5 reductase is one of the major partners of cytochrome c upon its release from mitochondria to the cytosol during apoptosis. Superoxide anion production and cytochrome c reduction are the consequences of the stimulated NADH consumption by cytochrome b5 reductase upon complex formation with cytochrome c and suggest a major role of this enzyme as an anti-apoptotic protein during cell death.

  17. The structure of Lactococcus lactis thioredoxin reductase reveals molecular features of photo-oxidative damage

    DEFF Research Database (Denmark)

    Skjoldager, Nicklas; Bang, Maria Blanner; Rykær, Martin

    2017-01-01

    The NADPH-dependent homodimeric flavoenzyme thioredoxin reductase (TrxR) provides reducing equivalents to thioredoxin, a key regulator of various cellular redox processes. Crystal structures of photo-inactivated thioredoxin reductase (TrxR) from the Gram-positive bacterium Lactococcus lactis have...... been determined. These structures reveal novel molecular features that provide further insight into the mechanisms behind the sensitivity of this enzyme toward visible light. We propose that a pocket on the si-face of the isoalloxazine ring accommodates oxygen that reacts with photo-excited FAD...... thus be a widespread feature among bacterial TrxR with the described characteristics, which affords applications in clinical photo-therapy of drug-resistant bacteria....

  18. Keratin23 (KRT23 knockdown decreases proliferation and affects the DNA damage response of colon cancer cells.

    Directory of Open Access Journals (Sweden)

    Karin Birkenkamp-Demtröder

    Full Text Available Keratin 23 (KRT23 is strongly expressed in colon adenocarcinomas but absent in normal colon mucosa. Array based methylation profiling of 40 colon samples showed that the promoter of KRT23 was methylated in normal colon mucosa, while hypomethylated in most adenocarcinomas. Promoter methylation correlated with absent expression, while increased KRT23 expression in tumor samples correlated with promoter hypomethylation, as confirmed by bisulfite sequencing. Demethylation induced KRT23 expression in vitro. Expression profiling of shRNA mediated stable KRT23 knockdown in colon cancer cell lines showed that KRT23 depletion affected molecules of the cell cycle and DNA replication, recombination and repair. In vitro analyses confirmed that KRT23 depletion significantly decreased the cellular proliferation of SW948 and LS1034 cells and markedly decreased the expression of genes involved in DNA damage response, mainly molecules of the double strand break repair homologous recombination pathway. KRT23 knockdown decreased the transcript and protein expression of key molecules as e.g. MRE11A, E2F1, RAD51 and BRCA1. Knockdown of KRT23 rendered colon cancer cells more sensitive to irradiation and reduced proliferation of the KRT23 depleted cells compared to irradiated control cells.

  19. Hydroxyurea-resistant vaccinia virus: overproduction of ribonucleotide reductase

    International Nuclear Information System (INIS)

    Slabaugh, M.B.; Mathews, C.K.

    1986-01-01

    Repeated passage of vaccinia virus in increasing concentrations of hydroxyurea followed by plaque purification resulted in the isolation of variants capable of growth in 5 mM hydroxyurea, a drug concentration which inhibited the reproduction of wild-type vaccinia virus 1000-fold. Analyses of viral protein synthesis by using [ 35 S]methionine pulse-labeling at intervals throughout the infection cycle revealed that all isolates overproduced a 34,000-molecular-weight (MW) early polypeptide. Measurement of ribonucleoside-diphosphate reductase activity after infection indicated that 4- to 10-fold more activity was induced by hydroxyurea-resistant viruses than by the wild-type virus. A two-step partial purification resulted in a substantial enrichment for the 34,000-MW protein from extracts of wild-type and hydroxyurea-resistant-virus-infected, but not mock-infected, cells. In the presence of the drug, the isolates incorporated [ 3 H]thymidine into DNA earlier and a rate substantially greater than that of the wild type, although the onset of DNA synthesis was delayed in both cases. The drug resistance trait was markedly unstable in all isolates. In the absence of selective pressure, plaque-purified isolated readily segregated progeny that displayed a wide range of resistance phenotypes. The results of this study indicate that vaccinia virus encodes a subunit of ribonucleotide reductase which is 34,000-MW early protein whose overproduction confers hydroxyurea resistance on reproducing viruses

  20. YqhD. A broad-substrate range aldehyde reductase with various applications in production of biorenewable fuels and chemicals

    Energy Technology Data Exchange (ETDEWEB)

    Jarboe, Laura R. [Iowa State Univ., Ames, IA (United States). Dept. of Chemical and Biological Engineering

    2011-01-15

    The Escherichia coli NADPH-dependent aldehyde reductase YqhD has contributed to a variety of metabolic engineering projects for production of biorenewable fuels and chemicals. As a scavenger of toxic aldehydes produced by lipid peroxidation, YqhD has reductase activity for a broad range of short-chain aldehydes, including butyraldehyde, glyceraldehyde, malondialdehyde, isobutyraldehyde, methylglyoxal, propanealdehyde, acrolein, furfural, glyoxal, 3-hydroxypropionaldehyde, glycolaldehyde, acetaldehyde, and acetol. This reductase activity has proven useful for the production of biorenewable fuels and chemicals, such as isobutanol and 1,3- and 1,2-propanediol; additional capability exists for production of 1-butanol, 1-propanol, and allyl alcohol. A drawback of this reductase activity is the diversion of valuable NADPH away from biosynthesis. This YqhD-mediated NADPH depletion provides sufficient burden to contribute to growth inhibition by furfural and 5-hydroxymethyl furfural, inhibitory contaminants of biomass hydrolysate. The structure of YqhD has been characterized, with identification of a Zn atom in the active site. Directed engineering efforts have improved utilization of 3-hydroxypropionaldehyde and NADPH. Most recently, two independent projects have demonstrated regulation of yqhD by YqhC, where YqhC appears to function as an aldehyde sensor. (orig.)

  1. Boletus edulis Nitrite Reductase Reduces Nitrite Content of Pickles and Mitigates Intoxication in Nitrite-intoxicated Mice.

    Science.gov (United States)

    Zhang, Weiwei; Tian, Guoting; Feng, Shanshan; Wong, Jack Ho; Zhao, Yongchang; Chen, Xiao; Wang, Hexiang; Ng, Tzi Bun

    2015-10-08

    Pickles are popular in China and exhibits health-promoting effects. However, nitrite produced during fermentation adversely affects health due to formation of methemoglobin and conversion to carcinogenic nitrosamine. Fruiting bodies of the mushroom Boletus edulis were capable of inhibiting nitrite production during pickle fermentation. A 90-kDa nitrite reductase (NiR), demonstrating peptide sequence homology to fungal nitrite reductase, was isolated from B. edulis fruiting bodies. The optimum temperature and pH of the enzyme was 45 °C and 6.8, respectively. B. edulis NiR was capable of prolonging the lifespan of nitrite-intoxicated mice, indicating that it had the action of an antidote. The enzyme could also eliminate nitrite from blood after intragastric administration of sodium nitrite, and after packaging into capsule, this nitrite-eliminating activity could persist for at least 120 minutes thus avoiding immediate gastric degradation. B. edulis NiR represents the first nitrite reductase purified from mushrooms and may facilitate subsequent applications.

  2. Salt Sensitive Tet-Off-Like Systems to Knockdown Primordial Germ Cell Genes for Repressible Transgenic Sterilization in Channel Catfish, Ictalurus punctatus.

    Science.gov (United States)

    Li, Hanbo; Su, Baofeng; Qin, Guyu; Ye, Zhi; Alsaqufi, Ahmed; Perera, Dayan A; Shang, Mei; Odin, Ramjie; Vo, Khoi; Drescher, David; Robinson, Dalton; Zhang, Dan; Abass, Nermeen; Dunham, Rex A

    2017-05-31

    Repressible knockdown approaches were investigated for transgenic sterilization in channel catfish, Ictalurus punctatus . Two primordial germ cell (PGC) marker genes, nanos and dead end , were targeted for knockdown, and an off-target gene, vasa , was monitored. Two potentially salt sensitive repressible promoters, zebrafish adenylosuccinate synthase 2 (ADSS) and zebrafish racemase (Rm), were each coupled with four knockdown strategies: ds-sh RNA targeting the 5' end (N1) or 3' end (N2) of channel catfish nanos , full-length cDNA sequence of channel catfish nanos for overexpression (cDNA) and ds-sh RNA targeting channel catfish dead end (DND). Each construct had an untreated group and treated group with sodium chloride as the repressor compound. Spawning rates of full-sibling P₁ fish exposed or not exposed to the constructs as treated and untreated embryos were 93% and 59%, respectively, indicating potential sterilization of fish and repression of the constructs. Although the mRNA expression data of PGC marker genes were inconsistent in P₁ fish, most F₁ individuals were able to downregulate the target genes in untreated groups and repress the knockdown process in treated groups. The results indicate that repressible transgenic sterilization is feasible for reproductive control of fish, but more data from F₂ or F₃ are needed for evaluation.

  3. Streptococcus sanguinis Class Ib Ribonucleotide Reductase

    Science.gov (United States)

    Makhlynets, Olga; Boal, Amie K.; Rhodes, DeLacy V.; Kitten, Todd; Rosenzweig, Amy C.; Stubbe, JoAnne

    2014-01-01

    Streptococcus sanguinis is a causative agent of infective endocarditis. Deletion of SsaB, a manganese transporter, drastically reduces S. sanguinis virulence. Many pathogenic organisms require class Ib ribonucleotide reductase (RNR) to catalyze the conversion of nucleotides to deoxynucleotides under aerobic conditions, and recent studies demonstrate that this enzyme uses a dimanganese-tyrosyl radical (MnIII2-Y•) cofactor in vivo. The proteins required for S. sanguinis ribonucleotide reduction (NrdE and NrdF, α and β subunits of RNR; NrdH and TrxR, a glutaredoxin-like thioredoxin and a thioredoxin reductase; and NrdI, a flavodoxin essential for assembly of the RNR metallo-cofactor) have been identified and characterized. Apo-NrdF with FeII and O2 can self-assemble a diferric-tyrosyl radical (FeIII2-Y•) cofactor (1.2 Y•/β2) and with the help of NrdI can assemble a MnIII2-Y• cofactor (0.9 Y•/β2). The activity of RNR with its endogenous reductants, NrdH and TrxR, is 5,000 and 1,500 units/mg for the Mn- and Fe-NrdFs (Fe-loaded NrdF), respectively. X-ray structures of S. sanguinis NrdIox and MnII2-NrdF are reported and provide a possible rationale for the weak affinity (2.9 μm) between them. These streptococcal proteins form a structurally distinct subclass relative to other Ib proteins with unique features likely important in cluster assembly, including a long and negatively charged loop near the NrdI flavin and a bulky residue (Thr) at a constriction in the oxidant channel to the NrdI interface. These studies set the stage for identifying the active form of S. sanguinis class Ib RNR in an animal model for infective endocarditis and establishing whether the manganese requirement for pathogenesis is associated with RNR. PMID:24381172

  4. RNAi-mediated knockdown of FANCF suppresses cell proliferation, migration, invasion, and drug resistance potential of breast cancer cells

    Directory of Open Access Journals (Sweden)

    L. Zhao

    2014-01-01

    Full Text Available Fanconi anemia complementation group F protein (FANCF is a key factor, which maintains the function of FA/BRCA, a DNA damage response pathway. However, the functional role of FANCF in breast cancer has not been elucidated. We performed a specific FANCF-shRNA knockdown of endogenous FANCF in vitro. Cell viability was measured with a CCK-8 assay. DNA damage was assessed with an alkaline comet assay. Apoptosis, cell cycle, and drug accumulation were measured by flow cytometry. The expression levels of protein were determined by Western blot using specific antibodies. Based on these results, we used cell migration and invasion assays to demonstrate a crucial role for FANCF in those processes. FANCF shRNA effectively inhibited expression of FANCF. We found that proliferation of FANCF knockdown breast cancer cells (MCF-7 and MDA-MB-435S was significantly inhibited, with cell cycle arrest in the S phase, induction of apoptosis, and DNA fragmentation. Inhibition of FANCF also resulted in decreased cell migration and invasion. In addition, FANCF knockdown enhanced sensitivity to doxorubicin in breast cancer cells. These results suggest that FANCF may be a potential target for molecular, therapeutic intervention in breast cancer.

  5. RNAi-mediated knockdown of FANCF suppresses cell proliferation, migration, invasion, and drug resistance potential of breast cancer cells

    International Nuclear Information System (INIS)

    Zhao, L.; Li, N.; Yu, J.K.; Tang, H.T.; Li, Y.L.; He, M.; Yu, Z.J.; Bai, X.F.; Zheng, Z.H.; Wang, E.H.; Wei, M.J.

    2013-01-01

    Fanconi anemia complementation group F protein (FANCF) is a key factor, which maintains the function of FA/BRCA, a DNA damage response pathway. However, the functional role of FANCF in breast cancer has not been elucidated. We performed a specific FANCF-shRNA knockdown of endogenous FANCF in vitro. Cell viability was measured with a CCK-8 assay. DNA damage was assessed with an alkaline comet assay. Apoptosis, cell cycle, and drug accumulation were measured by flow cytometry. The expression levels of protein were determined by Western blot using specific antibodies. Based on these results, we used cell migration and invasion assays to demonstrate a crucial role for FANCF in those processes. FANCF shRNA effectively inhibited expression of FANCF. We found that proliferation of FANCF knockdown breast cancer cells (MCF-7 and MDA-MB-435S) was significantly inhibited, with cell cycle arrest in the S phase, induction of apoptosis, and DNA fragmentation. Inhibition of FANCF also resulted in decreased cell migration and invasion. In addition, FANCF knockdown enhanced sensitivity to doxorubicin in breast cancer cells. These results suggest that FANCF may be a potential target for molecular, therapeutic intervention in breast cancer

  6. RNAi-mediated knockdown of FANCF suppresses cell proliferation, migration, invasion, and drug resistance potential of breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, L.; Li, N.; Yu, J.K.; Tang, H.T.; Li, Y.L.; He, M.; Yu, Z.J.; Bai, X.F. [Department of Pharmacology, School of Pharmacy, China Medical University, Heping Ward, Shenyang City, Liaoning (China); Zheng, Z.H.; Wang, E.H. [Institute of Pathology and Pathophysiology, China Medical University, Heping Ward, Shenyang City, Liaoning (China); Wei, M.J. [Department of Pharmacology, School of Pharmacy, China Medical University, Heping Ward, Shenyang City, Liaoning (China)

    2013-12-12

    Fanconi anemia complementation group F protein (FANCF) is a key factor, which maintains the function of FA/BRCA, a DNA damage response pathway. However, the functional role of FANCF in breast cancer has not been elucidated. We performed a specific FANCF-shRNA knockdown of endogenous FANCF in vitro. Cell viability was measured with a CCK-8 assay. DNA damage was assessed with an alkaline comet assay. Apoptosis, cell cycle, and drug accumulation were measured by flow cytometry. The expression levels of protein were determined by Western blot using specific antibodies. Based on these results, we used cell migration and invasion assays to demonstrate a crucial role for FANCF in those processes. FANCF shRNA effectively inhibited expression of FANCF. We found that proliferation of FANCF knockdown breast cancer cells (MCF-7 and MDA-MB-435S) was significantly inhibited, with cell cycle arrest in the S phase, induction of apoptosis, and DNA fragmentation. Inhibition of FANCF also resulted in decreased cell migration and invasion. In addition, FANCF knockdown enhanced sensitivity to doxorubicin in breast cancer cells. These results suggest that FANCF may be a potential target for molecular, therapeutic intervention in breast cancer.

  7. Cancer cell death induced by phosphine gold(I) compounds targeting thioredoxin reductase.

    Science.gov (United States)

    Gandin, Valentina; Fernandes, Aristi Potamitou; Rigobello, Maria Pia; Dani, Barbara; Sorrentino, Francesca; Tisato, Francesco; Björnstedt, Mikael; Bindoli, Alberto; Sturaro, Alberto; Rella, Rocco; Marzano, Cristina

    2010-01-15

    The thioredoxin system, composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH (nicotinamide adenine dinucleotide phosphate), plays a central role in regulating cellular redox homeostasis and signaling pathways. TrxR, overexpressed in many tumor cells and contributing to drug resistance, has emerged as a new target for anticancer drugs. Gold complexes have been validated as potent TrxR inhibitors in vitro in the nanomolar range. In order to obtain potent and selective TrxR inhibitors, we have synthesized a series of linear, 'auranofin-like' gold(I) complexes all containing the [Au(PEt(3))](+) synthon and the ligands: Cl(-), Br(-), cyanate, thiocyanate, ethylxanthate, diethyldithiocarbamate and thiourea. Phosphine gold(I) complexes efficiently inhibited cytosolic and mitochondrial TrxR at concentrations that did not affect the two related oxidoreductases glutathione reductase (GR) and glutathione peroxidase (GPx). The inhibitory effect of the redox proteins was also observed intracellularly in cancer cells pretreated with gold(I) complexes. Gold(I) compounds were found to induce antiproliferative effects towards several human cancer cells some of which endowed with cisplatin or multidrug resistance. In addition, they were able to activate caspase-3 and induce apoptosis observed as nucleosome formation and sub-G1 cell accumulation. The complexes with thiocyanate and xanthate ligands were particularly effective in inhibiting thioredoxin reductase and inducing apoptosis. Pharmacodynamic studies in human ovarian cancer cells allowed for the correlation of intracellular drug accumulation with TrxR inhibition that leads to the induction of apoptosis via the mitochondrial pathway.

  8. Characterisation of PduS, the pdu metabolosome corrin reductase, and evidence of substructural organisation within the bacterial microcompartment.

    Directory of Open Access Journals (Sweden)

    Joshua B Parsons

    2010-11-01

    Full Text Available PduS is a corrin reductase and is required for the reactivation of the cobalamin-dependent diol dehydratase. It is one component encoded within the large propanediol utilisation (pdu operon, which is responsible for the catabolism of 1,2-propanediol within a self-assembled proteinaceous bacterial microcompartment. The enzyme is responsible for the reactivation of the cobalamin coenzyme required by the diol dehydratase. The gene for the cobalamin reductase from Citrobacter freundii (pduS has been cloned to allow the protein to be overproduced recombinantly in E. coli with an N-terminal His-tag. Purified recombinant PduS is shown to be a flavoprotein with a non-covalently bound FMN that also contains two coupled [4Fe-4S] centres. It is an NADH-dependent flavin reductase that is able to mediate the one-electron reductions of cob(IIIalamin to cob(IIalamin and cob(IIalamin to cob(Ialamin. The [4Fe-4S] centres are labile to oxygen and their presence affects the midpoint redox potential of flavin. Evidence is presented that PduS is able to bind cobalamin, which is inconsistent with the view that PduS is merely a flavin reductase. PduS is also shown to interact with one of the shell proteins of the metabolosome, PduT, which is also thought to contain an [Fe-S] cluster. PduS is shown to act as a corrin reductase and its interaction with a shell protein could allow for electron passage out of the bacterial microcompartment.

  9. X-ray crystal structure of GarR-tartronate semialdehyde reductase from Salmonella typhimurium.

    Science.gov (United States)

    Osipiuk, J; Zhou, M; Moy, S; Collart, F; Joachimiak, A

    2009-09-01

    Tartronate semialdehyde reductases (TSRs), also known as 2-hydroxy-3-oxopropionate reductases, catalyze the reduction of tartronate semialdehyde using NAD as cofactor in the final stage of D-glycerate biosynthesis. These enzymes belong to family of structurally and mechanically related beta-hydroxyacid dehydrogenases which differ in substrate specificity and catalyze reactions in specific metabolic pathways. Here, we present the crystal structure of GarR a TSR from Salmonella typhimurium determined by the single-wavelength anomalous diffraction method and refined to 1.65 A resolution. The active site of the enzyme contains L-tartrate which most likely mimics a position of a glycerate which is a product of the enzyme reaction. The analysis of the TSR structure shows also a putative NADPH binding site in the enzyme.

  10. [Aldose reductase gene polymorphism and rate of appearance of retinopathy in non insulin dependent diabetics].

    Science.gov (United States)

    Olmos, P; Acosta, A M; Schiaffino, R; Díaz, R; Alvarado, D; O'Brien, A; Muñoz, X; Arriagada, P; Claro, J C; Vega, R; Vollrath, V; Velasco, S; Emmerich, M; Maiz, A

    1999-04-01

    Recent studies suggest that polymorphisms associated to the aldose reductase gene could be related to early retinopathy in noninsulin dependent diabetics (NIDDM). There is also new interest on the genetic modulation of coagulation factors in relation to this complication. To look for a possible relationship between the rate of appearance of retinopathy and the genotype of (AC)n polymorphic marker associated to aldose reductase gene. A random sample of 27 NIDDM, aged 68.1 +/- 10.6 years, with a mean diabetes duration of 20.7 +/- 4.8 years and a mean glycosilated hemoglobin of 10.6 +/- 1.6%, was studied. The genotype of the (AC)n, polymorphic marker associated to the 5' end of the aldose reductase (ALR2) gene was determined by 32P-PCR plus sequenciation. Mutations of the factor XIII-A gene were studied by single stranded conformational polymorphism, sequenciation and restriction fragment length polymorphism. Four patients lacked the (AC)24 and had a higher rate of appearance of retinopathy than patients with the (AC)24 allele (0.0167 and 0.0907 score points per year respectively, p = 0.047). Both groups had similar glycosilated hemoglobin (11.7 +/- 0.2 and 10.5 +/- 1.6% respectively). Factor XIII gene mutations were not related to the rate of appearance of retinopathy. Our data suggest that the absence of the (AC)24 allele of the (AC)n polymorphic marker associated to the 5' end of the aldose reductase gene, is associated to a five fold reduction of retinopathy appearance rate.

  11. Nanoparticle-mediated knockdown of DNA repair sensitizes cells to radiotherapy and extends survival in a genetic mouse model of glioblastoma.

    Science.gov (United States)

    Kievit, Forrest M; Wang, Kui; Ozawa, Tatsuya; Tarudji, Aria W; Silber, John R; Holland, Eric C; Ellenbogen, Richard G; Zhang, Miqin

    2017-10-01

    Glioblastoma (GBM) remains incurable, and recurrent tumors rarely respond to standard-of-care radiation and chemo-therapies. Therefore, strategies that enhance the effects of these therapies should provide significant benefits to GBM patients. We have developed a nanoparticle delivery vehicle that can stably bind and protect nucleic acids for specific delivery into brain tumor cells. These nanoparticles can deliver therapeutic siRNAs to sensitize GBM cells to radiotherapy and improve GBM treatment via systemic administration. We show that nanoparticle-mediated knockdown of the DNA repair protein apurinic endonuclease 1 (Ape1) sensitizes GBM cells to radiotherapy and extend survival in a genetic mouse model of GBM. Specific knockdown of Ape1 activity by 30% in brain tumor tissue doubled the extended survival achieved with radiotherapy alone. Ape1 is a promising target for increasing the effectiveness of radiotherapy, and nanoparticle-mediated delivery of siRNA is a promising strategy for tumor specific knockdown of Ape1. Copyright © 2017. Published by Elsevier Inc.

  12. RNAi-mediated knockdown of pituitary tumor-transforming gene-1 (PTTG1) suppresses the proliferation and invasive potential of PC3 human prostate cancer cells

    International Nuclear Information System (INIS)

    Huang, S.Q.; Liao, Q.J.; Wang, X.W.; Xin, D.Q.; Chen, S.X.; Wu, Q.J.; Ye, G.

    2012-01-01

    Pituitary tumor-transforming gene-1 (PTTG1) is a proto-oncogene that promotes tumorigenesis and metastasis in numerous cell types and is overexpressed in a variety of human tumors. We have demonstrated that PTTG1 expression was up-regulated in both human prostate cancer specimens and prostate cancer cell lines. For a more direct assessment of the function of PTTG1 in prostate tumorigenesis, RNAi-mediated knockdown was used to selectively decrease PTTG1 expression in PC3 human prostate tumor cells. After three weeks of selection, colonies stably transfected with PTTG1-targeted RNAi (the knockdown PC3 cell line) or empty vector (the control PC3 cell line) were selected and expanded to investigate the role of PTTG1 expression in PC3 cell growth and invasion. Cell proliferation rate was significantly slower (28%) in the PTTG1 knockdown line after 6 days of growth as indicated by an MTT cell viability assay (P < 0.05). Similarly, a soft agar colony formation assay revealed significantly fewer (66.7%) PTTG1 knockdown PC3 cell colonies than control colonies after three weeks of growth. In addition, PTTG1 knockdown resulted in cell cycle arrest at G1 as indicated by fluorescence-activated cell sorting. The PTTG1 knockdown PC3 cell line also exhibited significantly reduced migration through Matrigel in a transwell assay of invasive potential, and down-regulation of PTTG1 could lead to increased sensitivity of these prostate cancer cells to a commonly used anticancer drug, taxol. Thus, PTTG1 expression is crucial for PC3 cell proliferation and invasion, and could be a promising new target for prostate cancer therapy

  13. HMG-CoA reductase inhibitory activity and phytocomponent investigation of Basella alba leaf extract as a treatment for hypercholesterolemia

    Directory of Open Access Journals (Sweden)

    Baskaran G

    2015-01-01

    Full Text Available Gunasekaran Baskaran,1 Shamala Salvamani,1 Siti Aqlima Ahmad,1 Noor Azmi Shaharuddin,1 Parveen Devi Pattiram,2 Mohd Yunus Shukor1 1Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, 2Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, Selangor, Malaysia Abstract: The enzyme 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA reductase is the key enzyme of the mevalonate pathway that produces cholesterol. Inhibition of HMG-CoA reductase reduces cholesterol biosynthesis in the liver. Synthetic drugs, statins, are commonly used for the treatment of hypercholesterolemia. Due to the side effects of statins, natural HMG-CoA reductase inhibitors of plant origin are needed. In this study, 25 medicinal plant methanol extracts were screened for anti-HMG-CoA reductase activity. Basella alba leaf extract showed the highest inhibitory effect at about 74%. Thus, B. alba was examined in order to investigate its phytochemical components. Gas chromatography with tandem mass spectrometry and reversed phase high-performance liquid chromatography analysis revealed the presence of phenol 2,6-bis(1,1-dimethylethyl, 1-heptatriacotanol, oleic acid, eicosyl ester, naringin, apigenin, luteolin, ascorbic acid, and a-tocopherol, which have been reported to possess antihypercholesterolemic effects. Further investigation of in vivo models should be performed in order to confirm its potential as an alternative treatment for hypercholesterolemia and related cardiovascular diseases. Keywords: HMG-CoA reductase, Basella alba, phytochemical, GC-MS/MS, RP-HPLC, hypercholesterolemia

  14. Genetic and Biochemical Analysis of Intragenic Complementation Events among Nitrate Reductase Apoenzyme-Deficient Mutants of Nicotiana Plumbaginifolia

    OpenAIRE

    Pelsy, F.; Gonneau, M.

    1991-01-01

    Intragenic complementation has been observed between apoenzyme nitrate reductase-deficient mutants (nia) of Nicotiana plumbaginifolia. In vivo as in vitro, the NADH-nitrate reductase (NR) activity in plants heterozygous for two different nia alleles was lower than in the wild type plant, but the plants were able to grow on nitrate as a sole nitrogen source. NR activity, absent in extracts of homozygous nia mutants was restored by mixing extracts from two complementing nia mutants. These obser...

  15. [Effect of NOR1 gene knockdown on the biological behavior of HeLa cells].

    Science.gov (United States)

    Tan, Yixin; Li, Wenjuan; Yi, Mei; Wang, Wei; Zheng, Pan; Zhang, Haijing; Xiang, Bo; Li, Guiyuan

    2014-08-01

    To explore the effect of the oxidored nitro domain containing protein 1 (NOR1) gene knockdown on the biological behavior of HeLa cells in cervical carcinoma. The recombinant plasmids pSUPER-shNOR1-1, pSUPER-shNOR1-2 and pSUPERscramble, which targeted to NOR1 gene, were constructed by pSUPER.neo+GFP vector, transfected into HeLa cells respectively using Lipofectamine 2000 reagent, and followed by G418 selection. The expression level of NOR1 mRNA and protein were determined by RT-PCR and Western blotting, respectively. Methyl thiazolyl tetrazolium (MTT) assay was performed to determine the growth curve of cell viability. The stable transfectants were treated with H₂O₂ and cell apoptosis was determined by Hoechst 33258 staining and terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL) assay. The expression levels of Bcl-2, cleaved caspase 9 and poly ADP-ribose polymerase (PARP) were measured by Western blot. NOR1- knockdown HeLa cells were successfully constructed by transfection of pSUPER-shNOR1-1 or pSUPER-shNOR1-2 plasmids into HeLa cells. MTT assay showed that the silence of endogenous NOR1 in HeLa cells could lead to the increase in cell viability and proliferation, and the inhibition of H₂O₂-induced apoptosis compared with the negative control. Western blot showed that the expression level of active caspase 9 and cleaved PARP was inhibited in NOR1-knockdown cells when they were treated with H₂O₂ while the expression level of Bcl-2 protein increased. Silence of endogenous NOR1 facilitates the cell viability and growth of HeLa cells, and attenuates HeLa cells apoptosis induced by H₂O₂, which might be mediated by up-regulation of Bcl-2 level and down-regulation of the cleaved caspase 9 cascade.

  16. Colour formation in fermented sausages by meat-associated staphylococci with different nitrite- and nitrate-reductase activities.

    Science.gov (United States)

    Gøtterup, Jacob; Olsen, Karsten; Knøchel, Susanne; Tjener, Karsten; Stahnke, Louise H; Møller, Jens K S

    2008-04-01

    Three Staphylococcus strains, S. carnosus, S. simulans and S. saprophyticus, selected due to their varying nitrite and/or nitrate-reductase activities, were used to initiate colour formation during sausage fermentation. During fermentation of sausages with either nitrite or nitrate added, colour was followed by L(∗)a(∗)b measurements and the content of nitrosylmyoglobin (MbFe(II)NO) quantified by electron spin resonance (ESR). MbFe(II)NO was rapidly formed in sausages with added nitrite independent of the presence of nitrite reducing bacteria, whereas the rate of MbFe(II)NO formation in sausages with added nitrate depended on the specific Staphylococcus strain. Strains with high nitrate-reductase activity showed a significantly faster rate of pigment formation, but other factors were of influence as well. Product stability for the sliced, packaged sausage was evaluated as surface colour and oxidation by autofluorescence and hexanal content, respectively. No significant direct effect of the Staphylococcus addition was observed, however, there was a clear correspondence between high initial amount of MbFe(II)NO in the different sausages and the colour stability during storage. Autofluorescence data correlated well with hexanal content, and may be used as predictive tools. Overall, nitrite- and nitrate-reductase activities of Staphylococcus strains in nitrite-cured sausages were of limited importance regarding colour development, while in nitrate-cured sausages strains with higher nitrate reductase activity were crucial for ensuring optimal colour formation during initial fermentation stages.

  17. Expression, purification, crystallization and preliminary X-ray analysis of maleylacetate reductase from Burkholderia sp. strain SJ98

    International Nuclear Information System (INIS)

    Chauhan, Archana; Islam, Zeyaul; Jain, Rakesh Kumar; Karthikeyan, Subramanian

    2009-01-01

    Purification and preliminary X-ray crystallographic analysis of maleylacetate reductase encoded by the pnpD gene is reported. Maleylacetate reductase (EC 1.3.1.32) is an important enzyme that is involved in the degradation pathway of aromatic compounds and catalyzes the reduction of maleylacetate to 3-oxoadipate. The gene pnpD encoding maleylacetate reductase in Burkholderia sp. strain SJ98 was cloned, expressed in Escherichia coli and purified by affinity chromatography. The enzyme was crystallized in both native and SeMet-derivative forms by the sitting-drop vapour-diffusion method using PEG 3350 as a precipitant at 293 K. The crystals belonged to space group P2 1 2 1 2, with unit-cell parameters a = 72.91, b = 85.94, c = 53.07 Å. X-ray diffraction data for the native and SeMet-derivative crystal were collected to 2.7 and 2.9 Å resolution, respectively

  18. REST/NRSF Knockdown Alters Survival, Lineage Differentiation and Signaling in Human Embryonic Stem Cells.

    Directory of Open Access Journals (Sweden)

    Kaushali Thakore-Shah

    Full Text Available REST (RE1 silencing transcription factor, also known as NRSF (neuron-restrictive silencer factor, is a well-known transcriptional repressor of neural genes in non-neural tissues and stem cells. Dysregulation of REST activity is thought to play a role in diverse diseases including epilepsy, cancer, Down's syndrome and Huntington's disease. The role of REST/NRSF in control of human embryonic stem cell (hESC fate has never been examined. To evaluate the role of REST in hESCs we developed an inducible REST knockdown system and examined both growth and differentiation over short and long term culture. Interestingly, we have found that altering REST levels in multiple hESC lines does not result in loss of self-renewal but instead leads to increased survival. During differentiation, REST knockdown resulted in increased MAPK/ERK and WNT signaling and increased expression of mesendoderm differentiation markers. Therefore we have uncovered a new role for REST in regulation of growth and early differentiation decisions in human embryonic stem cells.

  19. Simultaneous analysis of multiple Mycobacterium tuberculosis knockdown mutants in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Antje Blumenthal

    2010-12-01

    Full Text Available Mycobacterium tuberculosis (Mtb represents one of the most persistent bacterial threats to human health and new drugs are needed to limit its impact. Conditional knockdown mutants can help validate new drug targets, but the analysis of individual mutants is laborious and time consuming. Here, we describe quantitative DNA tags (qTags and their use to simultaneously analyze conditional Mtb knockdown mutants that allowed silencing the glyoxylate and methylcitrate cycles (via depletion of isocitrate lyase, ICL, the serine protease Rv3671c, and the core subunits of the mycobacterial proteasome, PrcB and PrcA. The impact of gene silencing in multi-strain cultures was determined by measuring the relative abundance of mutant-specific qTags with real-time PCR. This achieved accurate quantification over a broad range of qTag abundances and depletion of ICL, Rv3671c, or PrcBA resulted in the expected impairment of growth of Mtb with butyrate as the primary carbon source, survival during oxidative stress, acid stress and starvation. The impact of depleting ICL, Rv3671c, or PrcBA in multi-strain mouse infections was analyzed with two approaches. We first measured the relative abundance of mutant-specific qTags in total chromosomal DNA isolated from bacteria that were recovered from infected lungs on agar plates. We then developed a two-step amplification procedure, which allowed us to measure the abundances of individual mutants directly in infected lung tissue. Both strategies confirmed that inactivation of Rv3671c and PrcBA severely reduced persistence of Mtb in mice. The multi-strain infections furthermore suggested that silencing ICL not only prevented growth of Mtb during acute infections but also prevented survival of Mtb during chronic infections. Analyses of the ICL knockdown mutant in single-strain infections confirmed this and demonstrated that silencing of ICL during chronic infections impaired persistence of Mtb to the extent that the pathogen

  20. A novel twist on molecular interactions between thioredoxin and nicotinamide adenine dinucleotide phosphate-dependent thioredoxin reductase

    DEFF Research Database (Denmark)

    Kirkensgaard, Kristine Groth; Hägglund, Per; Shahpiri, Azar

    2013-01-01

    The ubiquitous disulfide reductase thioredoxin (Trx) regulates several important biological processes such as seed germination in plants. Oxidized cytosolic Trx is regenerated by nicotinamide adenine dinucleotide phosphate (NADPH)-dependent thioredoxin reductase (NTR) in a multistep transfer...... dinucleotide (FAD)-binding domain of HvNTR2 to strongly affect the interaction with Trx. In particular, Trp42 and Met43 play key roles for recognition of the endogenous HvTrxh2. Trx from Arabidopsis thaliana is also efficiently recycled by HvNTR2 but turnover in this case appears to be less dependent...

  1. Knockdown of MAGEA6 Activates AMP-Activated Protein Kinase (AMPK) Signaling to Inhibit Human Renal Cell Carcinoma Cells.

    Science.gov (United States)

    Ye, Xueting; Xie, Jing; Huang, Hang; Deng, Zhexian

    2018-01-01

    Melanoma antigen A6 (MAGEA6) is a cancer-specific ubiquitin ligase of AMP-activated protein kinase (AMPK). The current study tested MAGEA6 expression and potential function in renal cell carcinoma (RCC). MAGEA6 and AMPK expression in human RCC tissues and RCC cells were tested by Western blotting assay and qRT-PCR assay. shRNA method was applied to knockdown MAGEA6 in human RCC cells. Cell survival and proliferation were tested by MTT assay and BrdU ELISA assay, respectively. Cell apoptosis was tested by the TUNEL assay and single strand DNA ELISA assay. The 786-O xenograft in nude mouse model was established to test RCC cell growth in vivo. MAGEA6 is specifically expressed in RCC tissues as well as in the established (786-O and A498) and primary human RCC cells. MAGEA6 expression is correlated with AMPKα1 downregulation in RCC tissues and cells. It is not detected in normal renal tissues nor in the HK-2 renal epithelial cells. MAGEA6 knockdown by targeted-shRNA induced AMPK stabilization and activation, which led to mTOR complex 1 (mTORC1) in-activation and RCC cell death/apoptosis. AMPK inhibition, by AMPKα1 shRNA or the dominant negative AMPKα1 (T172A), almost reversed MAGEA6 knockdown-induced RCC cell apoptosis. Conversely, expression of the constitutive-active AMPKα1 (T172D) mimicked the actions by MAGEA6 shRNA. In vivo, MAGEA6 shRNA-bearing 786-O tumors grew significantly slower in nude mice than the control tumors. AMPKα1 stabilization and activation as well as mTORC1 in-activation were detected in MAGEA6 shRNA tumor tissues. MAGEA6 knockdown inhibits human RCC cells via activating AMPK signaling. © 2018 The Author(s). Published by S. Karger AG, Basel.

  2. Pichia stipitis xylose reductase helps detoxifying lignocellulosic hydrolysate by reducing 5-hydroxymethyl-furfural (HMF

    Directory of Open Access Journals (Sweden)

    Röder Anja

    2008-06-01

    Full Text Available Abstract Background Pichia stipitis xylose reductase (Ps-XR has been used to design Saccharomyces cerevisiae strains that are able to ferment xylose. One example is the industrial S. cerevisiae xylose-consuming strain TMB3400, which was constructed by expression of P. stipitis xylose reductase and xylitol dehydrogenase and overexpression of endogenous xylulose kinase in the industrial S. cerevisiae strain USM21. Results In this study, we demonstrate that strain TMB3400 not only converts xylose, but also displays higher tolerance to lignocellulosic hydrolysate during anaerobic batch fermentation as well as 3 times higher in vitro HMF and furfural reduction activity than the control strain USM21. Using laboratory strains producing various levels of Ps-XR, we confirm that Ps-XR is able to reduce HMF both in vitro and in vivo. Ps-XR overexpression increases the in vivo HMF conversion rate by approximately 20%, thereby improving yeast tolerance towards HMF. Further purification of Ps-XR shows that HMF is a substrate inhibitor of the enzyme. Conclusion We demonstrate for the first time that xylose reductase is also able to reduce the furaldehyde compounds that are present in undetoxified lignocellulosic hydrolysates. Possible implications of this newly characterized activity of Ps-XR on lignocellulosic hydrolysate fermentation are discussed.

  3. Impact of Subolesin and Cystatin Knockdown by RNA Interference in Adult Female Haemaphysalis longicornis (Acari: Ixodidae on Blood Engorgement and Reproduction

    Directory of Open Access Journals (Sweden)

    Md. Khalesur Rahman

    2018-04-01

    Full Text Available Currently, multi-antigenic vaccine use is the method of choice for the strategic control of ticks. Therefore, determining the efficacy of combined antigens is a promising avenue of research in the development of anti-tick vaccines. The antigen responsible for blood intake and reproduction has proven suitable as a vaccine antigen. It has been shown to silence Haemaphysalis longicornis salivary cystatin (HlSC-1 and subolesin by RNA interference. Adult unfed female ticks were injected with double-stranded RNA of (A subolesin, (B cystatin, (C subolesin plus cystatin, and (D injection buffer, then fed alongside normal unfed males up to spontaneous drop-down. The percentage of knockdowns was determined by real-time polymerase chain reaction. Sixty-three percent and 53% knockdown rates were observed in subolesin and cystatin double-stranded RNA-injected ticks respectively, while 32 and 26% knockdown rates of subolesin and cystatin transcript were observed in subolesin plus cystatin double-stranded RNA-injected ticks. Subolesin and/or cystatin knockdown causes a significant (p < 0.05 reduction in tick engorgement, egg mass weight, and egg conversion ratio. Most importantly, combined silencing did not act synergistically, but caused a similarly significant (p < 0.05 reduction in tick engorgement, egg mass weight, and egg conversion ratio. Therefore, the elucidation of multiple antigens may be helpful in the future of vaccines.

  4. Salt Sensitive Tet-Off-Like Systems to Knockdown Primordial Germ Cell Genes for Repressible Transgenic Sterilization in Channel Catfish, Ictalurus punctatus

    Directory of Open Access Journals (Sweden)

    Hanbo Li

    2017-05-01

    Full Text Available Repressible knockdown approaches were investigated for transgenic sterilization in channel catfish, Ictalurus punctatus. Two primordial germ cell (PGC marker genes, nanos and dead end, were targeted for knockdown, and an off-target gene, vasa, was monitored. Two potentially salt sensitive repressible promoters, zebrafish adenylosuccinate synthase 2 (ADSS and zebrafish racemase (Rm, were each coupled with four knockdown strategies: ds-sh RNA targeting the 5′ end (N1 or 3′ end (N2 of channel catfish nanos, full-length cDNA sequence of channel catfish nanos for overexpression (cDNA and ds-sh RNA targeting channel catfish dead end (DND. Each construct had an untreated group and treated group with sodium chloride as the repressor compound. Spawning rates of full-sibling P1 fish exposed or not exposed to the constructs as treated and untreated embryos were 93% and 59%, respectively, indicating potential sterilization of fish and repression of the constructs. Although the mRNA expression data of PGC marker genes were inconsistent in P1 fish, most F1 individuals were able to downregulate the target genes in untreated groups and repress the knockdown process in treated groups. The results indicate that repressible transgenic sterilization is feasible for reproductive control of fish, but more data from F2 or F3 are needed for evaluation.

  5. X-Ray crystal structure of GarR—tartronate semialdehyde reductase from Salmonella typhimurium

    Science.gov (United States)

    Osipiuk, J.; Zhou, M.; Moy, S.; Collart, F.

    2009-01-01

    Tartronate semialdehyde reductases (TSRs), also known as 2-hydroxy-3-oxopropionate reductases, catalyze the reduction of tartronate semialdehyde using NAD as cofactor in the final stage of D-glycerate biosynthesis. These enzymes belong to family of structurally and mechanically related β-hydroxyacid dehydrogenases which differ in substrate specificity and catalyze reactions in specific metabolic pathways. Here, we present the crystal structure of GarR a TSR from Salmonella typhimurium determined by the single-wavelength anomalous diffraction method and refined to 1.65 Å resolution. The active site of the enzyme contains L-tartrate which most likely mimics a position of a glycerate which is a product of the enzyme reaction. The analysis of the TSR structure shows also a putative NADPH binding site in the enzyme. PMID:19184529

  6. Normal bone density in male pseudohermaphroditism due to 5a- reductase 2 deficiency

    Directory of Open Access Journals (Sweden)

    Costa Elaine Maria Frade

    2001-01-01

    Full Text Available Bone is an androgen-dependent tissue, but it is not clear whether the androgen action in bone depends on testosterone or on dihydrotestosterone. Patients with 5alpha-reductase 2 deficiency present normal levels of testosterone and low levels of dihydrotestosterone, providing an in vivo human model for the analysis of the effect of testosterone on bone. OBJECTIVE: To analyze bone mineral density in 4 adult patients with male pseudohermaphroditism due to 5alpha-reductase 2 deficiency. RESULTS: Three patients presented normal bone mineral density of the lumbar column (L1-L4 and femur neck, and the other patient presented a slight osteopenia in the lumbar column. CONCLUSION: Patients with dihydrotestosterone deficiency present normal bone mineral density, suggesting that dihydrotestosterone is not the main androgen acting in bone.

  7. RNAi-mediated knock-down of Dab and Numb attenuate Aβ levels via γ-secretase mediated APP processing.

    Science.gov (United States)

    Xie, Zhongcong; Dong, Yuanlin; Maeda, Uta; Xia, Weiming; Tanzi, Rudolph E

    2012-03-22

    Amyloid-β-protein (Aβ), the key component of senile plaques in Alzheimer's disease (AD) brain, is produced from amyloid precursor protein (APP) by cleavage of β-secretase and then γ-secretase. APP adaptor proteins with phosphotyrosine-binding (PTB) domains, including Dab (gene: DAB) and Numb (gene: NUMB), can bind to and interact with the conserved YENPTY-motif in the APP C-terminus. Here we describe, for the first time, the effects of RNAi knock-down of Dab and Numb expression on APP processing and Aβ production. RNAi knock-down of Dab and Numb in H4 human neuroglioma cells stably transfected to express either FL-APP (H4-FL-APP cells) or APP-C99 (H4-APP-C99 cells) increased levels of APP-C-terminal fragments (APP-CTFs) and lowered Aβ levels in both cell lines by inhibiting γ-secretase cleavage of APP. Finally, RNAi knock-down of APP also reduced levels of Numb in H4-APP cells. These findings suggest that pharmacologically blocking interaction of APP with Dab and Numb may provide novel therapeutic strategies of AD. The notion of attenuating γ-secretase cleavage of APP via the APP adaptor proteins, Dab and Numb, is particularly attractive with regard to therapeutic potential, given that side effects of γ-secretase inhibition owing to impaired proteolysis of other γ-secretase substrates, e.g. Notch, might be avoided.

  8. RNAi-mediated knockdown of the voltage gated sodium ion channel TcNav causes mortality in Tribolium castaneum.

    Science.gov (United States)

    Abd El Halim, Hesham M; Alshukri, Baida M H; Ahmad, Munawar S; Nakasu, Erich Y T; Awwad, Mohammed H; Salama, Elham M; Gatehouse, Angharad M R; Edwards, Martin G

    2016-07-14

    The voltage-gated sodium ion channel (VGSC) belongs to the largest superfamily of ion channels. Since VGSCs play key roles in physiological processes they are major targets for effective insecticides. RNA interference (RNAi) is widely used to analyse gene function, but recently, it has shown potential to contribute to novel strategies for selectively controlling agricultural insect pests. The current study evaluates the delivery of dsRNA targeted to the sodium ion channel paralytic A (TcNav) gene in Tribolium castaneum as a viable means of controlling this insect pest. Delivery of TcNav dsRNA caused severe developmental arrest with larval mortalities up to 73% post injection of dsRNA. Injected larvae showed significant (p < 0.05) knockdown in gene expression between 30-60%. Expression was also significantly (p < 0.05) reduced in pupae following injection causing 30% and 42% knockdown for early and late pupal stages, respectively. Oral delivery of dsRNA caused dose-dependant mortalities of between 19 and 51.34%; this was accompanied by significant (p < 0.05) knockdown in gene expression following 3 days of continuous feeding. The majority of larvae injected with, or fed, dsRNA died during the final larval stage prior to pupation. This work provides evidence of a viable RNAi-based strategy for insect control.

  9. RNAi-mediated knock-down of Dab and Numb attenuate Aβ levels via γ-secretase mediated APP processing

    Science.gov (United States)

    2012-01-01

    Amyloid-β-protein (Aβ), the key component of senile plaques in Alzheimer's disease (AD) brain, is produced from amyloid precursor protein (APP) by cleavage of β-secretase and then γ-secretase. APP adaptor proteins with phosphotyrosine-binding (PTB) domains, including Dab (gene: DAB) and Numb (gene: NUMB), can bind to and interact with the conserved YENPTY-motif in the APP C-terminus. Here we describe, for the first time, the effects of RNAi knock-down of Dab and Numb expression on APP processing and Aβ production. RNAi knock-down of Dab and Numb in H4 human neuroglioma cells stably transfected to express either FL-APP (H4-FL-APP cells) or APP-C99 (H4-APP-C99 cells) increased levels of APP-C-terminal fragments (APP-CTFs) and lowered Aβ levels in both cell lines by inhibiting γ-secretase cleavage of APP. Finally, RNAi knock-down of APP also reduced levels of Numb in H4-APP cells. These findings suggest that pharmacologically blocking interaction of APP with Dab and Numb may provide novel therapeutic strategies of AD. The notion of attenuating γ-secretase cleavage of APP via the APP adaptor proteins, Dab and Numb, is particularly attractive with regard to therapeutic potential, given that side effects of γ-secretase inhibition owing to impaired proteolysis of other γ-secretase substrates, e.g. Notch, might be avoided. PMID:23211096

  10. Knockdown of human serine/threonine kinase 33 suppresses human small cell lung carcinoma by blocking RPS6/BAD signaling transduction.

    Science.gov (United States)

    Sun, E L; Liu, C X; Ma, Z X; Mou, X Y; Mu, X A; Ni, Y H; Li, X L; Zhang, D; Ju, Y R

    2017-01-01

    Small cell lung cancer (SCLC) is characterized by rapid growth rate and a tendency to metastasize to distinct sites of patients' bodies. The human serine/threonine kinase 33 (STK33) gene has shown its potency as a therapeutic target for prevention of lung carcinomas including non-small cell lung cancer (NSCLC), but its function in the oncogenesis and development of SCLC remains unrevealed. In the current study, it was hypothesized that STK33 played a key role in the proliferation, survival, and invasion of SCLC cells. The expression of STK33 in human SCLC cell lines NCI-H466 and DMS153 was inhibited by specific shRNA. The cell proliferation, cell apoptosis, and cell invasion of the cells were assessed with a series of in vitro assays. To explore the mechanism through which STK33 gene exerted its function in the carcinogenesis of SCLC cells, the effect of STK33 knockdown on the activity of S6K1/RPS6/BAD signaling was detected. Then the results were further confirmed with STK33 inhibitor ML281 and in vivo assays. The results demonstrated that inhibition of STK33 in SCLC cells suppressed the cell proliferation and invasion while induced cell apoptosis. Associated with the change in the phenotypic features, knockdown of STK33 also decreased the phosphorylation of RPS6 and BAD while increased the expression of cleaved caspase 9, indicating that apoptosis induced by STK33 suppression was mediated via mitochondrial pathway. Similar to the results of STK33 knockdown, incubating NCI-H466 cells with STK33 inhibitor also reduced the cell viability by suppressing RPS6/BAD pathways. Additionally, STK33 knockdown also inhibited tumor growth and RPS6/BAD activity in mice models. Findings outlined in our study were different from that in NSCLC to some extent: knockdown of STK33 in SCLC cells induced the apoptosis through mitochondrial pathway but independent of S6K1 function, inferring that the function of STK33 might be cancer type specific.

  11. Knockdown of human deubiquitinase PSMD14 induces cell cycle arrest and senescence

    Energy Technology Data Exchange (ETDEWEB)

    Byrne, Ann; McLaren, Rajashree P.; Mason, Paul; Chai, Lilly; Dufault, Michael R.; Huang, Yinyin; Liang, Beirong; Gans, Joseph D.; Zhang, Mindy; Carter, Kara; Gladysheva, Tatiana B.; Teicher, Beverly A.; Biemann, Hans-Peter N.; Booker, Michael; Goldberg, Mark A.; Klinger, Katherine W.; Lillie, James [Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701 (United States); Madden, Stephen L., E-mail: steve.madden@genzyme.com [Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701 (United States); Jiang, Yide, E-mail: yide.jiang@genzyme.com [Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701 (United States)

    2010-01-15

    The PSMD14 (POH1, also known as Rpn11/MPR1/S13/CepP1) protein within the 19S complex (19S cap; PA700) is responsible for substrate deubiquitination during proteasomal degradation. The role of PSMD14 in cell proliferation and senescence was explored using siRNA knockdown in carcinoma cell lines. Our results reveal that down-regulation of PSMD14 by siRNA transfection had a considerable impact on cell viability causing cell arrest in the G0-G1 phase, ultimately leading to senescence. The molecular events associated with decreased cell proliferation, cell cycle arrest and senescence include down-regulation of cyclin B1-CDK1-CDC25C, down-regulation of cyclin D1 and up-regulation of p21{sup /Cip} and p27{sup /Kip1}. Most notably, phosphorylation of the retinoblastoma protein was markedly reduced in PSMD14 knockdown cells. A comparative study with PSMB5, a subunit of the 20S proteasome, revealed that PSMB5 and PSMD14 have different effects on cell cycle, senescence and associated molecular events. These data support the view that the 19S and 20S subunits of the proteasome have distinct biological functions and imply that targeting 19S and 20S would have distinct molecular consequences on tumor cells.

  12. Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR

    International Nuclear Information System (INIS)

    Hou, Feng; Miyakawa, Takuya; Kataoka, Michihiko; Takeshita, Daijiro; Kumashiro, Shoko; Uzura, Atsuko; Urano, Nobuyuki; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2014-01-01

    Highlights: • Crystal structure of AtQR has been determined at 1.72 Å. • NADH binding induces the formation of substrate binding site. • AtQR possesses a conserved hydrophobic wall for stereospecific binding of substrate. • Additional Glu197 residue is critical to the high binding affinity. - Abstract: (R)-3-Quinuclidinol, a useful compound for the synthesis of various pharmaceuticals, can be enantioselectively produced from 3-quinuclidinone by 3-quinuclidinone reductase. Recently, a novel NADH-dependent 3-quinuclidionone reductase (AtQR) was isolated from Agrobacterium tumefaciens, and showed much higher substrate-binding affinity (>100 fold) than the reported 3-quinuclidionone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of AtQR at 1.72 Å. Three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the α7 helix. This helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity. AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydrophobic wall for the enantioselective reduction of 3-quinuclidinone as well as RrQR. An additional residue on the α7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation and enhancement of substrate-binding affinity. Mutational analyses also support that Glu197 is an indispensable residue for the activity

  13. Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR

    Energy Technology Data Exchange (ETDEWEB)

    Hou, Feng; Miyakawa, Takuya [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Kataoka, Michihiko [Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 559-8531 (Japan); Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Takeshita, Daijiro [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Kumashiro, Shoko [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Uzura, Atsuko [Research and Development Center, Nagase and Co., Ltd., 2-2-3 Muratani, Nishi-ku, Kobe 651-2241 (Japan); Urano, Nobuyuki [Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 559-8531 (Japan); Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Nagata, Koji [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Shimizu, Sakayu [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Faculty of Bioenvironmental Science, Kyoto Gakuen University, Sogabe-cho, Kameoka 621-8555 (Japan); Tanokura, Masaru, E-mail: amtanok@mail.ecc.u-tokyo.ac.jp [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan)

    2014-04-18

    Highlights: • Crystal structure of AtQR has been determined at 1.72 Å. • NADH binding induces the formation of substrate binding site. • AtQR possesses a conserved hydrophobic wall for stereospecific binding of substrate. • Additional Glu197 residue is critical to the high binding affinity. - Abstract: (R)-3-Quinuclidinol, a useful compound for the synthesis of various pharmaceuticals, can be enantioselectively produced from 3-quinuclidinone by 3-quinuclidinone reductase. Recently, a novel NADH-dependent 3-quinuclidionone reductase (AtQR) was isolated from Agrobacterium tumefaciens, and showed much higher substrate-binding affinity (>100 fold) than the reported 3-quinuclidionone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of AtQR at 1.72 Å. Three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the α7 helix. This helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity. AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydrophobic wall for the enantioselective reduction of 3-quinuclidinone as well as RrQR. An additional residue on the α7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation and enhancement of substrate-binding affinity. Mutational analyses also support that Glu197 is an indispensable residue for the activity.

  14. ADP-ribosylation of dinitrogenase reductase in Rhodobacter capsulatus

    International Nuclear Information System (INIS)

    Jouanneau, Y.; Roby, C.; Meyer, C.M.; Vignais, P.M.

    1989-01-01

    In the photosynthetic bacterium Rhodobacter capsulatus, nitrogenase is regulated by a reversible covalent modification of Fe protein or dinitrogenase reductase (Rc2). The linkage of the modifying group to inactive Rc2 was found to be sensitive to alkali and to neutral hydroxylamine. Complete release of the modifying group was achieved by incubation of inactive Rc2 in 0.4 or 1 M hydroxylamine. After hydroxylamine treatment of the Rc2 preparation, the modifying group could be isolated and purified by affinity chromatography and ion-exchange HPLC. The modifying group comigrated with ADP-ribose on both ion-exchange HPLC and thin-layer chromatography. Analyses by 31 P NMR spectroscopy and mass spectrometry provided further evidence that the modifying group was ADP-ribose. The NMR spectrum of inactive Rc2 exhibited signals characteristic of ADP-ribose; integration of these signals allowed calculation of a molar ration ADP-ribose/Rc2 of 0.63. A hexapeptide carrying the ADP-ribose moiety was purified from a subtilisin digest of inactive Rc2. The structure of this peptide, determined by amino acid analysis and sequencing, is Gly-Arg(ADP-ribose)-Gly-Val-Ile-Thr. This structure allows identification of the binding site for ADP-ribose as Arg 101 of the polypeptide chain of Rc2. It is concluded that nitrogenase activity in R. capsulatus is regulated by reversible ADP-ribosylation of a specific arginyl residue of dinitrogenase reductase

  15. A genetic screen reveals a periplasmic copper chaperone required for nitrite reductase activity in pathogenic Neisseria.

    Science.gov (United States)

    Jen, Freda E-C; Djoko, Karrera Y; Bent, Stephen J; Day, Christopher J; McEwan, Alastair G; Jennings, Michael P

    2015-09-01

    Under conditions of low oxygen availability, Neisseria meningitidis and Neisseria gonorrhoeae are able to respire via a partial denitrification pathway in which nitrite is converted to nitrous oxide. In this process, nitrite reductase (AniA), a copper (Cu)-containing protein converts nitrite to NO, and this product is converted to nitrous oxide by nitric oxide reductase (NorB). NorB also confers protection against toxic NO, and so we devised a conditional lethal screen, using a norB mutant, to identify mutants that were resistant to nitrite-dependent killing. After random-deletion mutagenesis of N. meningitidis, this genetic screen identified a gene encoding a Cu chaperone that is essential for AniA function, AccA. Purified AccA binds one Cu (I) ion and also possesses a second binding site for Cu (II). This novel periplasmic Cu chaperone (AccA) appears to be essential for provision of Cu ions to AniA of pathogenic Neisseria to generate an active nitrite reductase. Apart from the Neisseria genus, AccA is distributed across a wide range of environmental Proteobacteria species. © FASEB.

  16. Progranulin modulates zebrafish motoneuron development in vivo and rescues truncation defects associated with knockdown of Survival motor neuron 1

    Directory of Open Access Journals (Sweden)

    Bateman Andrew

    2010-10-01

    Full Text Available Abstract Background Progranulin (PGRN encoded by the GRN gene, is a secreted glycoprotein growth factor that has been implicated in many physiological and pathophysiological processes. PGRN haploinsufficiency caused by autosomal dominant mutations within the GRN gene leads to progressive neuronal atrophy in the form of frontotemporal lobar degeneration (FTLD. This form of the disease is associated with neuronal inclusions that bear the ubiquitinated TAR DNA Binding Protein-43 (TDP-43 molecular signature (FTLD-U. The neurotrophic properties of PGRN in vitro have recently been reported but the role of PGRN in neurons is not well understood. Here we document the neuronal expression and functions of PGRN in spinal cord motoneuron (MN maturation and branching in vivo using zebrafish, a well established model of vertebrate embryonic development. Results Whole-mount in situ hybridization and immunohistochemical analyses of zebrafish embryos revealed that zfPGRN-A is expressed within the peripheral and central nervous systems including the caudal primary (CaP MNs within the spinal cord. Knockdown of zfPGRN-A mRNA translation mediated by antisense morpholino oligonucleotides disrupted normal CaP MN development resulting in both truncated MNs and inappropriate early branching. Ectopic over-expression of zfPGRN-A mRNA resulted in increased MN branching and rescued the truncation defects brought about by knockdown of zfPGRN-A expression. The ability of PGRN to interact with established MN developmental pathways was tested. PGRN over-expression was found to reverse the truncation defect resulting from knockdown of Survival of motor neuron 1 (smn1. This is involved in small ribonucleoprotein biogenesis RNA processing, mutations of which cause Spinal Muscular Atrophy (SMA in humans. It did not reverse the MN defects caused by interfering with the neuronal guidance pathway by knockdown of expression of NRP-1, a semaphorin co-receptor. Conclusions Expression of

  17. Homology building as a means to define antigenic epitopes on dihydrofolate reductase (DHFR) from Plasmodium falciparum

    DEFF Research Database (Denmark)

    Alifrangis, Michael; Christensen, Inge T; Jørgensen, Flemming S

    2004-01-01

    in the gene coding for Pf-DHFR. Furthermore, we wanted to study the potential use of homology models in general and of Pf-DHFR in particular in predicting antigenic malarial surface epitopes. METHODS: A homology model of Pf-DHFR domain was employed to define an epitope for the development of site...

  18. Aldose Reductase-Deficient Mice Develop Nephrogenic Diabetes Insipidus

    Science.gov (United States)

    Ho, Horace T. B.; Chung, Sookja K.; Law, Janice W. S.; Ko, Ben C. B.; Tam, Sidney C. F.; Brooks, Heddwen L.; Knepper, Mark A.; Chung, Stephen S. M.

    2000-01-01

    Aldose reductase (ALR2) is thought to be involved in the pathogenesis of various diseases associated with diabetes mellitus, such as cataract, retinopathy, neuropathy, and nephropathy. However, its physiological functions are not well understood. We developed mice deficient in this enzyme and found that they had no apparent developmental or reproductive abnormality except that they drank and urinated significantly more than their wild-type littermates. These ALR2-deficient mice exhibited a partially defective urine-concentrating ability, having a phenotype resembling that of nephrogenic diabetes insipidus. PMID:10913167

  19. X-ray structural studies of quinone reductase 2 nanomolar range inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Pegan, Scott D.; Sturdy, Megan; Ferry, Gilles; Delagrange, Philippe; Boutin, Jean A.; Mesecar, Andrew D. (IdRS); (Purdue); (Colorado); (UIC)

    2011-09-06

    Quinone reductase 2 (QR2) is one of two members comprising the mammalian quinone reductase family of enzymes responsible for performing FAD mediated reductions of quinone substrates. In contrast to quinone reductase 1 (QR1) which uses NAD(P)H as its co-substrate, QR2 utilizes a rare group of hydride donors, N-methyl or N-ribosyl nicotinamide. Several studies have linked QR2 to the generation of quinone free radicals, several neuronal degenerative diseases, and cancer. QR2 has been also identified as the third melatonin receptor (MT3) through in cellulo and in vitro inhibition of QR2 by traditional MT3 ligands, and through recent X-ray structures of human QR2 (hQR2) in complex with melatonin and 2-iodomelatonin. Several MT3 specific ligands have been developed that exhibit both potent in cellulo inhibition of hQR2 nanomolar, affinity for MT3. The potency of these ligands suggest their use as molecular probes for hQR2. However, no definitive correlation between traditionally obtained MT3 ligand affinity and hQR2 inhibition exists limiting our understanding of how these ligands are accommodated in the hQR2 active site. To obtain a clearer relationship between the structures of developed MT3 ligands and their inhibitory properties, in cellulo and in vitro IC{sub 50} values were determined for a representative set of MT3 ligands (MCA-NAT, 2-I-MCANAT, prazosin, S26695, S32797, and S29434). Furthermore, X-ray structures for each of these ligands in complex with hQR2 were determined allowing for a structural evaluation of the binding modes of these ligands in relation to the potency of MT3 ligands.

  20. Ebselen: A thioredoxin reductase-dependent catalyst for α-tocopherol quinone reduction

    International Nuclear Information System (INIS)

    Fang Jianguo; Zhong Liangwei; Zhao Rong; Holmgren, Arne

    2005-01-01

    The thioredoxin system, composed of thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH, is a powerful protein disulfide reductase system with a broad substrate specificity. Recently the selenazol drug ebselen was shown to be a substrate for both mammalian TrxR and Trx. We examined if α-tocopherol quinone (TQ), a product of α-tocopherol oxidation, is reduced by ebselen in the presence of TrxR, since TQ was not a substrate for the enzyme itself. Ebselen reduction of TQ in the presence of TrxR was caused by ebselen selenol, generated from fast reduction of ebselen by the enzyme. TQ has no intrinsic antioxidant activity, while the product of reduction of TQ, α-tocopherolhydroquinone (TQH 2 ), is a potent antioxidant. The thioredoxin system dependence of ebselen to catalyze reduction of other oxidized species, such as hydrogen peroxide, dehydroascorbate, and peroxynitrite, is discussed. The ability of ebselen to reduce TQ via the thioredoxin system is a novel mechanism to explain the effects of the drug as an antioxidant in vivo

  1. A botanical containing freeze dried açai pulp promotes healthy aging and reduces oxidative damage in sod1 knockdown flies

    OpenAIRE

    Laslo, Mara; Sun, Xiaoping; Hsiao, Cheng-Te; Wu, Wells W.; Shen, Rong-Fong; Zou, Sige

    2012-01-01

    Superoxide dismutase 1 (SOD1), a critical enzyme against oxidative stress, is implicated in aging and degenerative diseases. We previously showed that a nutraceutical containing freeze-dried açai pulp promotes survival of flies fed a high-fat diet or sod1 knockdown flies fed a standard diet. Here, we investigated the effect of açai supplementation initiated at the early or late young adulthood on lifespan, physiological function, and oxidative damage in sod1 knockdown flies. We found that Aça...

  2. Hypersensitivity of mouse NEIL1-knockdown cells to hydrogen peroxide during S phase

    International Nuclear Information System (INIS)

    Yamamoto, Ryohei; Ohshiro, Yukari; Shimotani, Tatsuhiko; Yamamoto, Mizuki; Matsuyama, Satoshi; Ide, Hiroshi; Kubo, Kihei

    2014-01-01

    Oxidative base damage occurs spontaneously due to reactive oxygen species generated as byproducts of respiration and other pathological processes in mammalian cells. Many oxidized bases are mutagenic and/or toxic, and most are repaired through the base excision repair pathway. Human endonuclease VIII-like protein 1 (hNEIL1) is thought to play an important role during the S phase of the cell cycle by removing oxidized bases in DNA replication fork-like (bubble) structures, and the protein level of hNEIL1 is increased in S phase. Compared with hNEIL1, there is relatively little information on the properties of the mouse ortholog mNEIL1. Since mouse cell nuclei lack endonuclease III-like protein (NTH) activity, in contrast to human cell nuclei, mNEIL1 is a major DNA glycosylase for repair of oxidized pyrimidines in mouse nuclei. In this study, we made mNEIL1-knockdown cells using an shRNA expression vector and examined the cell cycle-related variation in hydrogen peroxide (H 2 O 2 ) sensitivity. Hypersensitivity to H 2 O 2 caused by mNEIL1 knockdown was more significant in S phase than in G1 phase, suggesting that mNEIL1 has an important role during S phase, similarly to hNEIL1

  3. Protective effect of Pterocarpus marsupium bark extracts against cataract through the inhibition of aldose reductase activity in streptozotocin-induced diabetic male albino rats.

    Science.gov (United States)

    Xu, YanLi; Zhao, Yongxia; Sui, YaNan; Lei, XiaoJun

    2018-04-01

    The present study was aimed to investigate the protective effect of Pterocarpus marsupium bark extracts against cataract in streptozotocin-induced diabetic male albino rats. Aldose reductase is a key enzyme in the intracellular polyol pathway, which plays a major role in the development of diabetic cataract. Rats were divided into five groups as normal control, diabetic control, and diabetic control treated with different concentrations of Pterocarpus marsupium bark extracts. Presence of major constituents in Pterocarpus marsupium bark extract was performed by qualitative analysis. Body weight changes, blood glucose, blood insulin, and reduced glutathione (GSH) and aldose reductase mRNA and protein expression were determined. Rat body weight gain was noted following treatment with bark extracts. The blood glucose was reduced up to 36% following treatment with bark extracts. The blood insulin and tissue GSH contents were substantially increased more than 100% in diabetic rats following treatment with extracts. Aldose reductase activity was reduced up to 79.3% in diabetic rats following treatment with extracts. V max , K m , and K i of aldose reductase were reduced in the lens tissue homogenate compared to the diabetic control. Aldose reductase mRNA and protein expression were reduced more than 50% following treatment with extracts. Treatment with Pterocarpus marsupium bark was able to normalize these levels. Taking all these data together, it is concluded that the use of Pterocarpus marsupium bark extracts could be the potential therapeutic approach for the reduction of aldose reductase against diabetic cataract.

  4. OAZ1 knockdown enhances viability and inhibits ER and LHR transcriptions of granulosa cells in geese.

    Directory of Open Access Journals (Sweden)

    Bo Kang

    Full Text Available An increasing number of studies suggest that ornithine decarboxylase antizyme 1 (OAZ1, which is regarded as a tumor suppressor gene, regulates follicular development, ovulation, and steroidogenesis. The granulosa cells in the ovary play a critical role in these ovarian functions. However, the action of OAZ1 mediating physiological functions of granulosa cells is obscure. OAZ1 knockdown in granulosa cells of geese was carried out in the current study. The effect of OAZ1 knockdown on polyamine metabolism, cell proliferation, apoptosis, and hormone receptor transcription of primary granulosa cells in geese was measured. The viability of granulosa cells transfected with the shRNA OAZ1 at 48 h was significantly higher than the control (p<0.05. The level of putrescine and spermidine in granulosa cells down-regulating OAZ1 was 7.04- and 2.11- fold higher compared with the control, respectively (p<0.05. The CCND1, SMAD1, and BCL-2 mRNA expression levels in granulosa cells down-regulating OAZ1 were each significantly higher than the control, respectively (p<0.05, whereas the PCNA and CASPASE 3 expression levels were significantly lower than the control (p<0.05. The estradiol concentration, ER and LHR mRNA expression levels were significantly lower in granulosa cells down-regulating OAZ1 compared with the control (p<0.05. Taken together, our results indicated that OAZ1 knockdown elevated the putrescine and spermidine contents and enhanced granulosa cell viability and inhibited ER and LHR transcriptions of granulosa cells in geese.

  5. Repair of 8-methoxypsoralen + UVA-induced damage in specific sequences in chromosomal and episomal DNA in human cells

    Energy Technology Data Exchange (ETDEWEB)

    Dean, S.W.

    1989-07-01

    A study of the repair of DNA damage in the dihydrofolate reductase (dhfr) gene of SV40-transformed human fibroblasts after treatment with 8-methoxypsoralen (8MOP) and UVA is described. 8MOP+UVA-induced cross-links in the dhfr gene were completely repaired by 12 h in one normal and one Fanconi's anaemia (FA) group A cell line. In contrast, approximately 35% of cross-links in an episomally maintained Epstein--Barr virus derived plasmid remained unrepaired even after 48 h. Cross-linkable monoadducts in the dhfr gene were repaired more slowly than cross-links, and there was no detectable repair of cross-linkable monoadducts in the plasmid. Thus the ability of a cell to repair 8MOP+UVA-induced cross-links or cross-linkable monoadducts in an episome does not reflect its capacity to repair such lesions in genomic DNA.

  6. Repair of 8-methoxypsoralen + UVA-induced damage in specific sequences in chromosomal and episomal DNA in human cells

    International Nuclear Information System (INIS)

    Dean, S.W.

    1989-01-01

    A study of the repair of DNA damage in the dihydrofolate reductase (dhfr) gene of SV40-transformed human fibroblasts after treatment with 8-methoxypsoralen (8MOP) and UVA is described. 8MOP+UVA-induced cross-links in the dhfr gene were completely repaired by 12 h in one normal and one Fanconi's anaemia (FA) group A cell line. In contrast, ∼35% of cross-links in an episomally maintained Epstein-Barr virus derived plasmid remained unrepaired even after 48 h. Cross-linkable monoadducts in the dhfr gene were repaired more slowly than cross-links, and there was no detectable repair of cross-linkable monoadducts in the plasmid. Thus the ability of a cell to repair 8MOP+UVA-induced cross-links or cross-linkable monoadducts in an episome does not reflect its capacity to repair such lesions in genomic DNA. (author)

  7. Development of a Rapid Real-Time PCR Assay for Quantitation of Pneumocystis carinii f. sp. Carinii

    DEFF Research Database (Denmark)

    Larsen, Hans Henrik; Kovacs, Joseph A; Stock, Frida

    2002-01-01

    ) PCR assay for detecting P. carinii f. sp. carinii, the subspecies of P. carinii commonly used in research models of PCP. The assay was based on the single-copy dihydrofolate reductase gene and was able to detect r = 0.99) over...... 6 log values for standards containing > or =5 copies/tube. Application of the assay to a series of 10-fold dilutions of P. carinii organisms isolated from rat lung demonstrated that it was reproducibly quantitative over 5 log values (r = 0.99). The assay was applied to a recently reported in vitro....... In conclusion, a rapid, sensitive, and reproducible quantitative PCR assay for P. carinii f. sp. carinii has been developed and is applicable to in vivo as well as in vitro systems. The assay should prove useful for conducting studies in which quantification of organism burden or growth assessment is critical...

  8. NADPH-dependent D-aldose reductases and xylose fermentation in Fusarium oxysporum

    DEFF Research Database (Denmark)

    Panagiotou, Gianni; Christakopoulos, P.

    2004-01-01

    Two aldose (xylose) reductases (ARI and ARII) from Fusarium oxysporum were purified and characterized. The native ARI was a monomer with M-r 41000, pI 5.2 and showed a 52-fold preference for NADPH over NADH, while ARII was homodimeric with a subunit of M-r 37000, pI 3.6 and a 60-fold preference...

  9. Influence of acute and chronic administration of methadone hydrochloride on NADPH-cytochrome c reductase and cytochrome P-450 of mouse liver microsomes.

    Science.gov (United States)

    Datta, R K; Johnson, E A; Bhattacharjee, G; Stenger, R J

    1976-03-01

    Administration of a single acute dose (20 mg/kg body weight) of methadone hydrochloride to both male and female mice increased the specific activity of NADPH-cytochrome c reductase and did not change much the content of cytochrome P-450 of their liver microsomes. Administration of multiple acute doses of methadone in male mice increased the specific activity of cytochrome c reductase and the content of cytochrome P-450 of their liver microsomes. Chronic administration of progressively increasing doses of methadone (up to 40 mg/kg body weight) to male mice increased the specific activity of c reductase. Similar chronic administration of methadone up to 28 mg/kg body weight also increased the microsomal content of P-450, but with higher doses of methadone, the content of P-450 declined and finally dropped slightly below control levels. The levels of c reductase activity and P-450 content returned to normal about two weeks after discontinuation of methadone administration.

  10. Unexpected ethical dilemmas in sex assignment in 46,XY DSD due to 5-alpha reductase type 2 deficiency.

    Science.gov (United States)

    Byers, Heather M; Mohnach, Lauren H; Fechner, Patricia Y; Chen, Ming; Thomas, Inas H; Ramsdell, Linda A; Shnorhavorian, Margarett; McCauley, Elizabeth A; Amies Oelschlager, Anne-Marie E; Park, John M; Sandberg, David E; Adam, Margaret P; Keegan, Catherine E

    2017-06-01

    Sex assignment at birth remains one of the most clinically challenging and controversial topics in 46,XY disorders of sexual development (DSD). This is particularly challenging in deficiency of 5-alpha reductase type 2 given that external genitalia are typically undervirilized at birth but typically virilize at puberty to a variable degree. Historically, most individuals with 5-alpha reductase deficiency were raised females. However, reports that over half of patients who underwent a virilizing puberty adopted an adult male gender identity have challenged this practice. Consensus guidelines on assignment of sex of rearing at birth are equivocal or favor male assignment in the most virilized cases. While a male sex of rearing assignment may avoid lifelong hormonal therapy and/or allow the potential for fertility, female sex assignment may be more consistent with external anatomy in the most severely undervirilized cases. Herein, we describe five patients with 46,XY DSD due 5-alpha-reductase type 2 deficiency, all with a severe phenotype. An inter-disciplinary DSD medical team at one of two academic centers evaluated each patient. This case series illustrates the complicated decision-making process of assignment of sex of rearing at birth in 5-alpha reductase type 2 deficiency and the challenges that arise when the interests of the child, parental wishes, recommendations of the medical team, and state law collide. © 2017 Wiley Periodicals, Inc.

  11. RNAi-mediated knock-down of Dab and Numb attenuate Aβ levels via γ-secretase mediated APP processing

    Directory of Open Access Journals (Sweden)

    Xie Zhongcong

    2012-03-01

    Full Text Available Abstract Amyloid-β-protein (Aβ, the key component of senile plaques in Alzheimer's disease (AD brain, is produced from amyloid precursor protein (APP by cleavage of β-secretase and then γ-secretase. APP adaptor proteins with phosphotyrosine-binding (PTB domains, including Dab (gene: DAB and Numb (gene: NUMB, can bind to and interact with the conserved YENPTY-motif in the APP C-terminus. Here we describe, for the first time, the effects of RNAi knock-down of Dab and Numb expression on APP processing and Aβ production. RNAi knock-down of Dab and Numb in H4 human neuroglioma cells stably transfected to express either FL-APP (H4-FL-APP cells or APP-C99 (H4-APP-C99 cells increased levels of APP-C-terminal fragments (APP-CTFs and lowered Aβ levels in both cell lines by inhibiting γ-secretase cleavage of APP. Finally, RNAi knock-down of APP also reduced levels of Numb in H4-APP cells. These findings suggest that pharmacologically blocking interaction of APP with Dab and Numb may provide novel therapeutic strategies of AD. The notion of attenuating γ-secretase cleavage of APP via the APP adaptor proteins, Dab and Numb, is particularly attractive with regard to therapeutic potential, given that side effects of γ-secretase inhibition owing to impaired proteolysis of other γ-secretase substrates, e.g. Notch, might be avoided.

  12. Knockdown of UbcH10 Enhances the Chemosensitivity of Dual Drug Resistant Breast Cancer Cells to Epirubicin and Docetaxel

    Directory of Open Access Journals (Sweden)

    Cheng Wang

    2015-03-01

    Full Text Available Breast cancer is one of the most common and lethal cancers in women. As a hub gene involved in a diversity of tumors, the ubiquitin-conjugating enzyme H10 (UbcH10, may also play some roles in the genesis and development of breast cancer. In the current study, we found that the expression of UbcH10 was up-regulated in some breast cancer tissues and five cell lines. We established a dual drug resistant cell line MCF-7/EPB (epirubicin/TXT (docetaxel and a lentiviral system expressing UbcH10 shRNA to investigate the effects of UbcH10 knockdown on the chemosensitivity of MCF-7/EPB/TXT cells to epirubicin and docetaxel. The knockdown of UbcH10 inhibited the proliferation of both MCF-7 and MCF-7/EPB/TXT cells, due to the G1 phase arrest in cell cycle. Furthermore, UbcH10 knockdown increased the sensitivity of MCF-7/EPB/TXT cells to epirubicin and docetaxel and promoted the apoptosis induced by these two drugs. Protein detection showed that, in addition to inhibiting the expression of Ki67 and cyclin D1, UbcH10 RNAi also impaired the increased BCL-2 and MDR-1 expression levels in MCF-7/EPB/TXT cells, which may contribute to abating the drug resistance in the breast cancer cells. Our research in the current study demonstrated that up-regulation of UbcH10 was involved in breast cancer and its knockdown can inhibit the growth of cancer cells and increase the chemosensitivity of the dual drug resistant breast cancer cells to epirubicin and docetaxel, suggesting that UbcH10 may be a promising target for the therapy of breast cancer.

  13. Variation and inheritance of iron reductase activity in the roots of common bean (Phaseolus vulgaris L.) and association with seed iron accumulation QTL.

    Science.gov (United States)

    Blair, Matthew W; Knewtson, Sharon Jb; Astudillo, Carolina; Li, Chee-Ming; Fernandez, Andrea C; Grusak, Michael A

    2010-10-05

    Iron deficiency anemia is a global problem which often affects women and children of developing countries. Strategy I plants, such as common bean (Phaseolus vulgaris L.) take up iron through a process that involves an iron reduction mechanism in their roots; this reduction is required to convert ferric iron to ferrous iron. Root absorbed iron is critical for the iron nutrition of the plant, and for the delivery of iron to the shoot and ultimately the seeds. The objectives of this study were to determine the variability and inheritance for iron reductase activity in a range of genotypes and in a low × high seed iron cross (DOR364 x G19833), to identify quantitative trait loci (QTL) for this trait, and to assess possible associations with seed iron levels. The experiments were carried out with hydroponically grown plants provided different amounts of iron varying between 0 and 20 μM Fe(III)-EDDHA. The parents, DOR364 and G19833, plus 13 other cultivated or wild beans, were found to differ in iron reductase activity. Based on these initial experiments, two growth conditions (iron limited and iron sufficient) were selected as treatments for evaluating the DOR364 × G19833 recombinant inbred lines. A single major QTL was found for iron reductase activity under iron-limited conditions (1 μM Fe) on linkage group b02 and another major QTL was found under iron sufficient conditions (15 μM Fe) on linkage group b11. Associations between the b11 QTL were found with several QTL for seed iron. Genes conditioning iron reductase activity in iron sufficient bean plants appear to be associated with genes contributing to seed iron accumulation. Markers for bean iron reductase (FRO) homologues were found with in silico mapping based on common bean synteny with soybean and Medicago truncatula on b06 and b07; however, neither locus aligned with the QTL for iron reductase activity. In summary, the QTL for iron reductase activity under iron limited conditions may be useful in

  14. Variation and inheritance of iron reductase activity in the roots of common bean (Phaseolus vulgaris L. and association with seed iron accumulation QTL

    Directory of Open Access Journals (Sweden)

    Fernandez Andrea C

    2010-10-01

    Full Text Available Abstract Background Iron deficiency anemia is a global problem which often affects women and children of developing countries. Strategy I plants, such as common bean (Phaseolus vulgaris L. take up iron through a process that involves an iron reduction mechanism in their roots; this reduction is required to convert ferric iron to ferrous iron. Root absorbed iron is critical for the iron nutrition of the plant, and for the delivery of iron to the shoot and ultimately the seeds. The objectives of this study were to determine the variability and inheritance for iron reductase activity in a range of genotypes and in a low × high seed iron cross (DOR364 × G19833, to identify quantitative trait loci (QTL for this trait, and to assess possible associations with seed iron levels. Results The experiments were carried out with hydroponically grown plants provided different amounts of iron varying between 0 and 20 μM Fe(III-EDDHA. The parents, DOR364 and G19833, plus 13 other cultivated or wild beans, were found to differ in iron reductase activity. Based on these initial experiments, two growth conditions (iron limited and iron sufficient were selected as treatments for evaluating the DOR364 × G19833 recombinant inbred lines. A single major QTL was found for iron reductase activity under iron-limited conditions (1 μM Fe on linkage group b02 and another major QTL was found under iron sufficient conditions (15 μM Fe on linkage group b11. Associations between the b11 QTL were found with several QTL for seed iron. Conclusions Genes conditioning iron reductase activity in iron sufficient bean plants appear to be associated with genes contributing to seed iron accumulation. Markers for bean iron reductase (FRO homologues were found with in silico mapping based on common bean synteny with soybean and Medicago truncatula on b06 and b07; however, neither locus aligned with the QTL for iron reductase activity. In summary, the QTL for iron reductase activity

  15. Knockdown of ARK5 Expression Suppresses Invasion and Metastasis of Gastric Cancer

    Directory of Open Access Journals (Sweden)

    Dehu Chen

    2017-06-01

    Full Text Available Background/Aims: Gastric cancer (GC is a common and lethal malignancy, and AMP-activated protein kinase-related kinase 5 (ARK5 has been discovered to promote cancer metastasis in certain types of cancer. In this study, we explored the role of ARK5 in GC invasion and metastasis. Methods: ARK5 and epithelial-mesenchymal transition (EMT-related markers were determined by immunohistochemistry and western blot in GC specimens. Other methods including stably transfected against ARK5 into SGC7901 and AGS cells, western blot, migration and invasion assays in vitro and nude mice tumorigenicity in vivo were also employed. Results: The results demonstrated that ARK5 expression was increased and positively correlated with metastasis, EMT-related markers and poor prognosis in patients with GC. Knockdown of ARK5 expression remarkably suppressed GC cells invasion and metastasis via regulating EMT, rather than proliferation in vitro and in vivo. And knockdown of ARK5 expression in GC cells resulted in the down-regulation of the mTOR/p70S6k signals, Slug and SIP1. Conclusion: The elevated ARK5 expression was closely associated with cancer metastasis and patient survival, and it seemed to function in GC cells migration and invasion via EMT alteration, together with the alteration of the mTOR/p70S6k signals, Slug and SIP1, thus providing a potential therapeutic target for GC.

  16. Design of 8-ft-Diameter Barrel Test Article Attachment Rings for Shell Buckling Knockdown Factor Project

    Science.gov (United States)

    Lovejoy, Andrew E.; Hilburger, Mark W.

    2010-01-01

    The Shell Buckling Knockdown Factor (SBKF) project includes the testing of sub-scale cylinders to validate new shell buckling knockdown factors for use in the design of the Ares-I and Ares-V launch vehicles. Test article cylinders represent various barrel segments of the Ares-I and Ares-V vehicles, and also include checkout test articles. Testing will be conducted at Marshall Space Flight Center (MSFC) for test articles having an eight-foot diameter outer mold line (OML) and having lengths that range from three to ten feet long. Both ends of the test articles will be connected to the test apparatus using attachment rings. Three multiple-piece and one single-piece design for the attachment rings were developed and analyzed. The single-piece design was chosen and will be fabricated from either steel or aluminum (Al) depending on the required safety factors (SF) for test hardware. This report summarizes the design and analysis of these attachment ring concepts.

  17. TREX1 Knockdown Induces an Interferon Response to HIV that Delays Viral Infection in Humanized Mice

    Directory of Open Access Journals (Sweden)

    Lee Adam Wheeler

    2016-05-01

    Full Text Available Despite their antiviral effect, the in vivo effect of interferons on HIV transmission is difficult to predict, because interferons also activate and recruit HIV-susceptible cells to sites of infection. HIV does not normally induce type I interferons in infected cells, but does if TREX1 is knocked down. Here, we investigated the effect of topical TREX1 knockdown and local interferon production on HIV transmission in human cervicovaginal explants and humanized mice. In explants in which TREX1 was knocked down, HIV induced interferons, which blocked infection. In humanized mice, even though TREX1 knockdown increased infiltrating immune cells, it delayed viral replication for 3–4 weeks. Similarly intravaginal application of type I interferons the day before HIV infection induced interferon responsive genes, reduced inflammation, and decreased viral replication. However, intravenous interferon enhanced inflammation and infection. Thus, in models of human sexual transmission, a localized interferon response inhibits HIV transmission but systemic interferons do not.

  18. Fatty acyl-CoA reductases of birds

    Directory of Open Access Journals (Sweden)

    Hellenbrand Janine

    2011-12-01

    Full Text Available Abstract Background Birds clean and lubricate their feathers with waxes that are produced in the uropygial gland, a holocrine gland located on their back above the tail. The type and the composition of the secreted wax esters are dependent on the bird species, for instance the wax ester secretion of goose contains branched-chain fatty acids and unbranched fatty alcohols, whereas that of barn owl contains fatty acids and alcohols both of which are branched. Alcohol-forming fatty acyl-CoA reductases (FAR catalyze the reduction of activated acyl groups to fatty alcohols that can be esterified with acyl-CoA thioesters forming wax esters. Results cDNA sequences encoding fatty acyl-CoA reductases were cloned from the uropygial glands of barn owl (Tyto alba, domestic chicken (Gallus gallus domesticus and domestic goose (Anser anser domesticus. Heterologous expression in Saccharomyces cerevisiae showed that they encode membrane associated enzymes which catalyze a NADPH dependent reduction of acyl-CoA thioesters to fatty alcohols. By feeding studies of transgenic yeast cultures and in vitro enzyme assays with membrane fractions of transgenic yeast cells two groups of isozymes with different properties were identified, termed FAR1 and FAR2. The FAR1 group mainly synthesized 1-hexadecanol and accepted substrates in the range between 14 and 18 carbon atoms, whereas the FAR2 group preferred stearoyl-CoA and accepted substrates between 16 and 20 carbon atoms. Expression studies with tissues of domestic chicken indicated that FAR transcripts were not restricted to the uropygial gland. Conclusion The data of our study suggest that the identified and characterized avian FAR isozymes, FAR1 and FAR2, can be involved in wax ester biosynthesis and in other pathways like ether lipid synthesis.

  19. Fatty acyl-CoA reductases of birds

    Science.gov (United States)

    2011-01-01

    Background Birds clean and lubricate their feathers with waxes that are produced in the uropygial gland, a holocrine gland located on their back above the tail. The type and the composition of the secreted wax esters are dependent on the bird species, for instance the wax ester secretion of goose contains branched-chain fatty acids and unbranched fatty alcohols, whereas that of barn owl contains fatty acids and alcohols both of which are branched. Alcohol-forming fatty acyl-CoA reductases (FAR) catalyze the reduction of activated acyl groups to fatty alcohols that can be esterified with acyl-CoA thioesters forming wax esters. Results cDNA sequences encoding fatty acyl-CoA reductases were cloned from the uropygial glands of barn owl (Tyto alba), domestic chicken (Gallus gallus domesticus) and domestic goose (Anser anser domesticus). Heterologous expression in Saccharomyces cerevisiae showed that they encode membrane associated enzymes which catalyze a NADPH dependent reduction of acyl-CoA thioesters to fatty alcohols. By feeding studies of transgenic yeast cultures and in vitro enzyme assays with membrane fractions of transgenic yeast cells two groups of isozymes with different properties were identified, termed FAR1 and FAR2. The FAR1 group mainly synthesized 1-hexadecanol and accepted substrates in the range between 14 and 18 carbon atoms, whereas the FAR2 group preferred stearoyl-CoA and accepted substrates between 16 and 20 carbon atoms. Expression studies with tissues of domestic chicken indicated that FAR transcripts were not restricted to the uropygial gland. Conclusion The data of our study suggest that the identified and characterized avian FAR isozymes, FAR1 and FAR2, can be involved in wax ester biosynthesis and in other pathways like ether lipid synthesis. PMID:22151413

  20. Use of 5-alpha-reductase inhibitors did not increase the risk of cardiovascular diseases in patients with benign prostate hyperplasia: a five-year follow-up study.

    Directory of Open Access Journals (Sweden)

    Teng-Fu Hsieh

    Full Text Available This nationwide population-based study investigated the risk of cardiovascular diseases after 5-alpha-reductase inhibitor therapy for benign prostate hyperplasia (BPH using the National Health Insurance Research Database (NHIRD in Taiwan.In total, 1,486 adult patients newly diagnosed with BPH and who used 5-alpha-reductase inhibitors were recruited as the study cohort, along with 9,995 subjects who did not use 5-alpha-reductase inhibitors as a comparison cohort from 2003 to 2008. Each patient was monitored for 5 years, and those who subsequently had cardiovascular diseases were identified. A Cox proportional hazards model was used to compare the risk of cardiovascular diseases between the study and comparison cohorts after adjusting for possible confounding risk factors.The patients who received 5-alpha-reductase inhibitor therapy had a lower cumulative rate of cardiovascular diseases than those who did not receive 5-alpha-reductase inhibitor therapy during the 5-year follow-up period (8.4% vs. 11.2%, P=0.003. In subgroup analysis, the 5-year cardiovascular event hazard ratio (HR was lower among the patients older than 65 years with 91 to 365 cumulative defined daily dose (cDDD 5-alpha-reductase inhibitor use (HR=0.63, 95% confidence interval (CI 0.42 to 0.92; P=0.018, however there was no difference among the patients with 28 to 90 and more than 365 cDDD 5-alpha-reductase inhibitor use (HR=1.14, 95% CI 0.77 to 1.68; P=0.518 and HR=0.83, 95% CI 0.57 to 1.20; P=0.310, respectively.5-alpha-reductase inhibitor therapy did not increase the risk of cardiovascular events in the BPH patients in 5 years of follow-up. Further mechanistic research is needed.

  1. Crystallization and preliminary crystallographic analysis of selenomethionine-labelled progesterone 5β-reductase from Digitalis lanata Ehrh

    Energy Technology Data Exchange (ETDEWEB)

    Egerer-Sieber, Claudia [Lehrstuhl für Biotechnik, Institut für Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Henkestrasse 91, D-91052 Erlangen (Germany); Herl, Vanessa; Müller-Uri, Frieder; Kreis, Wolfgang [Lehrstuhl für Pharmazeutische Biologie, Institut für Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstrasse 5, D-91058 Erlangen (Germany); Muller, Yves A., E-mail: ymuller@biologie.uni-erlangen.de [Lehrstuhl für Biotechnik, Institut für Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Henkestrasse 91, D-91052 Erlangen (Germany)

    2006-03-01

    Progesterone 5β-reductase is the first stereospecific enzyme in the pathway for the synthesis of cardenolides. To elucidate the structural mechanism of this reaction, we crystallized the selenomethionine-labelled enzyme from D. lanata and report the preliminary analysis of a MAD data set collected from these crystals. Progesterone 5β-reductase (5β-POR) catalyzes the reduction of progesterone to 5β-pregnane-3,20-dione and is the first stereospecific enzyme in the putative biosynthetic pathway of Digitalis cardenolides. Selenomethionine-derivatized 5β-POR from D. lanata was successfully overproduced and crystallized. The crystals belong to space group P4{sub 3}2{sub 1}2, with unit-cell parameters a = 71.73, c = 186.64 Å. A MAD data set collected at 2.7 Å resolution allowed the identification of six out of eight possible Se-atom positions. A first inspection of the MAD-phased electron-density map shows that 5β-POR is a Rossmann-type reductase and the quality of the map is such that it is anticipated that a complete atomic model of 5β-POR will readily be built.

  2. Brevetoxin-2, is a unique inhibitor of the C-terminal redox center of mammalian thioredoxin reductase-1.

    Science.gov (United States)

    Chen, Wei; Tuladhar, Anupama; Rolle, Shantelle; Lai, Yanhao; Rodriguez Del Rey, Freddy; Zavala, Cristian E; Liu, Yuan; Rein, Kathleen S

    2017-08-15

    Karenia brevis, the Florida red tide dinoflagellate produces a suite of neurotoxins known as the brevetoxins. The most abundant of the brevetoxins PbTx-2, was found to inhibit the thioredoxin-thioredoxin reductase system, whereas the PbTx-3 has no effect on this system. On the other hand, PbTx-2 activates the reduction of small disulfides such as 5,5'-dithio-bis-(2-nitrobenzoic acid) by thioredoxin reductase. PbTx-2 has an α, β-unsaturated aldehyde moiety which functions as an efficient electrophile and selenocysteine conjugates are readily formed. PbTx-2 blocks the inhibition of TrxR by the inhibitor curcumin, whereas curcumin blocks PbTx-2 activation of TrxR. It is proposed that the mechanism of inhibition of thioredoxin reduction is via the formation of a Michael adduct between selenocysteine and the α, β-unsaturated aldehyde moiety of PbTx-2. PbTx-2 had no effect on the rates of reactions catalyzed by related enzymes such as glutathione reductase, glutathione peroxidase or glutaredoxin. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. The 5 Alpha-Reductase Isozyme Family: A Review of Basic Biology and Their Role in Human Diseases

    Directory of Open Access Journals (Sweden)

    Faris Azzouni

    2012-01-01

    Full Text Available Despite the discovery of 5 alpha-reduction as an enzymatic step in steroid metabolism in 1951, and the discovery that dihydrotestosterone is more potent than testosterone in 1968, the significance of 5 alpha-reduced steroids in human diseases was not appreciated until the discovery of 5 alpha-reductase type 2 deficiency in 1974. Affected males are born with ambiguous external genitalia, despite normal internal genitalia. The prostate is hypoplastic, nonpalpable on rectal examination and approximately 1/10th the size of age-matched normal glands. Benign prostate hyperplasia or prostate cancer does not develop in these patients. At puberty, the external genitalia virilize partially, however, secondary sexual hair remains sparse and male pattern baldness and acne develop rarely. Several compounds have been developed to inhibit the 5 alpha-reductase isozymes and they play an important role in the prevention and treatment of many common diseases. This review describes the basic biochemical properties, functions, tissue distribution, chromosomal location, and clinical significance of the 5 alpha-reductase isozyme family.

  4. Selective knockdown of ceramide synthases reveals complex interregulation of sphingolipid metabolism[S

    OpenAIRE

    Mullen, Thomas D.; Spassieva, Stefka; Jenkins, Russell W.; Kitatani, Kazuyuki; Bielawski, Jacek; Hannun, Yusuf A.; Obeid, Lina M.

    2011-01-01

    Mammalian ceramide synthases 1 to 6 (CerS1–6) generate Cer in an acyl-CoA-dependent manner, and expression of individual CerS has been shown to enhance the synthesis of ceramides with particular acyl chain lengths. However, the contribution of each CerS to steady-state levels of specific Cer species has not been evaluated. We investigated the knockdown of individual CerS in the MCF-7 human breast adenocarcinoma cell line by using small-interfering RNA (siRNA). We found that siRNA-induced down...

  5. Crystallization and preliminary characterization of dihydropteridine reductase from Dictyostelium discoideum

    International Nuclear Information System (INIS)

    Chen, Cong; Seo, Kyung Hye; Kim, Hye Lim; Zhuang, Ningning; Park, Young Shik; Lee, Kon Ho

    2008-01-01

    The dihydropteridine reductase from D. discoideum has been crystallized. Diffraction data were collected from a rectangular-shaped crystal to 2.16 Å resolution. Dihydropteridine reductase from Dictyostelium discoideum (dicDHPR) can produce d-threo-BH 4 [6R-(1′R,2′R)-5,6,7,8-tetrahydrobiopterin], a stereoisomer of l-erythro-BH 4 , in the last step of tetrahydrobiopterin (BH 4 ) recycling. In this reaction, DHPR uses NADH as a cofactor to reduce quinonoid dihydrobiopterin back to BH 4 . To date, the enzyme has been purified to homogeneity from many sources. In this report, the dicDHPR–NAD complex has been crystallized using the hanging-drop vapour-diffusion method with PEG 3350 as a precipitant. Rectangular-shaped crystals were obtained. Crystals grew to maximum dimensions of 0.4 × 0.6 × 0.1 mm. The crystal belonged to space group P2 1 , with unit-cell parameters a = 49.81, b = 129.90, c = 78.76 Å, β = 100.00°, and contained four molecules in the asymmetric unit, forming two closely interacting dicDHPR–NAD dimers. Diffraction data were collected to 2.16 Å resolution using synchrotron radiation. The crystal structure has been determined using the molecular-replacement method

  6. Inhibition of NADH-ubiquinone reductase activity by N,N'-dicyclohexylcarbodiimide and correlation of this inhibition with the occurrence of energy-coupling site 1 in various organisms

    International Nuclear Information System (INIS)

    Yagi, T.

    1987-01-01

    The NADH-ubiquinone reductase activity of the respiratory chains of several organisms was inhibited by the carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD). This inhibition correlated with the presence of an energy-transducing site in this segment of the respiratory chain. Where the NADH-quinone reductase segment involved an energy-coupling site (e.g., in bovine heart and rat liver mitochondria, and in Paracoccus denitrificans, Escherichia coli, and Thermus thermophilus HB-8 membranes), DCCD acted as an inhibitor of ubiquinone reduction by NADH. By contrast, where energy-coupling site 1 was absent (e.g., in Saccharomyces cerevisiae mitochondria and BacilLus subtilis membranes), there was no inhibition of NADH-ubiquinone reductase activity by DCCD. In the bovine and P. denitrificans systems, DCCD inhibition was pseudo first order with respect to incubation time, and reaction order with respect to inhibitor concentration was close to unity, indicating that inhibition resulted from the binding of one inhibitor molecule per active unit of NADH-ubiquinone reductase. In the bovine NADH-ubiquinone reductase complex (complex I), [ 14 C]DCCD was preferentially incorporated into two subunits of molecular weight 49,000 and 29,000. The time course of labeling of the 29,000 molecular weight subunit with [ 14 C]DCCD paralleled the time course of inhibition of NADH-ubiquinone reductase activity

  7. Inhibition of thioredoxin reductase but not of glutathione reductase by the major classes of alkylating and platinum-containing anticancer compounds.

    Science.gov (United States)

    Witte, Anne-Barbara; Anestål, Karin; Jerremalm, Elin; Ehrsson, Hans; Arnér, Elias S J

    2005-09-01

    Mammalian thioredoxin reductase (TrxR) is important for cell proliferation, antioxidant defense, and redox signaling. Together with glutathione reductase (GR) it is the main enzyme providing reducing equivalents to many cellular processes. GR and TrxR are flavoproteins of the same enzyme family, but only the latter is a selenoprotein. With the active site containing selenocysteine, TrxR may catalyze reduction of a wide range of substrates, but can at the same time easily be targeted by electrophilic compounds due to the extraordinarily high reactivity of a selenolate moiety. Here we addressed the inhibition of the enzyme by major anticancer alkylating agents and platinum-containing compounds and we compared it to that of GR. We confirmed prior studies suggesting that the nitrosourea carmustine can inhibit both GR and TrxR. We next found, however, that nitrogen mustards (chlorambucil and melphalan) and alkyl sulfonates (busulfan) efficiently inhibited TrxR while these compounds, surprisingly, did not inhibit GR. Inhibitions were concentration and time dependent and apparently irreversible. Anticancer anthracyclines (daunorubicin and doxorubicin) were, in contrast to the alkylating agents, not inhibitors but poor substrates of TrxR. We also found that TrxR, but not GR, was efficiently inhibited by both cisplatin, its monohydrated complex, and oxaliplatin. Carboplatin, in contrast, could not inhibit any of the two enzymes. These findings lead us to conclude that representative compounds of the major classes of clinically used anticancer alkylating agents and most platinum compounds may easily target TrxR, but not GR. The TrxR inhibition should thereby be considered as a factor that may contribute to the cytotoxicity seen upon clinical use of these drugs.

  8. Solution structure of an arsenate reductase-related protein, YffB, from Brucella melitensis, the etiological agent responsible for brucellosis

    International Nuclear Information System (INIS)

    Buchko, Garry W.; Hewitt, Stephen N.; Napuli, Alberto J.; Van Voorhis, Wesley C.; Myler, Peter J.

    2011-01-01

    B. melitensis is a NIAID Category B microorganism that is responsible for brucellosis and is a potential agent for biological warfare. Here, the solution structure of the 116-residue arsenate reductase-related protein Bm-YffB (BR0369) from this organism is reported. Brucella melitensis is the etiological agent responsible for brucellosis. Present in the B. melitensis genome is a 116-residue protein related to arsenate reductases (Bm-YffB; BR0369). Arsenate reductases (ArsC) convert arsenate ion (H 2 AsO 4 − ), a compound that is toxic to bacteria, to arsenite ion (AsO 2 − ), a product that may be efficiently exported out of the cell. Consequently, Bm-YffB is a potential drug target because if arsenate reduction is the protein’s major biological function then disabling the cell’s ability to reduce arsenate would make these cells more sensitive to the deleterious effects of arsenate. Size-exclusion chromatography and NMR spectroscopy indicate that Bm-YffB is a monomer in solution. The solution structure of Bm-YffB shows that the protein consists of two domains: a four-stranded mixed β-sheet flanked by two α-helices on one side and an α-helical bundle. The α/β domain is characteristic of the fold of thioredoxin-like proteins and the overall structure is generally similar to those of known arsenate reductases despite the marginal sequence similarity. Chemical shift perturbation studies with 15 N-labeled Bm-YffB show that the protein binds reduced glutathione at a site adjacent to a region similar to the HX 3 CX 3 R catalytic sequence motif that is important for arsenic detoxification activity in the classical arsenate-reductase family of proteins. The latter observation supports the hypothesis that the ArsC-YffB family of proteins may function as glutathione-dependent thiol reductases. However, comparison of the structure of Bm-YffB with the structures of proteins from the classical ArsC family suggest that the mechanism and possibly the function of Bm

  9. Knock-down and speed of kill of a combination of fipronil and permethrin for the prevention of Ctenocephalides felis flea infestation in dogs.

    Science.gov (United States)

    Halos, Lénaïg; Fourie, Josephus J; Fankhauser, Becky; Beugnet, Frederic

    2016-02-02

    A topical combination of fipronil + permethrin (Frontline Tri-Act/Frontect, Merial) has recently been developed to control fleas, ticks, mosquitoes, sandflies and stable flies on dogs. Two studies were conducted to assess its speed of kill and knock-down effect on Ctenocephalides felis fleas. The combination was compared to either fipronil alone or to a combination of permethrin, dinotefuran, and pyriproxyfen, In each study, 18 dogs were randomly allocated to one of three groups: (Group 1: untreated dog; Group 2: treated once on D0 with the combination of fipronil and permethrin; Group 3: treated once on D0 either with fipronil alone (study 1) or with a combination of permethrin, dinetofuran and pyriproxyfen (study 2)). Each dog was infested with 100 unfed adult C. felis fleas on Days 2 (study 2), 7, 14, 21 and 28. Fleas were collected from dogs at 1 h and 12 h post- infestations (PI) (study 1) or at 2 h and 6 h PI (study 2) to assess efficacy and from collection pans underneath cages 1 h (study 1) or 5 min (study 2) PI to assess knock-down effect. All treated dogs had significantly (p ≤ 0.01) lower flea counts than untreated dogs at every time point in both studies. For a whole month, a significant knock-down effect against infesting fleas is obtained in five minutes PI with the combination of permethrin and fipronil. Complete efficacy (>95%) was achieved in 1 h (study 1) or 2 h (study 2) PI for 14 days and by 6 h PI for all challenges conducted throughout the month. Efficacy remains >85% at 2 h PI for the whole month. A significantly higher efficacy of the fipronil + permethrin combination compared to other treatments was demonstrated at the earliest time points for the month (1 h knock-down effect and insecticidal efficacy compared to fipronil alone; 5 min knock-down effect compared to the combination of permethrin + dinetofuran + pyriproxyfen). The rapid flea knock-down effect and speed of kill demonstrated by the spot on combination of

  10. The knock-down of the expression of MdMLO19 reduces susceptibility to powdery mildew (Podosphaera leucotricha) in apple (Malus domestica).

    Science.gov (United States)

    Pessina, Stefano; Angeli, Dario; Martens, Stefan; Visser, Richard G F; Bai, Yuling; Salamini, Francesco; Velasco, Riccardo; Schouten, Henk J; Malnoy, Mickael

    2016-10-01

    Varieties resistant to powdery mildew (PM; caused by Podosphaera leucotricha) are a major component of sustainable apple production. Resistance can be achieved by knocking-out susceptibility S-genes to be singled out among members of the MLO (Mildew Locus O) gene family. Candidates are MLO S-genes of phylogenetic clade V up-regulated upon PM inoculation, such as MdMLO11 and 19 (clade V) and MdMLO18 (clade VII). We report the knock-down through RNA interference of MdMLO11 and 19, as well as the complementation of resistance with MdMLO18 in the Arabidopsis thaliana triple mlo mutant Atmlo2/6/12. The knock-down of MdMLO19 reduced PM disease severity by 75%, whereas the knock-down of MdMLO11, alone or in combination with MdMLO19, did not result in any reduction or additional reduction of susceptibility compared with MdMLO19 alone. The test in A. thaliana excluded a role for MdMLO18 in PM susceptibility. Cell wall appositions (papillae) were present in both PM-resistant and PM-susceptible plants, but were larger in resistant lines. No obvious negative phenotype was observed in plants with mlo genes knocked down. Apparently, MdMLO19 plays the pivotal role in apple PM susceptibility and its knock-down induces a very significant level of resistance. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  11. Raman spectroscopic study of keratin 8 knockdown oral squamous cell carcinoma derived cells

    Science.gov (United States)

    Singh, S. P.; Alam, Hunain; Dmello, Crismita; Vaidya, Milind M.; Krishna, C. Murali

    2012-03-01

    Keratins are one of most widely used markers for oral cancers. Keratin 8 and 18 are expressed in simple epithelia and perform both mechanical and regulatory functions. Their expression are not seen in normal oral tissues but are often expressed in oral squamous cell carcinoma. Aberrant expression of keratins 8 and 18 is most common change in human oral cancer. Optical-spectroscopic methods are sensitive to biochemical changes and being projected as novel diagnostic tools for cancer diagnosis. Aim of this study was to evaluate potentials of Raman spectroscopy in detecting minor changes associated with differential level of keratin expression in tongue-cancer-derived AW13516 cells. Knockdown clones for K8 were generated and synchronized by growing under serum-free conditions. Cell pellets of three independent experiments in duplicate were used for recording Raman spectra with fiberoptic-probe coupled HE-785 Raman-instrument. A total of 123 and 96 spectra from knockdown clones and vector controls respectively in 1200-1800 cm-1 region were successfully utilized for classification using LDA. Two separate clusters with classification-efficiency of ~95% were obtained. Leave-one-out cross-validation yielded ~63% efficiency. Findings of the study demonstrate the potentials of Raman spectroscopy in detecting even subtle changes such as variations in keratin expression levels. Future studies towards identifying Raman signals from keratin in oral cells can help in precise cancer diagnosis.

  12. Multiple origins of knockdown resistance mutations in the Afrotropical mosquito vector Anopheles gambiae.

    Directory of Open Access Journals (Sweden)

    João Pinto

    2007-11-01

    Full Text Available How often insecticide resistance mutations arise in natural insect populations is a fundamental question for understanding the evolution of resistance and also for modeling its spread. Moreover, the development of resistance is regarded as a favored model to study the molecular evolution of adaptive traits. In the malaria vector Anopheles gambiae two point mutations (L1014F and L1014S in the voltage-gated sodium channel gene, that confer knockdown resistance (kdr to DDT and pyrethroid insecticides, have been described. In order to determine whether resistance alleles result from single or multiple mutation events, genotyping of the kdr locus and partial sequencing of the upstream intron-1 was performed on a total of 288 A. gambiae S-form collected from 28 localities in 15 countries. Knockdown resistance alleles were found to be widespread in West Africa with co-occurrence of both 1014S and 1014F in West-Central localities. Differences in intron-1 haplotype composition suggest that kdr alleles may have arisen from at least four independent mutation events. Neutrality tests provided evidence for a selective sweep acting on this genomic region, particularly in West Africa. The frequency and distribution of these kdr haplotypes varied geographically, being influenced by an interplay between different mutational occurrences, gene flow and local selection. This has important practical implications for the management and sustainability of malaria vector control programs.

  13. The role of quinone reductase (NQO1) and quinone chemistry in quercetin cytotoxicity

    NARCIS (Netherlands)

    Gliszczynska-Swiglo, A.; Woude, van der H.; Haan, de L.H.J.; Tyrakowska, B.; Aarts, J.M.M.J.G.; Rietjens, I.M.C.M.

    2003-01-01

    The effects of quercetin on viability and proliferation of Chinese Hamster Ovary (CHO) cells and CHO cells overexpressing human quinone reductase (CHO+NQO1) were studied to investigate the involvement of the pro-oxidant quinone chemistry of quercetin. The toxicity of menadione was significantly

  14. Positive correlation between decreased cellular uptake, NADPH-glutathione reductase activity and adriamycin resistance in Ehrlich ascites tumor lines.

    Science.gov (United States)

    Scheulen, M E; Hoensch, H; Kappus, H; Seeber, S; Schmidt, C G

    1987-01-01

    From a wild type strain of Ehrlich ascites tumor (EATWT) sublines resistant to daunorubicin (EATDNM), etoposide (EATETO), and cisplatinum (EATCIS) have been developed in vivo. Increase in survival and cure rate caused by adriamycin (doxorubicin) have been determined in female NMRI mice which were inoculated i.p. with EAT cells. Adriamycin concentrations causing 50% inhibition of 3H-thymidine (ICT) and 3H-uridine incorporation (ICU) and intracellular adriamycin steady-state concentrations (SSC) were measured in vitro. Adriamycin resistance increased and SSC decreased in the following sequence: EATWT - EATCIS - EATDNM - EATETO. When ICT and ICU were corrected for intracellular adriamycin concentrations in consideration of the different SSC (ICTc, ICUc), ICTc and ICUc still varied up to the 3.2 fold in EATCIS, EATDNM and EATETO in comparison to EATWT. Thus, in addition to different SSC other factors must be responsible for adriamycin resistance. Therefore, enzymes which may play a role in the cytotoxicity related to adriamycin metabolism (NADPH-cytochrome P-450 reductase, NADPH-glutathione reductase, NADP-glucose-6-phosphate dehydrogenase, NADP-isocitrate dehydrogenase) were measured. In contrast to the other parameters determined, NADPH-glutathione reductase was significantly (p less than 0.01) increased up to the 3.2 fold parallel to adriamycin resistance as determined by increase in life span, cure rate, ICTc, and ICUc, respectively. It is concluded that high activities of NADPH-glutathione reductase may contribute to an increase in adriamycin resistance of malignant tumors.

  15. 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.

  16. Thermophilic enzymes and their applications in biocatalysis: a robust aldo-keto reductase.

    Science.gov (United States)

    Willies, Simon; Isupov, Misha; Littlechild, Jennifer

    2010-09-01

    Extremophiles are providing a good source of novel robust enzymes for use in biocatalysis for the synthesis of new drugs. This is particularly true for the enzymes from thermophilic organisms which are more robust than their mesophilic counterparts to the conditions required for industrial bio-processes. This paper describes a new aldo-keto reductase enzyme from a thermophilic eubacteria, Thermotoga maritima which can be used for the production of primary alcohols. The enzyme has been cloned and over-expressed in Escherichia coli and has been purified and subjected to full biochemical characterization. The aldo-keto reductase can be used for production of primary alcohols using substrates including benzaldehyde, 1,2,3,6-tetrahydrobenzaldehyde and para-anisaldehyde. It is stable up to 80 degrees C, retaining over 60% activity for 5 hours at this temperature. The enzyme at pH 6.5 showed a preference for the forward, carbonyl reduction. The enzyme showed moderate stability with organic solvents, and retained 70% activity in 20% (v/v) isopropanol or DMSO. These properties are favourable for its potential industrial applications.

  17. Reduction of azo dyes by flavin reductase from Citrobacter freundii A1

    Directory of Open Access Journals (Sweden)

    Mohd Firdaus Abdul-Wahab

    2012-12-01

    Full Text Available Citrobacter freundii A1 isolated from a sewage treatment facility was demonstrated to be able to effectively decolorize azo dyes as pure and mixed culture. This study reports on the investigation on the enzymatic systems involved. An assay performed suggested the possible involvement of flavin reductase (Fre as an azo reductase. A heterologouslyexpressed recombinant Fre from C. freundii A1 was used to investigate its involvement in the azo reduction process. Three model dyes were used, namely Acid Red 27 (AR27, Direct Blue 15 (DB15 and Reactive Black 5 (RB5. AR27 was found to be reduced the fastest by Fre, followed by RB5, and lastly DB15. Redox mediators nicotinamide adenine dinucleotide (NADH and riboflavin enhance the reduction, suggesting the redox activity of the enzyme. The rate and extent of reduction of the model dyes correlate well with the reduction potentials (Ep. The data presented here strongly suggest that Fre is one of the enzymes responsible for azo reduction in C. freundii A1, acting via an oxidation-reduction reaction.

  18. Resonance Raman spectra of the copper-sulfur chromophores in Achromobacter cycloclastes nitrite reductase.

    Science.gov (United States)

    Dooley, D M; Moog, R S; Liu, M Y; Payne, W J; LeGall, J

    1988-10-15

    Resonance Raman spectroscopy at ambient temperature and 77 K has been used to probe the structures of the copper sites in Achromobacter cycloclastes nitrite reductase. This enzyme contains three copper ions per protein molecule and has two principal electronic absorption bands with lambda max values of 458 and 585 nm. Comparisons between the resonance Raman spectra of nitrite reductase and blue copper proteins establish that both the 458 and 585 nm bands are associated with Cu(II)-S(Cys) chromophores. A histidine ligand probably is also present. Different sets of vibrational frequencies are observed with 457.9 nm (ambient) or 476.1 nm (77 K) excitation as compared with 590 nm (ambient) or 593 nm (77 K) excitation. Excitation profiles indicate that the 458 and 585 nm absorption bands are associated with separate [Cu(II)-S(Cys)N(His)] sites or with inequivalent and uncoupled cysteine ligands in the same site. The former possibility is considered to be more likely.

  19. Molecular cloning and characterization of Fasciola gigantica thioredoxin-glutathione reductase.

    Science.gov (United States)

    Changklungmoa, Narin; Kueakhai, Pornanan; Sangpairoj, Kant; Chaichanasak, Pannigan; Jaikua, Wipaphorn; Riengrojpitak, Suda; Sobhon, Prasert; Chaithirayanon, Kulathida

    2015-06-01

    The Fasciola gigantica thioredoxin-glutathione reductase (FgTGR) gene is a fusion between thioredoxin reductase (TR) and a glutaredoxin (Grx) gene. FgTGR was cloned by polymerase chain reaction (PCR) from adult complementary DNA (cDNA), and its sequences showed two isoforms, i.e., the cytosolic and mitochondrial FgTGR. Cytosolic FgTGR (cytFgTGR) was composed of 2370 bp, and its peptide had no signal sequence and hence was not a secreted protein. Mitochondrial FgTGR (mitFgTGR) was composed of 2506 bp with a signal peptide of 43 amino acids; therefore, it was a secreted protein. The putative cytFgTGR and mitFgTGR peptides comprised of 598 and 641 amino acids, respectively, with a molecular weight of 65.8 kDa for cytFgTGR and mitFgTGR, with a conserved sequence (CPYC) of TR, and ACUG and CVNVGC of Grx domains. The recombinant FgTGR (rFgTGR) was expressed in Escherichia coli BL21 (DE3) and used for production for a polyclonal antibody in rabbits (anti-rFgTGR). The FgTGR protein expression, estimated by indirect ELISA using the rabbit anti-rFgTGR as probe, showed high levels of expression in eggs, and 2- and 4-week-old juveniles and adults. The rFgTGR exhibited specific activities in the 5,5'-dithiobis (2-nitro-benzoic acid) (DTNB) reductase assay for TR activity and in β-hydroxyethul disulfide (HED) for Grx activity. When analyzed by immunoblotting and immunohistochemistry, rabbit anti-rFgTGR reacted with natural FgTGR at a molecular weight of 66 kDa from eggs, whole body fraction (WB) of metacercariae, NEJ, 2- and 4-week-old juveniles and adults, and the tegumental antigen (TA) of adult. The FgTGR protein was expressed at high levels in the tegument of 2- and 4-week-old juveniles. The FgTGR may be one of the major factors acting against oxidative stresses that can damage the parasite; hence, it could be considered as a novel vaccine or a drug target.

  20. Knockdown of hypocretin attenuates extended access of cocaine self-administration in rats.

    Science.gov (United States)

    Schmeichel, Brooke E; Matzeu, Alessandra; Koebel, Pascale; Vendruscolo, Leandro F; Sidhu, Harpreet; Shahryari, Roxana; Kieffer, Brigitte L; Koob, George F; Martin-Fardon, Rémi; Contet, Candice

    2018-04-06

    The hypocretin/orexin (HCRT) neuropeptide system regulates feeding, arousal state, stress responses, and reward, especially under conditions of enhanced motivational relevance. In particular, HCRT neurotransmission facilitates drug-seeking behavior in circumstances that demand increased effort and/or motivation to take the drug. The present study used a shRNA-encoding adeno-associated viral vector to knockdown Hcrt expression throughout the dorsal hypothalamus in adult rats and determine the role of HCRT in cocaine self-administration. Chronic Hcrt silencing did not impact cocaine self-administration under short-access conditions, but robustly attenuated cocaine intake under extended access conditions, a model that mimics key features of compulsive cocaine taking. In addition, Hcrt silencing decreased motivation for both cocaine and a highly palatable food reward (i.e., sweetened condensed milk; SCM) under a progressive ratio schedule of reinforcement, but did not alter responding for SCM under a fixed ratio schedule. Importantly, Hcrt silencing did not affect food or water consumption, and had no consequence for general measures of arousal and stress reactivity. At the molecular level, chronic Hcrt knockdown reduced the number of neurons expressing dynorphin (DYN), and to a smaller extent melanin-concentrating hormone (MCH), in the dorsal hypothalamus. These original findings support the hypothesis that HCRT neurotransmission promotes operant responding for both drug and non-drug rewards, preferentially under conditions requiring a high degree of motivation. Furthermore, the current study provides compelling evidence for the involvement of the HCRT system in cocaine self-administration also under low-effort conditions in rats allowed extended access, possibly via functional interactions with DYN and MCH signaling.

  1. Comparison of the CAS-POL and IOM samplers for determining the knockdown efficiencies of water sprays on float coal dust.

    Science.gov (United States)

    Seaman, Clara E; Shahan, Michael R; Beck, Timothy W; Mischler, Steven E

    2018-03-01

    Float coal dust, generated by mining operations, is distributed throughout mine airways by ventilating air designed to purge gases and respirable dust. Float coal dust poses an explosion hazard in the event of a methane ignition. Current regulation requires the application of inert rock dust in areas subjected to float coal dust in order to mitigate the hazard. An alternate method using water sprays, which have been effective in controlling respirable dust hazards, has been proposed as a way to control float coal dust generated on longwall faces. However, the knockdown efficiency of the proposed water sprays on float coal dust needs to be verified. This study used gravimetric isokinetic Institute of Occupational Medicine (IOM) samplers alongside a real-time aerosol monitor (Cloud Aerosol Spectrometer with polarization; CAS-POL) to study the effects of spray type, operating pressure, and spray orientation on knockdown efficiencies for seven different water sprays. Because the CAS-POL has not been used to study mining dust, the CAS-POL measurements were validated with respect to the IOM samplers. This study found that the CAS-POL was able to resolve the same trends measured by the IOM samplers, while providing additional knockdown information for specific particle size ranges and locations in the test area. In addition, the CAS-POL data was not prone to the same process errors, which may occur due to the handling of the IOM filter media, and was able to provide a faster analysis of the data after testing. This study also determined that pressure was the leading design criteria influencing spray knockdown efficiency, with spray type also having some effect and orientation having little to no effect. The results of this study will be used to design future full-scale float coal dust capture tests involving multiple sprays, which will be evaluated using the CAS-POL.

  2. Knockdown of astrocyte elevated gene-1 inhibits tumor growth and modifies microRNAs expression profiles in human colorectal cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Sujun [East Department of Gastroenterology, Institute of Geriatrics, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong 510080 (China); Southern Medical University, Guangzhou, Guangdong 510515 (China); Wu, Binwen, E-mail: wubinwengd@aliyun.com [East Department of Gastroenterology, Institute of Geriatrics, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong 510080 (China); Li, Dongfeng; Zhou, Weihong; Deng, Gang; Zhang, Kaijun; Li, Youjia [East Department of Gastroenterology, Institute of Geriatrics, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong 510080 (China)

    2014-02-14

    Highlights: • AEG-1 expression in CRC cell lines and down-regulation or upregulation of AEG-1 in vitro. • Knockdown of AEG-1 inhibits cell proliferation, colony formation and invasion. • Upregulation of AEG-1 enhances proliferation, invasion and colony formation. • Knockdown of AEG-1 accumulates G0/G1-phase cells and promotes apoptosis in CRC cells. • AEG-1 knockdown increases 5-FU cytotoxicity. - Abstract: Astrocyte elevated gene-1 (AEG-1), upregulated in various types of malignancies including colorectal cancer (CRC), has been reported to be associated with the carcinogenesis. MicroRNAs (miRNAs) are widely involved in the initiation and progression of cancer. However, the functional significance of AEG-1 and the relationship between AEG-1 and microRNAs in human CRC remains unclear. The aim of this study was to investigate whether AEG-1 could serve as a potential therapeutic target of human CRC and its possible mechanism. We adopted a strategy of ectopic overexpression or RNA interference to upregulate or downregulate expression of AEG-1 in CRC models. Their phenotypic changes were analyzed by Western blot, MTT and transwell matrix penetration assays. MicroRNAs expression profiles were performed using microarray analysis followed by validation using qRT-PCR. Knockdown of AEG-1 could significantly inhibit colon cancer cell proliferation, colony formation, invasion and promotes apoptosis. Conversely, upregulation of AEG-1 could significantly enhance cell proliferation, invasion and reduced apoptisis. AEG-1 directly contributes to resistance to chemotherapeutic drug. Targeted downregulation of AEG-1 might improve the expression of miR-181a-2{sup ∗}, -193b and -193a, and inversely inhibit miR-31 and -9{sup ∗}. Targeted inhibition of AEG-1 can lead to modification of key elemental characteristics, such as miRNAs, which may become a potential effective therapeutic strategy for CRC.

  3. Confusion, knock-down and kill of Aedes aegypti using metofluthrin in domestic settings: a powerful tool to prevent dengue transmission?

    Science.gov (United States)

    Ritchie, Scott A; Devine, Gregor J

    2013-09-11

    Dengue control methods are reliant upon control of the vector, primarily Aedes aegypti. Current adulticiding methods in North Queensland include treating premises with residual synthetic pyrethroid insecticides (interior residual spraying; IRS), a laborious, intrusive task. The vapor active synthetic pyrethroid metofluthrin might offer an efficient alternative as some studies indicate that it prevents biting and has strong knockdown effects. However, its expellant and/or irritant effects, longevity, residual activity and the speed with which biting behavior is disrupted have not yet been characterized. We exposed cohorts of Cairns colony (F2-4) Ae. aegypti to rooms (17-24 m3) treated with 5% and 10% AI metofluthrin emanators. Using free-flying and caged populations we measured biting (human landing rate), expulsion through unscreened windows, knockdown and death over periods ranging between a few minutes and 24 hrs. Observations of the behavior of single female Ae. aegypti exposed to metofluthrin were also made. Female Ae. aegypti exposed to 5% or 10% metofluthrin formulations were almost entirely inhibited from biting. This was the result of rapid knockdown and mortality (80-90% in less than one hour) and to the behavioral impacts of exposure that, within minutes, caused female Ae. aegypti to become disoriented, stop landing on hosts, and seek resting sites. Exposed mosquitoes did not exhibit any increased propensity to exit treated rooms and the 10% AI resin remained fully active for at least 20 days. The new, high-dose, resin formulations of metofluthrin act quickly to prevent biting and to knockdown and kill free-flying female Ae. aegypti in our experimental rooms. There was no evidence that metofluthrin induced escape from treated areas. Resin-based metofluthrin emanators show great potential as a replacement for labor intensive IRS for dengue vector control.

  4. Knockdown of astrocyte elevated gene-1 inhibits tumor growth and modifies microRNAs expression profiles in human colorectal cancer cells

    International Nuclear Information System (INIS)

    Huang, Sujun; Wu, Binwen; Li, Dongfeng; Zhou, Weihong; Deng, Gang; Zhang, Kaijun; Li, Youjia

    2014-01-01

    Highlights: • AEG-1 expression in CRC cell lines and down-regulation or upregulation of AEG-1 in vitro. • Knockdown of AEG-1 inhibits cell proliferation, colony formation and invasion. • Upregulation of AEG-1 enhances proliferation, invasion and colony formation. • Knockdown of AEG-1 accumulates G0/G1-phase cells and promotes apoptosis in CRC cells. • AEG-1 knockdown increases 5-FU cytotoxicity. - Abstract: Astrocyte elevated gene-1 (AEG-1), upregulated in various types of malignancies including colorectal cancer (CRC), has been reported to be associated with the carcinogenesis. MicroRNAs (miRNAs) are widely involved in the initiation and progression of cancer. However, the functional significance of AEG-1 and the relationship between AEG-1 and microRNAs in human CRC remains unclear. The aim of this study was to investigate whether AEG-1 could serve as a potential therapeutic target of human CRC and its possible mechanism. We adopted a strategy of ectopic overexpression or RNA interference to upregulate or downregulate expression of AEG-1 in CRC models. Their phenotypic changes were analyzed by Western blot, MTT and transwell matrix penetration assays. MicroRNAs expression profiles were performed using microarray analysis followed by validation using qRT-PCR. Knockdown of AEG-1 could significantly inhibit colon cancer cell proliferation, colony formation, invasion and promotes apoptosis. Conversely, upregulation of AEG-1 could significantly enhance cell proliferation, invasion and reduced apoptisis. AEG-1 directly contributes to resistance to chemotherapeutic drug. Targeted downregulation of AEG-1 might improve the expression of miR-181a-2 ∗ , -193b and -193a, and inversely inhibit miR-31 and -9 ∗ . Targeted inhibition of AEG-1 can lead to modification of key elemental characteristics, such as miRNAs, which may become a potential effective therapeutic strategy for CRC

  5. Sqstm1 knock-down causes a locomotor phenotype ameliorated by rapamycin in a zebrafish model of ALS/FTLD.

    Science.gov (United States)

    Lattante, Serena; de Calbiac, Hortense; Le Ber, Isabelle; Brice, Alexis; Ciura, Sorana; Kabashi, Edor

    2015-03-15

    Mutations in SQSTM1, encoding for the protein SQSTM1/p62, have been recently reported in 1-3.5% of patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration (ALS/FTLD). Inclusions positive for SQSTM1/p62 have been detected in patients with neurodegenerative disorders, including ALS/FTLD. In order to investigate the pathogenic mechanisms induced by SQSTM1 mutations in ALS/FTLD, we developed a zebrafish model. Knock-down of the sqstm1 zebrafish ortholog, as well as impairment of its splicing, led to a specific phenotype, consisting of behavioral and axonal anomalies. Here, we report swimming deficits associated with shorter motor neuronal axons that could be rescued by the overexpression of wild-type human SQSTM1. Interestingly, no rescue of the loss-of-function phenotype was observed when overexpressing human SQSTM1 constructs carrying ALS/FTLD-related mutations. Consistent with its role in autophagy regulation, we found increased mTOR levels upon knock-down of sqstm1. Furthermore, treatment of zebrafish embryos with rapamycin, a known inhibitor of the mTOR pathway, yielded an amelioration of the locomotor phenotype in the sqstm1 knock-down model. Our results suggest that loss-of-function of SQSTM1 causes phenotypic features characterized by locomotor deficits and motor neuron axonal defects that are associated with a misregulation of autophagic processes. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  6. Molecular Diagnosis of 5α-Reductase Type II Deficiency in Brazilian Siblings with 46,XY Disorder of Sex Development

    Directory of Open Access Journals (Sweden)

    Maricilda Palandi de Mello

    2011-12-01

    Full Text Available The steroid 5α-reductase type II enzyme catalyzes the conversion of testosterone (T to dihydrotestosterone (DHT, and its deficiency leads to undervirilization in 46,XY individuals, due to an impairment of this conversion in genital tissues. Molecular analysis in the steroid 5α-reductase type II gene (SRD5A2 was performed in two 46,XY female siblings. SRD5A2 gene sequencing revealed that the patients were homozygous for p.Gln126Arg missense mutation, which results from the CGA > CAA nucleotide substitution. The molecular result confirmed clinical diagnosis of 46,XY disorder of sex development (DSD for the older sister and directed the investigation to other family members. Studies on SRD5A2 protein structure showed severe changes at NADPH binding region indicating that structural modeling analysis can be useful to evaluate the deleterious role of a mutation as causing 5α-reductase type II enzyme deficiency.

  7. A small interfering RNA screen of genes involved in DNA repair identifies tumor-specific radiosensitization by POLQ knockdown

    DEFF Research Database (Denmark)

    Higgins, Geoff S; Prevo, Remko; Lee, Yin-Fai

    2010-01-01

    The effectiveness of radiotherapy treatment could be significantly improved if tumor cells could be rendered more sensitive to ionizing radiation (IR) without altering the sensitivity of normal tissues. However, many of the key therapeutically exploitable mechanisms that determine intrinsic tumor...... radiosensitivity are largely unknown. We have conducted a small interfering RNA (siRNA) screen of 200 genes involved in DNA damage repair aimed at identifying genes whose knockdown increased tumor radiosensitivity. Parallel siRNA screens were conducted in irradiated and unirradiated tumor cells (SQ20B......) and irradiated normal tissue cells (MRC5). Using gammaH2AX foci at 24 hours after IR, we identified several genes, such as BRCA2, Lig IV, and XRCC5, whose knockdown is known to cause increased cell radiosensitivity, thereby validating the primary screening end point. In addition, we identified POLQ (DNA...

  8. Knockdown of versican 1 blocks cigarette-induced loss of insoluble elastin in human lung fibroblasts.

    Science.gov (United States)

    Xu, Lu-lu; Lu, Yun-tao; Zhang, Jing; Wu, Lian; Merrilees, Mervyn J; Qu, Jie-ming

    2015-08-15

    COPD lung is characterized by loss of alveolar elastic fibers and an increase in the chondroitin sulfate (CS) matrix proteoglycan versican V1 (V1). V1 is a known inhibitor of elastic fiber deposition and this study investigates the effects of knockdown of V1, and add-back of CS, on CCL-210 lung fibroblasts treated with cigarette smoke extract (CSE) as a model for COPD. CSE inhibited fibroblast proliferation, viability, tropoelastin synthesis, and elastin deposition, and increased V1 synthesis and secretion. V1 siRNA decreased V1 and constituent CS, did not affect tropoelastin production, but blocked the CSE-induced loss in insoluble elastin. Exogenous CS reduced insoluble elastin, even in the presence of V1 siRNA. These findings confirm that V1 and CS impair the assembly of tropoelastin monomers into insoluble fibers, and further demonstrate that specific knockdown of V1 alleviates the impaired assembly of elastin seen in cultures of pulmonary fibroblasts exposed to CSE, indicating a regulatory role for this protein in the pathophysiology of COPD. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Methylenetetrahydrofolate reductase (MTHFR) deficiency presenting as a rash.

    LENUS (Irish Health Repository)

    Crushell, Ellen

    2012-09-01

    We report on the case of a 2-year-old girl recently diagnosed with Methylenetetrahydrofolate reductase (MTHFR) deficiency who originally presented in the neonatal period with a distinctive rash. At 11 weeks of age she developed seizures, she had acquired microcephaly and developmental delay. The rash deteriorated dramatically following commencement of phenobarbitone; both rash and seizures abated following empiric introduction of pyridoxine and folinic acid as treatment of possible vitamin responsive seizures. We postulate that phenobarbitone in combination with MTHFR deficiency may have caused her rash to deteriorate and subsequent folinic acid was helpful in treating the rash and preventing further acute neurological decline as commonly associated with this condition.

  10. Purification, crystallization and preliminary X-ray analysis of 3-hydroxy-3-methylglutaryl-coenzyme A reductase of Streptococcus pneumoniae

    International Nuclear Information System (INIS)

    Zhang, Liping; Feng, Lingling; Zhou, Li; Gui, Jie; Wan, Jian; Hu, Xiaopeng

    2010-01-01

    3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase of Streptococcus pneumoniae has been cloned, overexpressed and purified to homogeneity using Ni–NTA affinity chromatography. Crystals were obtained using the hanging-drop vapour-diffusion method. Class II 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases are potential targets for novel antibiotic development. In order to obtain a precise structural model for use in virtual screening and inhibitor design, HMG-CoA reductase of Streptococcus pneumoniae was cloned, overexpressed and purified to homogeneity using Ni–NTA affinity chromatography. Crystals were obtained using the hanging-drop vapour-diffusion method. A complete data set was collected from a single frozen crystal on a home X-ray source. The crystal diffracted to 2.3 Å resolution and belonged to the orthorhombic space group C222 1 , with unit-cell parameters a = 773.4836, b = 90.3055, c = 160.5592 Å, α = β = γ = 90°. Assuming the presence of two molecules in the asymmetric unit, the solvent content was estimated to be 54.1% (V M = 2.68 Å 3 Da −1 )

  11. Homology modeling of dissimilatory APS reductases (AprBA of sulfur-oxidizing and sulfate-reducing prokaryotes.

    Directory of Open Access Journals (Sweden)

    Birte Meyer

    Full Text Available BACKGROUND: The dissimilatory adenosine-5'-phosphosulfate (APS reductase (cofactors flavin adenine dinucleotide, FAD, and two [4Fe-4S] centers catalyzes the transformation of APS to sulfite and AMP in sulfate-reducing prokaryotes (SRP; in sulfur-oxidizing bacteria (SOB it has been suggested to operate in the reverse direction. Recently, the three-dimensional structure of the Archaeoglobus fulgidus enzyme has been determined in different catalytically relevant states providing insights into its reaction cycle. METHODOLOGY/PRINCIPAL FINDINGS: Full-length AprBA sequences from 20 phylogenetically distinct SRP and SOB species were used for homology modeling. In general, the average accuracy of the calculated models was sufficiently good to allow a structural and functional comparison between the beta- and alpha-subunit structures (78.8-99.3% and 89.5-96.8% of the AprB and AprA main chain atoms, respectively, had root mean square deviations below 1 A with respect to the template structures. Besides their overall conformity, the SRP- and SOB-derived models revealed the existence of individual adaptations at the electron-transferring AprB protein surface presumably resulting from docking to different electron donor/acceptor proteins. These structural alterations correlated with the protein phylogeny (three major phylogenetic lineages: (1 SRP including LGT-affected Archaeoglobi and SOB of Apr lineage II, (2 crenarchaeal SRP Caldivirga and Pyrobaculum, and (3 SOB of the distinct Apr lineage I and the presence of potential APS reductase-interacting redox complexes. The almost identical protein matrices surrounding both [4Fe-4S] clusters, the FAD cofactor, the active site channel and center within the AprB/A models of SRP and SOB point to a highly similar catalytic process of APS reduction/sulfite oxidation independent of the metabolism type the APS reductase is involved in and the species it has been originated from. CONCLUSIONS: Based on the comparative

  12. Elucidating the role of free polycations in gene knockdown by siRNA polyplexes

    DEFF Research Database (Denmark)

    Klauber, Thomas Christopher Bogh; Søndergaard, Rikke Vicki; Sawant, Rupa R.

    2016-01-01

    capability, but are very different regarding siRNA decondensation, cellular internalization and induction of reporter gene knockdown. Lipid conjugation of bPEI 1.8. kDa improves the siRNA delivery properties, but with markedly different formulation requirements and mechanisms of action compared...... today.A major reason for the lack of progress is insufficient understanding of cell-polyplex interaction. We investigate siRNA delivery using polyethyleneimine (PEI) based vectors and examine how crucial formulation parameters determine the challenges associated with PEI as a delivery vector. We further...

  13. CERKL knockdown causes retinal degeneration in zebrafish.

    Directory of Open Access Journals (Sweden)

    Marina Riera

    Full Text Available The human CERKL gene is responsible for common and severe forms of retinal dystrophies. Despite intense in vitro studies at the molecular and cellular level and in vivo analyses of the retina of murine knockout models, CERKL function remains unknown. In this study, we aimed to approach the developmental and functional features of cerkl in Danio rerio within an Evo-Devo framework. We show that gene expression increases from early developmental stages until the formation of the retina in the optic cup. Unlike the high mRNA-CERKL isoform multiplicity shown in mammals, the moderate transcriptional complexity in fish facilitates phenotypic studies derived from gene silencing. Moreover, of relevance to pathogenicity, teleost CERKL shares the two main human protein isoforms. Morpholino injection has been used to generate a cerkl knockdown zebrafish model. The morphant phenotype results in abnormal eye development with lamination defects, failure to develop photoreceptor outer segments, increased apoptosis of retinal cells and small eyes. Our data support that zebrafish Cerkl does not interfere with proliferation and neural differentiation during early developmental stages but is relevant for survival and protection of the retinal tissue. Overall, we propose that this zebrafish model is a powerful tool to unveil CERKL contribution to human retinal degeneration.

  14. Biocatalysis with thermostable enzymes: structure and properties of a thermophilic 'ene'-reductase related to old yellow enzyme.

    Science.gov (United States)

    Adalbjörnsson, Björn V; Toogood, Helen S; Fryszkowska, Anna; Pudney, Christopher R; Jowitt, Thomas A; Leys, David; Scrutton, Nigel S

    2010-01-25

    We report the crystal structure of a thermophilic "ene" reductase (TOYE) isolated from Thermoanaerobacter pseudethanolicus E39. The crystal structure reveals a tetrameric enzyme and an active site that is relatively large compared to most other structurally determined and related Old Yellow Enzymes. The enzyme adopts higher order oligomeric states (octamers and dodecamers) in solution, as revealed by sedimentation velocity and multiangle laser light scattering. Bead modelling indicates that the solution structure is consistent with the basic tetrameric structure observed in crystallographic studies and electron microscopy. TOYE is stable at high temperatures (T(m)>70 degrees C) and shows increased resistance to denaturation in water-miscible organic solvents compared to the mesophilic Old Yellow Enzyme family member, pentaerythritol tetranitrate reductase. TOYE has typical ene-reductase properties of the Old Yellow Enzyme family. There is currently major interest in using Old Yellow Enzyme family members in the preparative biocatalysis of a number of activated alkenes. The increased stability of TOYE in organic solvents is advantageous for biotransformations in which water-miscible organic solvents and biphasic reaction conditions are required to both deliver novel substrates and minimize product racemisation.

  15. LOXL4 knockdown enhances tumor growth and lung metastasis through collagen-dependent extracellular matrix changes in triple-negative breast cancer.

    Science.gov (United States)

    Choi, Sul Ki; Kim, Hoe Suk; Jin, Tiefeng; Moon, Woo Kyung

    2017-02-14

    Lysyl oxidase (LOX) family genes catalyze collagen cross-link formation. To determine the effects of lysyl oxidase-like 4 (LOXL4) expression on breast tumor formation and metastasis, we evaluated primary tumor growth and lung metastasis in mice injected with LOXL4-knockdown MDA-MB-231 triple-negative human breast cancer cells. In addition, we analyzed overall survival in breast cancer patients based on LOXL4 expression using a public online database. In the mouse xenograft model, LOXL4 knockdown increased primary tumor growth and lung colonization as well as collagen I and IV, lysine hydroxylase 1 and 2, and prolyl 4-hydroxylase subunit alpha 1 and 2 levels. Second harmonic generation imaging revealed that LOXL4 knockdown resulted in the thickening of collagen bundles within tumors. In addition, weak LOXL4 expression was associated with poor overall survival in breast cancer patients from the BreastMark dataset, and this association was strongest in triple-negative breast cancer patients. These results demonstrate that weak LOXL4 expression leads to remodeling of the extracellular matrix through induction of collagen synthesis, deposition, and structural changes. These alterations in turn promote tumor growth and metastasis and are associated with poor clinical outcomes in triple-negative breast cancer.

  16. Functional properties and structural characterization of rice δ1-pyrroline-5-carboxylate reductase

    Directory of Open Access Journals (Sweden)

    Giuseppe eForlani

    2015-07-01

    Full Text Available The majority of plant species accumulate high intracellular levels of proline to cope with hyperosmotic stress conditions. Proline synthesis from glutamate is tightly regulated at both the transcriptional and the translational levels, yet little is known about the mechanisms for post-translational regulation of the enzymatic activities involved. The gene coding in rice (Oryza sativa L. for δ1-pyrroline-5-carboxylate (P5C reductase, the enzyme that catalyzes the second and final step in this pathway, was isolated and expressed in E. coli. The structural and functional properties of the affinity-purified protein were characterized. As for most species, rice P5C reductase was able to use in vitro either NADH or NADPH as the electron donor. However, strikingly different effects of cations and anions were found depending on the pyridine nucleotide used, namely inhibition of NADH-dependent activity and stimulation of NADPH-dependent activity. Moreover, physiological concentrations of proline and NADP+ were strongly inhibitory for the NADH-dependent reaction, whereas the NADPH-dependent activity was mildly affected. Our results suggest that only NADPH may be used in vivo and that stress-dependent variations in ion homeostasis and NADPH/NADP+ ratio could modulate enzyme activity, being functional in promoting proline accumulation and potentially also adjusting NADPH consumption during the defense against hyperosmotic stress. The apparent molecular weight of the native protein observed in size exclusion chromatography indicated a high oligomerization state. We also report the first crystal structure of a plant P5C reductase at 3.40-Å resolution, showing a decameric quaternary assembly. Based on the structure, it was possible to identify dynamic structural differences among rice, human and bacterial enzymes.

  17. Sunflower (Helianthus annuus) fatty acid synthase complex: enoyl-[acyl carrier protein]-reductase genes.

    Science.gov (United States)

    González-Thuillier, Irene; Venegas-Calerón, Mónica; Garcés, Rafael; von Wettstein-Knowles, Penny; Martínez-Force, Enrique

    2015-01-01

    Enoyl-[acyl carrier protein]-reductases from sunflower. A major factor contributing to the amount of fatty acids in plant oils are the first steps of their synthesis. The intraplastidic fatty acid biosynthetic pathway in plants is catalysed by type II fatty acid synthase (FAS). The last step in each elongation cycle is carried out by the enoyl-[ACP]-reductase, which reduces the dehydrated product of β-hydroxyacyl-[ACP] dehydrase using NADPH or NADH. To determine the mechanisms involved in the biosynthesis of fatty acids in sunflower (Helianthus annuus) seeds, two enoyl-[ACP]-reductase genes have been identified and cloned from developing seeds with 75 % identity: HaENR1 (GenBank HM021137) and HaENR2 (HM021138). The two genes belong to the ENRA and ENRB families in dicotyledons, respectively. The genetic duplication most likely originated after the separation of di- and monocotyledons. RT-qPCR revealed distinct tissue-specific expression patterns. Highest expression of HaENR1 was in roots, stems and developing cotyledons whereas that of H a ENR2 was in leaves and early stages of seed development. Genomic DNA gel blot analyses suggest that both are single-copy genes. In vivo activity of the ENR enzymes was tested by complementation experiments with the JP1111 fabI(ts) E. coli strain. Both enzymes were functional demonstrating that they interacted with the bacterial FAS components. That different fatty acid profiles resulted infers that the two Helianthus proteins have different structures, substrate specificities and/or reaction rates. The latter possibility was confirmed by in vitro analysis with affinity-purified heterologous-expressed enzymes that reduced the crotonyl-CoA substrate using NADH with different V max.

  18. Purine biosynthesis in L1210 leukemia cells is inhibited by 7-hydroxymethotrexate (7-OH-MTX) polyglutamates (PGS)

    International Nuclear Information System (INIS)

    Seither, R.L.; Matherly, L.H.; Goldman, I.D.

    1986-01-01

    The biochemical basis for 7-OH-MTX cytotoxicity was examined in L1210 tumor cells. Cells were exposed to 100 μM 7-OH-MTX (approx. 50% growth inhibition) or 10 μM methotrexate (MTX) (approx. 95% growth inhibition) for 6 hrs to allow high levels of PGS to accumulate. Dihydrofolate reductase (DHFR) activity was assessed by dihydrofolate (FH 2 ) pools labeled with 5-formyl-[ 3 H]-tetrahydrofolate (5μM) or 3 H-folic acid (1 μM). FH 2 was not elevated above control levels in 7-OH-MTX treated cells, in contrast to MTX treated cells in which FH 2 increased 4- to 7-fold. 3 H-Deoxyuridine incorporation into DNA was not inhibited in cells containing high levels (11.5 nmol/g dry wt.) of 7-OH-MTX tetraglutamate (7-OH-4-NH 2 -10-CH 3 -PteGlu 4 ), well in excess of the DHFR-binding capacity (7.3 +/- 0.9 nmol/g), indicating a normal rate of thymidylate synthesis. Although small amounts of 7-OH-MTX and its PGS were bound to DHFR in L1210 cells, as assessed by gel filtration, there was evidence for the preferential binding of 7-OH-MTX tetraglutamate. In all cases this was well below the DHFR binding capacity, consistent with normal rates of deoxyuridine metabolism and FH 2 levels in the cell. Incorporation of 14 C-formate (60 min) into thymidylate and amino acids was unaffected by 7-OH-MTX, yet incorporation into purines was inhibited over 50%, supporting a block(s) in de novo purine biosynthesis

  19. Crystal structure of conjugated polyketone reductase (CPR-C1) from Candida parapsilosis IFO 0708 complexed with NADPH.

    Science.gov (United States)

    Qin, Hui-Min; Yamamura, Akihiro; Miyakawa, Takuya; Kataoka, Michihiko; Maruoka, Shintaro; Ohtsuka, Jun; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2013-11-01

    Conjugated polyketone reductase (CPR-C1) from Candida parapsilosis IFO 0708 is a member of the aldo-keto reductase (AKR) superfamily and reduces ketopantoyl lactone to d-pantoyl lactone in a NADPH-dependent and stereospecific manner. We determined the crystal structure of CPR-C1.NADPH complex at 2.20 Å resolution. CPR-C1 adopted a triose-phosphate isomerase (TIM) barrel fold at the core of the structure in which Thr25 and Lys26 of the GXGTX motif bind uniquely to the adenosine 2'-phosphate group of NADPH. This finding provides a novel structural basis for NADPH binding of the AKR superfamily. Copyright © 2013 Wiley Periodicals, Inc.

  20. Benzothiazole analogues: Synthesis, characterization, MO calculations with PM6 and DFT, in silico studies and in vitro antimalarial as DHFR inhibitors and antimicrobial activities.

    Science.gov (United States)

    Thakkar, Sampark S; Thakor, Parth; Ray, Arabinda; Doshi, Hiren; Thakkar, Vasudev R

    2017-10-15

    Benzothiazole analogues are of interest due to their potential activity against malarial and microbial infections. In search of suitable antimicrobial and antimalarial agents, we report here the synthesis, characterization and biological activities of benzothiazole analogues (J 1-J 10). The molecules were characterized by IR, Mass, 1 H NMR, 13 C NMR and elemental analysis. The in vitro antimicrobial activity was investigated against pathogenic strains; the results were explained with the help of DFT and PM6 molecular orbital calculations. In vitro cytotoxicity and genotoxicity of the molecules were studied against S. pombe cells. In vitro antimalarial activity was studied. The active compounds J 1, J 2, J 3, J 5 and J 6 were further evaluated for enzyme inhibition efficacy against the receptor Pf-DHFR, computational and in vitro studies were carried out to examine their candidatures as lead dihydrofolate reductase inhibitors. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. HER2 specific delivery of methotrexate by dendrimer conjugated anti-HER2 mAb

    International Nuclear Information System (INIS)

    Shukla, Rameshwer; Thomas, Thommey P; Desai, Ankur M; Kotlyar, Alina; Park, Steve J; Baker, James R Jr

    2008-01-01

    Herceptin, a humanized monoclonal antibody that binds to human growth factor receptor-2 (HER2), was covalently attached to a fifth-generation (G5) polyamidoamine dendrimer containing the cytotoxic drug methotrexate. The specific binding and internalization of this conjugate labeled with FITC was clearly demonstrated in cell lines overexpressing HER2 by flow cytometry as well as confocal microscopic analysis. In addition, binding and uptake of antibody conjugated dendrimers was completely blocked by excess non-conjugated herceptin. The dendrimer conjugate was also shown to inhibit the dihydrofolate reductase with similar activity to methotrexate. Co-localization experiments with lysotracker red indicate that antibody conjugate, although internalized efficiently into cells, has an unusually long residence time in the lysosome. Somewhat lower cytotoxicity of the conjugate in comparison to free methotrexate was attributed to the slow release of methotrexate from the conjugate and its long retention in the lysosomal pocket

  2. Chemical Morphing of DNA Containing Four Noncanonical Bases.

    Science.gov (United States)

    Eremeeva, Elena; Abramov, Michail; Margamuljana, Lia; Rozenski, Jef; Pezo, Valerie; Marlière, Philippe; Herdewijn, Piet

    2016-06-20

    The ability of alternative nucleic acids, in which all four nucleobases are substituted, to replicate in vitro and to serve as genetic templates in vivo was evaluated. A nucleotide triphosphate set of 5-chloro-2'-deoxyuridine, 7-deaza-2'-deoxyadenosine, 5-fluoro-2'-deoxycytidine, and 7-deaza-2'deoxyguanosine successfully underwent polymerase chain reaction (PCR) amplification using templates of different lengths (57 or 525mer) and Taq or Vent (exo-) DNA polymerases as catalysts. Furthermore, a fully morphed gene encoding a dihydrofolate reductase was generated by PCR using these fully substituted nucleotides and was shown to transform and confer trimethoprim resistance to E. coli. These results demonstrated that fully modified templates were accurately read by the bacterial replication machinery and provide the first example of a long fully modified DNA molecule being functional in vivo. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Properties of latent and thiol-activated rat hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase and regulation of enzyme activity.

    Science.gov (United States)

    Dotan, I; Shechter, I

    1983-10-15

    The effect of the thiols glutathione (GSH), dithiothreitol (DTT), and dithioerythritol (DTE) on the conversion of an inactive, latent form (El) of rat liver 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) to a catalyticaly active form (Ea) is examined. Latent hepatic microsomal HMG-CoA reductase is activated to a similar degree of activation by DTT and DTE and to a lower extent by GSH. All three thiols affect both Km and Vmax values of the enzyme toward HMG-CoA and NADPH. Studies of the effect of DTT on the affinity binding of HMG-CoA reductase to agarose-hexane-HMG-CoA (AG-HMG-CoA) resin shows that thiols are necessary for the binding of the enzyme to the resin. Removal of DTT from AG-HMG-CoA-bound soluble Ea (active enzyme) does not cause dissociation of the enzyme from the resin at low salt concentrations. Substitution of DTT by NADPH does not promote binding of soluble El (latent enzyme) to AG-HMG-CoA. The enzymatic activity of Ea in the presence of DTT and GSH indicates that these thiols compete for the same binding site on the enzyme. Diethylene glycol disulfide (ESSE) and glutathione disulfide (GSSG) inhibit the activity of Ea. ESSE is more effective for the inhibition of Ea than GSSG, causing a higher degree of maximal inhibition and affecting the enzymatic activity at lower concentrations. A method is described for the rapid conversion of soluble purified Ea to El using gel-filtration chromatography on Bio-Gel P-4 columns. These combined results point to the importance of the thiol/disulfide ratio for the modulation of hepatic HMG-CoA reductase activity.

  4. A Rational Approach to Identify Inhibitors of Mycobacterium tuberculosis Enoyl Acyl Carrier Protein Reductase

    Czech Academy of Sciences Publication Activity Database

    Chhabria, M. T.; Parmar, K. B.; Brahmkshatriya, Pathik

    2013-01-01

    Roč. 19, č. 21 (2013), s. 3878-3883 ISSN 1381-6128 Institutional support: RVO:61388963 Keywords : mycobacterium tuberculosis * enoyl acyl carrier protein reductase * pharmacophore modeling * molecular docking * binding interactions Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 3.288, year: 2013

  5. AHR2 morpholino knockdown reduces the toxicity of total particulate matter to zebrafish embryos

    Energy Technology Data Exchange (ETDEWEB)

    Massarsky, Andrey, E-mail: andrey.massarsky@duke.edu [Nicholas School of the Environment, Duke University, Durham, NC 27708 (United States); Bone, Audrey J. [Nicholas School of the Environment, Duke University, Durham, NC 27708 (United States); Dong, Wu [Nicholas School of the Environment, Duke University, Durham, NC 27708 (United States); School of Animal Science and Technology, Inner Mongolia Provincial Key Laboratory for Toxicants and Animal Disease, Inner Mongolia University for the Nationalities, Tongliao, Inner Mongolia 028000 (China); Hinton, David E. [Nicholas School of the Environment, Duke University, Durham, NC 27708 (United States); Prasad, G.L. [RAI Services Company, Winston-Salem, NC 27101 (United States); Di Giulio, Richard T. [Nicholas School of the Environment, Duke University, Durham, NC 27708 (United States)

    2016-10-15

    The zebrafish embryo has been proposed as a ‘bridge model’ to study the effects of cigarette smoke on early development. Previous studies showed that exposure to total particulate matter (TPM) led to adverse effects in developing zebrafish, and suggested that the antioxidant and aryl hydrocarbon receptor (AHR) pathways play important roles. This study investigated the roles of these two pathways in mediating TPM toxicity. The study consisted of four experiments. In experiment I, zebrafish embryos were exposed from 6 h post fertilization (hpf) until 96 hpf to TPM{sub 0.5} and TPM{sub 1.0} (corresponding to 0.5 and 1.0 μg/mL equi-nicotine units) in the presence or absence of an antioxidant (N-acetyl cysteine/NAC) or a pro-oxidant (buthionine sulfoximine/BSO). In experiment II, TPM exposures were performed in embryos that were microinjected with nuclear factor erythroid 2-related factor 2 (Nrf2), AHR2, cytochrome P450 1A (CYP1A), or CYP1B1 morpholinos, and deformities were assessed. In experiment III, embryos were exposed to TPM, and embryos/larvae were collected at 24, 48, 72, and 96 hpf to assess several genes associated with the antioxidant and AHR pathways. Lastly, experiment IV assessed the activity and protein levels of CYP1A and CYP1B1 after exposure to TPM. We demonstrate that the incidence of TPM-induced deformities was generally not affected by NAC/BSO treatments or Nrf2 knockdown. In contrast, AHR2 knockdown reduced, while CYP1A or CYP1B1 knockdowns elevated the incidence of some deformities. Moreover, as shown by gene expression the AHR pathway, but not the antioxidant pathway, was induced in response to TPM exposure, providing further evidence for its importance in mediating TPM toxicity. - Highlights: • Total particulate matter (TPM) is the particulate phase of cigarette smoke. • Zebrafish is proposed as a ‘bridge model’ to study the effects of TPM. • We investigate the roles of antioxidant and aryl hydrocarbon receptor (AHR) pathways.

  6. Evidence for a Ustilago maydis Steroid 5α-Reductase by Functional Expression in Arabidopsis det2-1 Mutants1

    Science.gov (United States)

    Basse, Christoph W.; Kerschbamer, Christine; Brustmann, Markus; Altmann, Thomas; Kahmann, Regine

    2002-01-01

    We have identified a gene (udh1) in the basidiomycete Ustilago maydis that is induced during the parasitic interaction with its host plant maize (Zea mays). udh1 encodes a protein with high similarity to mammalian and plant 5α-steroid reductases. Udh1 differs from those of known 5α-steroid reductases by six additional domains, partially predicted to be membrane-spanning. A fusion protein of Udh1 and the green fluorescent protein provided evidence for endoplasmic reticulum localization in U. maydis. The function of the Udh1 protein was demonstrated by complementing Arabidopsis det2-1 mutants, which display a dwarf phenotype due to a mutation in the 5α-steroid reductase encoding DET2 gene. det2-1 mutant plants expressing either the udh1 or the DET2 gene controlled by the cauliflower mosaic virus 35S promoter differed from wild-type Columbia plants by accelerated stem growth, flower and seed development and a reduction in size and number of rosette leaves. The accelerated growth phenotype of udh1 transgenic plants was stably inherited and was favored under reduced light conditions. Truncation of the N-terminal 70 amino acids of the Udh1 protein abolished the ability to restore growth in det2-1 plants. Our results demonstrate the existence of a 5α-steroid reductase encoding gene in fungi and suggest a common ancestor between fungal, plant, and mammalian proteins. PMID:12068114

  7. Evidence that the intra-amoebal Legionella drancourtii acquired a sterol reductase gene from eukaryotes

    Directory of Open Access Journals (Sweden)

    Fournier Pierre-Edouard

    2009-03-01

    Full Text Available Abstract Background Free-living amoebae serve as a natural reservoir for some bacteria that have evolved into «amoeba-resistant» bacteria. Among these, some are strictly intra-amoebal, such as Candidatus "Protochlamydia amoebophila" (Candidatus "P. amoebophila", whose genomic sequence is available. We sequenced the genome of Legionella drancourtii (L. drancourtii, another recently described intra-amoebal bacterium. By comparing these two genomes with those of their closely related species, we were able to study the genetic characteristics specific to their amoebal lifestyle. Findings We identified a sterol delta-7 reductase-encoding gene common to these two bacteria and absent in their relatives. This gene encodes an enzyme which catalyses the last step of cholesterol biosynthesis in eukaryotes, and is probably functional within L. drancourtii since it is transcribed. The phylogenetic analysis of this protein suggests that it was acquired horizontally by a few bacteria from viridiplantae. This gene was also found in the Acanthamoeba polyphaga Mimivirus genome, a virus that grows in amoebae and possesses the largest viral genome known to date. Conclusion L. drancourtii acquired a sterol delta-7 reductase-encoding gene of viridiplantae origin. The most parsimonious hypothesis is that this gene was initially acquired by a Chlamydiales ancestor parasite of plants. Subsequently, its descendents transmitted this gene in amoebae to other intra-amoebal microorganisms, including L. drancourtii and Coxiella burnetii. The role of the sterol delta-7 reductase in prokaryotes is as yet unknown but we speculate that it is involved in host cholesterol parasitism.

  8. Pharmacogenetics of aldo-keto reductase 1C (AKR1C) enzymes.

    Science.gov (United States)

    Alshogran, Osama Y

    2017-10-01

    Genetic variation in metabolizing enzymes contributes to variable drug response and disease risk. Aldo-keto reductase type 1C (AKR1C) comprises a sub-family of reductase enzymes that play critical roles in the biotransformation of various drug substrates and endogenous compounds such as steroids. Several single nucleotide polymorphisms have been reported among AKR1C encoding genes, which may affect the functional expression of the enzymes. Areas covered: This review highlights and comprehensively discusses previous pharmacogenetic reports that have examined genetic variations in AKR1C and their association with disease development, drug disposition, and therapeutic outcomes. The article also provides information about the effect of AKR1C genetic variants on enzyme function in vitro. Expert opinion: The current evidence that links the effect of AKR1C gene polymorphisms to disease progression and development is inconsistent and needs further validation, despite of the tremendous knowledge available. Information about association of AKR1C genetic variants and drug efficacy, safety, and pharmacokinetics is limited, thus, future studies that advance our understanding about these relationships and their clinical relevance are needed. It is imperative to achieve consistent findings before the potential translation and adoption of AKR1C genetic variants in clinical practice.

  9. Evaluation of constitutive iron reductase (AtFRO2 expression on mineral accumulation and distribution in soybean (Glycine max. L

    Directory of Open Access Journals (Sweden)

    Marta Wilton Vasconcelos

    2014-04-01

    Full Text Available Iron is an important micronutrient in human and plant nutrition. Adequate iron nutrition during crop production is central for assuring appropriate iron concentrations in the harvestable organs, for human food or animal feed. The whole-plant movement of iron involves several processes, including the reduction of ferric to ferrous iron at several locations throughout the plant, prior to transmembrane trafficking of ferrous iron. In this study, soybean plants that constitutively expressed the AtFRO2 iron reductase gene were analyzed for leaf iron reductase activity, as well as the effect of this transgene's expression on root, leaf, pod wall, and seed mineral concentrations. High Fe supply, in combination with the constitutive expression of AtFRO2, resulted in significantly higher concentrations of different minerals in roots (K, P, Zn, Ca, Ni, Mg and Mo, pod walls (Fe, K, P, Cu and Ni, leaves (Fe, P, Cu, Ca, Ni and Mg and seeds (Fe, Zn, Cu and Ni. Leaf and pod wall iron concentrations increased as much as 500% in transgenic plants, while seed iron concentrations only increased by 10%, suggesting that factors other than leaf and pod wall reductase activity were limiting the translocation of iron to seeds. Protoplasts isolated from transgenic leaves had three-fold higher reductase activity than controls. Expression levels of the iron storage protein, ferritin, were higher in the transgenic leaves than in wild-type, suggesting that the excess iron may be stored as ferritin in the leaves and therefore unavailable for phloem loading and delivery to the seeds. Also, citrate and malate levels in the roots and leaves of transgenic plants were significantly higher than in wild-type, suggesting that organic acid production could be related to the increased accumulation of minerals in roots, leaves and pod walls, but not in the seeds. All together, these results suggest a more ubiquitous role for the iron reductase in whole-plant mineral accumulation and

  10. Purification of a NAD(P) reductase-like protein from the thermogenic appendix of the Sauromatum guttatum inflorescence.

    Science.gov (United States)

    Skubatz, Hanna; Howald, William N

    2013-03-01

    A NAD(P) reductase-like protein with a molecular mass of 34.146 ± 34 Da was purified to homogeneity from the appendix of the inflorescence of the Sauromatum guttatum. On-line liquid chromatography/electrospray ionization-mass spectrometry was used to isolate and quantify the protein. For the identification of the protein, liquid chromatography/electrospray ionization-tandem mass spectrometry analysis of tryptic digests of the protein was carried out. The acquired mass spectra were used for database searching, which led to the identification of a single tryptic peptide. The 12 amino acid tryptic peptide (FLPSEFGNDVDR) was found to be identical to amino acid residues at the positions 108-120 of isoflavone reductase in the Arabidopsis genome. A BLAST search identified this sequence region as unique and specific to a class of NAD(P)-dependent reductases involved in phenylpropanoid biosynthesis. Edman degradation revealed that the protein was N-terminally blocked. The amount of the protein (termed RL, NAD(P) reductase-like protein) increased 60-fold from D-4 (4 days before inflorescence-opening, designated as D-day) to D-Day, and declined the following day, when heat-production ceased. When salicylic acid, the endogenous trigger of heat-production in the Sauromatum appendix, was applied to premature appendices, a fivefold decrease in the amount of RL was detected in the treated section relative to the non-treated section. About 40 % of RL was found in the cytoplasm. Another 30 % was detected in Percoll-purified mitochondria and the rest, about 30 % was associated with a low speed centrifugation pellet due to nuclei and amyloplast localization. RL was also found in other thermogenic plants and detected in Arabidopsis leaves. The function of RL in thermogenic and non-thermogenic plants requires further investigation.

  11. Effect of sulfasalazine on human neuroblastoma: analysis of sepiapterin reductase (SPR) as a new therapeutic target

    International Nuclear Information System (INIS)

    Yco, Lisette P.; Geerts, Dirk; Mocz, Gabor; Koster, Jan; Bachmann, André S.

    2015-01-01

    Neuroblastoma (NB) is an aggressive childhood malignancy in children up to 5 years of age. High-stage tumors frequently relapse even after aggressive multimodal treatment, and then show therapy resistance, typically resulting in patient death. New molecular-targeted compounds that effectively suppress tumor growth and prevent relapse with more efficacy are urgently needed. We and others previously showed that polyamines (PA) like spermidine and spermine are essential for NB tumorigenesis and that DFMO, an inhibitor of the key PA synthesis gene product ODC, is effective both in vitro and in vivo, securing its evaluation in NB clinical trials. To find additional compounds interfering with PA biosynthesis, we tested sulfasalazine (SSZ), an FDA-approved salicylate-based anti-inflammatory and immune-modulatory drug, recently identified to inhibit sepiapterin reductase (SPR). We earlier presented evidence for a physical interaction between ODC and SPR and we showed that RNAi-mediated knockdown of SPR expression significantly reduced native ODC enzyme activity and impeded NB cell proliferation. Human NB mRNA expression datasets in the public domain were analyzed using the R2 platform. Cell viability, isobologram, and combination index analyses as a result of SSZ treatment with our without DFMO were carried out in NB cell cultures. Molecular protein-ligand docking was achieved using the GRAMM algorithm. Statistical analyses were performed with the Kruskal-Wallis test, 2log Pearson test, and Student’s t test. In this study, we show the clinical relevance of SPR in human NB tumors. We found that high SPR expression is significantly correlated to unfavorable NB characteristics like high age at diagnosis, MYCN amplification, and high INSS stage. SSZ inhibits the growth of NB cells in vitro, presumably due to the inhibition of SPR as predicted by computational docking of SSZ into SPR. Importantly, the combination of SSZ with DFMO produces synergistic antiproliferative effects

  12. Effect of sulfasalazine on human neuroblastoma: analysis of sepiapterin reductase (SPR) as a new therapeutic target.

    Science.gov (United States)

    Yco, Lisette P; Geerts, Dirk; Mocz, Gabor; Koster, Jan; Bachmann, André S

    2015-06-21

    Neuroblastoma (NB) is an aggressive childhood malignancy in children up to 5 years of age. High-stage tumors frequently relapse even after aggressive multimodal treatment, and then show therapy resistance, typically resulting in patient death. New molecular-targeted compounds that effectively suppress tumor growth and prevent relapse with more efficacy are urgently needed. We and others previously showed that polyamines (PA) like spermidine and spermine are essential for NB tumorigenesis and that DFMO, an inhibitor of the key PA synthesis gene product ODC, is effective both in vitro and in vivo, securing its evaluation in NB clinical trials. To find additional compounds interfering with PA biosynthesis, we tested sulfasalazine (SSZ), an FDA-approved salicylate-based anti-inflammatory and immune-modulatory drug, recently identified to inhibit sepiapterin reductase (SPR). We earlier presented evidence for a physical interaction between ODC and SPR and we showed that RNAi-mediated knockdown of SPR expression significantly reduced native ODC enzyme activity and impeded NB cell proliferation. Human NB mRNA expression datasets in the public domain were analyzed using the R2 platform. Cell viability, isobologram, and combination index analyses as a result of SSZ treatment with our without DFMO were carried out in NB cell cultures. Molecular protein-ligand docking was achieved using the GRAMM algorithm. Statistical analyses were performed with the Kruskal-Wallis test, 2log Pearson test, and Student's t test. In this study, we show the clinical relevance of SPR in human NB tumors. We found that high SPR expression is significantly correlated to unfavorable NB characteristics like high age at diagnosis, MYCN amplification, and high INSS stage. SSZ inhibits the growth of NB cells in vitro, presumably due to the inhibition of SPR as predicted by computational docking of SSZ into SPR. Importantly, the combination of SSZ with DFMO produces synergistic antiproliferative effects

  13. CXCL5 knockdown expression inhibits human bladder cancer T24 cells proliferation and migration

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Jiajia [Department of Laboratory Medicine, Peking University Third Hospital, Beijing (China); Zhu, Xi [Department of Urology, Beijing Friendship Hospital Affiliated to Capital Medical University, Beijing (China); Zhang, Jie, E-mail: zhangjiebjmu@163.com [Department of Laboratory Medicine, Peking University Third Hospital, Beijing (China)

    2014-03-28

    Highlights: • We first demonstrated CXCL5 is highly expressed in human bladder tumor tissues and cells. • CXCL5 knockdown inhibits proliferation, migration and promotes apoptosis in T24 cells. • CXCL5 knockdown inhibits Snail, PI3K-AKT and ERK1/2 signaling pathways in T24 cells. • CXCL5 is critical for bladder tumor growth and progression. - Abstract: CXCL5 (epithelial neutrophil activating peptide-78) which acts as a potent chemoattractant and activator of neutrophil function was reported to play a multifaceted role in tumorigenesis. To investigate the role of CXCL5 in bladder cancer progression, we examined the CXCL5 expression in bladder cancer tissues by real-time PCR and Western blot, additionally, we used shRNA-mediated silencing to generate stable CXCL5 silenced bladder cancer T24 cells and defined its biological functions. Our results demonstrated that mRNA and protein of CXCL5 is increased in human bladder tumor tissues and cell lines, down-regulation of CXCL5 in T24 cells resulted in significantly decreased cell proliferation, migration and increased cell apoptosis in vitro through Snail, PI3K-AKT and ERK1/2 signaling pathways. These data suggest that CXCL5 is critical for bladder tumor growth and progression, it may represent a potential application in cancer diagnosis and therapy.

  14. CXCL5 knockdown expression inhibits human bladder cancer T24 cells proliferation and migration

    International Nuclear Information System (INIS)

    Zheng, Jiajia; Zhu, Xi; Zhang, Jie

    2014-01-01

    Highlights: • We first demonstrated CXCL5 is highly expressed in human bladder tumor tissues and cells. • CXCL5 knockdown inhibits proliferation, migration and promotes apoptosis in T24 cells. • CXCL5 knockdown inhibits Snail, PI3K-AKT and ERK1/2 signaling pathways in T24 cells. • CXCL5 is critical for bladder tumor growth and progression. - Abstract: CXCL5 (epithelial neutrophil activating peptide-78) which acts as a potent chemoattractant and activator of neutrophil function was reported to play a multifaceted role in tumorigenesis. To investigate the role of CXCL5 in bladder cancer progression, we examined the CXCL5 expression in bladder cancer tissues by real-time PCR and Western blot, additionally, we used shRNA-mediated silencing to generate stable CXCL5 silenced bladder cancer T24 cells and defined its biological functions. Our results demonstrated that mRNA and protein of CXCL5 is increased in human bladder tumor tissues and cell lines, down-regulation of CXCL5 in T24 cells resulted in significantly decreased cell proliferation, migration and increased cell apoptosis in vitro through Snail, PI3K-AKT and ERK1/2 signaling pathways. These data suggest that CXCL5 is critical for bladder tumor growth and progression, it may represent a potential application in cancer diagnosis and therapy

  15. Sex hormones reduce NNK detoxification through inhibition of short-chain dehydrogenases/reductases and aldo-keto reductases in vitro.

    Science.gov (United States)

    Stapelfeld, Claudia; Maser, Edmund

    2017-10-01

    Carbonyl reduction is an important metabolic pathway for endogenous and xenobiotic substances. The tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, nicotine-derived nitrosamine ketone) is classified as carcinogenic to humans (IARC, Group 1) and considered to play the most important role in tobacco-related lung carcinogenesis. Detoxification of NNK through carbonyl reduction is catalyzed by members of the AKR- and the SDR-superfamilies which include AKR1B10, AKR1C1, AKR1C2, AKR1C4, 11β-HSD1 and CBR1. Because some reductases are also involved in steroid metabolism, five different hormones were tested for their inhibitory effect on NNK carbonyl reduction. Two of those hormones were estrogens (estradiol and ethinylestradiol), another two hormones belong to the gestagen group (progesterone and drospirenone) and the last tested hormone was an androgen (testosterone). Furthermore, one of the estrogens (ethinylestradiol) and one of the gestagens (drospirenone) are synthetic hormones, used as hormonal contraceptives. Five of six NNK reducing enzymes (AKR1B10, AKR1C1, AKR1C2, AKR1C4 and 11β-HSD1) were significantly inhibited by the tested sex hormones. Only NNK reduction catalyzed by CBR1 was not significantly impaired. In the case of the other five reductases, gestagens had remarkably stronger inhibitory effects at a concentration of 25 μM (progesterone: 66-88% inhibition; drospirenone: 26-87% inhibition) in comparison to estrogens (estradiol: 17-51% inhibition; ethinylestradiol: 14-79% inhibition) and androgens (14-78% inhibition). Moreover, in most cases the synthetic hormones showed a greater ability to inhibit NNK reduction than the physiologic derivatives. These results demonstrate that male and female sex hormones have different inhibitory potentials, thus indicating that there is a varying detoxification capacity of NNK in men and women which could result in a different risk for developing lung cancer. Copyright © 2017 Elsevier B

  16. Short-hairpin Mediated Myostatin Knockdown Resulted in Altered Expression of Myogenic Regulatory Factors with Enhanced Myoblast Proliferation in Fetal Myoblast Cells of Goats.

    Science.gov (United States)

    Kumar, Rohit; Singh, Satyendra Pal; Mitra, Abhijit

    2018-01-02

    Myostatin (MSTN) is a well-known negative regulator of skeletal muscle development. Reduced expression due to natural mutations in the coding region and knockout as well as knockdown of MSTN results in an increase in the muscle mass. In the present study, we demonstrated as high as 60 and 52% downregulation (p < 0.01) of MSTN mRNA and protein in the primary fetal myoblast cells of goats using synthetic shRNAs (n = 3), without any interferon response. We, for the first time, evaluated the effect of MSTN knockdown on the expression of MRFs (namely, MyoD, Myf5), follistatin (FST), and IGFs (IGF-1 & IGF-2) in goat myoblast cells. MSTN knockdown caused an upregulation (p < 0.05) of MyoD and downregulation (p < 0.01) of MYf5 and FST expression. Moreover, we report up to ∼four fold (p < 0.001) enhanced proliferation in myoblasts after four days of culture. The anti-MSTN shRNA demonstrated in the present study could be used for the production of transgenic goats to increase the muscle mass.

  17. Direct antioxidant properties of bilirubin andbiliverdin. Is there a role for biliverdin reductase?

    Directory of Open Access Journals (Sweden)

    Thomas eJansen

    2012-03-01

    Full Text Available Reactive oxygen species (ROS and signaling events are involved in the pathogenesis of endothelial dysfunction and represent a major contribution to vascular regulation. Molecular signaling is highly dependent on reactive oxygen species. But depending on the amount of ROS production it might have toxic or protective effects. Despite a large number of negative outcomes in large clinical trials (e.g. HOPE, HOPE-TOO, antioxidant molecules and agents are important players to influence the critical balance between production and elimination of RONS. However, chronic systemic antioxidant therapy lacks clinical efficacy, probably by interfering with important physiological redox signaling pathways. Therefore, it may be a much more promising attempt to induce intrinsic antioxidant pathways in order to increase the antioxidants not systemically but at the place of oxidative stress and complications. Among others, heme oxygenase (HO has been shown to be important for attenuating the overall production of ROS in a broad range of disease states through its ability to degrade heme and to produce carbon monoxide (CO, biliverdin/bilirubin, and the release of free iron with subsequent ferritin induction. With the present review we would like to highlight the important antioxidant role of the heme oxygenase system and especially discuss the contribution of the biliverdin, bilirubin and biliverdin reductase to these beneficial effects. The bilierdin reductase was reported to confer an antioxidant redox amplification cycle by which low, physiological bilirubin concentrations confer potent antioxidant protection via recycling of biliverdin from oxidized bilirubin by the biliverdin reductase, linking this sink for oxidants to the NADPH pool. To date the existence and role of this antioxidant redox cycle is still under debate and we present and discuss the pros and cons as well as our own findings on this topic.

  18. Identification of HMG-CoA Reductase Inhibitor Active Compound in Medicinal Forest Plants

    Directory of Open Access Journals (Sweden)

    Shelly Rahmania

    2017-08-01

    Full Text Available Cardiovascular disease is a leading cause of death worldwide, hypercholesterolemia is one of the causes. Three medicinal forest plants are potential natural resources to be developed as cholesterol-reducing herbal product, but scientific informations on their mechanism is still limited. The objective of this research is to explore the potency of the leaf of Jati Belanda (Guazuma ulmifolia, Jabon (Antocephalus macrophyllus, and Mindi (Melia azedarach as inhibitor of HMG-CoA reductase (HMGR, a key enzyme in the regulation of cholesterol biosynthesis. Samples were macerated in ethanol 96% and the filtrate was partitioned using n-hexane and chloroform to obtain the ethanolic flavonoid extract. The effect of each extracts on the HMG-CoA reductase activity were analyzed using HMGR assay kit. At concentration of 10 ppm the G.ulmifolia ethanolic extract showed the highest inhibitory activity as well as pravastatin control inhibitor.  The phenolic content of the ethanolic extracts of G.ulmifolia, A.macrophyllus, and M.azedarach were: 11.00, 34.83, and 13.67 mg gallic acid AE/g dried leaves, respectively. The flavonoid content of the ethanolic extracts of G.ulmifolia, A.macrophyllus, and M.azedarach were: 0.22, 0.64, and 0.78 mg QE/g dried leaves, respectively. Interestingly, G.ulmifolia extract the lowest concentration of phenolic and flavonoid content. HPLC analysis showed that all samples contain quercetin at similiar small concentrations (6.7%, 6.6%, and 7.0% for G.ulmifolia, A.macrophyllus, and M.azedarach, respectively. This indicating other active compounds may play some roles in this inhibitory action on HMG-CoA reductase activity. Further identification using LC-MS/MS showed that G.ulmifolia flavonoid extract contained an unidetified coumpound with molecural weight of 380.0723 Da.  

  19. Kinetic properties and inhibition of Trypanosoma cruzi 3-hydroxy-3-methylglutaryl CoA reductase

    DEFF Research Database (Denmark)

    Hurtado-Guerrrero, Ramón; Pena Diaz, Javier; Montalvetti, Andrea

    2002-01-01

    A detailed kinetic analysis of the recombinant soluble enzyme 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) from Trypanosoma cruzi has been performed. The enzyme catalyzes the normal anabolic reaction and the reductant is NADPH. It also catalyzes the oxidation of mevalonate but at a lower propo...

  20. A soluble 3-hydroxy-3-methylglutaryl-CoA reductase in the protozoan Trypanosoma cruzi

    DEFF Research Database (Denmark)

    Pena Diaz, Javier; Montalvetti, A; Camacho, A

    1997-01-01

    of the genes described from eukaryotic organisms and the deduced amino acid sequence could be aligned with the C-terminal half of animal and plant reductases exhibiting pronounced similarity to other eukaryotic counterparts. Further examination of the 5' flanking region by cDNA analysis and establishment...

  1. Survival and Psychomotor Development With Early Betaine Treatment in Patients With Severe Methylenetetrahydrofolate Reductase Deficiency

    NARCIS (Netherlands)

    Diekman, Eugene F.; de Koning, Tom J.; Verhoeven-Duif, Nanda M.; Rovers, Maroeska M.; van Hasselt, Peter M.

    IMPORTANCE The impact of betaine treatment on outcome in patients with severe methylenetetrahydrofolate reductase (MTHFR) deficiency is presently unclear. OBJECTIVE To investigate the effect of betaine treatment on development and survival in patients with severe MTHFR deficiency. DATA SOURCES

  2. Survival and psychomotor development with early betaine treatment in patients with severe methylenetetrahydrofolate reductase deficiency

    NARCIS (Netherlands)

    Diekman, E.F.; Koning, T.J. de; Verhoeven-Duif, N.M.; Rovers, M.M.; Hasselt, P.M. van

    2014-01-01

    IMPORTANCE The impact of betaine treatment on outcome in patients with severe methylenetetrahydrofolate reductase (MTHFR) deficiency is presently unclear. OBJECTIVE To investigate the effect of betaine treatment on development and survival in patients with severe MTHFR deficiency. DATA SOURCES

  3. STARD4 knockdown in HepG2 cells disrupts cholesterol trafficking associated with the plasma membrane, ER, and ERC

    DEFF Research Database (Denmark)

    Garbarino, J.; Pan, M. H.; Chin, H. F.

    2012-01-01

    small hairpin RNA knockdown technology to reduce STARD4 expression in HepG2 cells. In a cholesterol-poor environment, we found that a reduction in STARD4 expression leads to retention of cholesterol at the plasma membrane, reduction of endoplasmic reticulum-associated cholesterol, and decreased ACAT...... synthesized cholesteryl esters. Furthermore, D4 KD cells exhibited a reduced rate of sterol transport to the endocytic recycling compartment after cholesterol repletion. Although these cells displayed normal endocytic trafficking in cholesterol-poor and replete conditions, cell surface low density lipoprotein...... membrane and the endocytic recycling compartment to the endoplasmic reticulum and perhaps other intracellular compartments as well. -Garbarino, J., M. Pan, H.F. Chin, F.W. Lund, F.R. Maxfield, and J.L. Breslow. STARD4 knockdown in HepG2 cells disrupts cholesterol trafficking associated with the plasma...

  4. The Inhibitory Effect of Prunella vulgaris L. on Aldose Reductase and Protein Glycation

    Directory of Open Access Journals (Sweden)

    Hong Mei Li

    2012-01-01

    Full Text Available To evaluate the aldose reductase (AR enzyme inhibitory ability of Prunella vulgaris L. extract, six compounds were isolated and tested for their effects. The components were subjected to in vitro bioassays to investigate their inhibitory assays using rat lens aldose reductase (rAR and human recombinant AR (rhAR. Among them, caffeic acid ethylene ester showed the potent inhibition, with the IC50 values of rAR and rhAR at 3.2±0.55 μM and 12.58±0.32 μM, respectively. In the kinetic analyses using Lineweaver-Burk plots of 1/velocity and 1/concentration of substrate, this compound showed noncompetitive inhibition against rhAR. Furthermore, it inhibited galactitol formation in a rat lens incubated with a high concentration of galactose. Also it has antioxidative as well as advanced glycation end products (AGEs inhibitory effects. As a result, this compound could be offered as a leading compound for further study as a new natural products drug for diabetic complications.

  5. Structure and biocatalytic scope of thermophilic flavin-dependent halogenase and flavin reductase enzymes.

    Science.gov (United States)

    Menon, Binuraj R K; Latham, Jonathan; Dunstan, Mark S; Brandenburger, Eileen; Klemstein, Ulrike; Leys, David; Karthikeyan, Chinnan; Greaney, Michael F; Shepherd, Sarah A; Micklefield, Jason

    2016-10-04

    Flavin-dependent halogenase (Fl-Hal) enzymes have been shown to halogenate a range of synthetic as well as natural aromatic compounds. The exquisite regioselectively of Fl-Hal enzymes can provide halogenated building blocks which are inaccessible using standard halogenation chemistries. Consequently, Fl-Hal are potentially useful biocatalysts for the chemoenzymatic synthesis of pharmaceuticals and other valuable products, which are derived from haloaromatic precursors. However, the application of Fl-Hal enzymes, in vitro, has been hampered by their poor catalytic activity and lack of stability. To overcome these issues, we identified a thermophilic tryptophan halogenase (Th-Hal), which has significantly improved catalytic activity and stability, compared with other Fl-Hal characterised to date. When used in combination with a thermostable flavin reductase, Th-Hal can efficiently halogenate a number of aromatic substrates. X-ray crystal structures of Th-Hal, and the reductase partner (Th-Fre), provide insights into the factors that contribute to enzyme stability, which could guide the discovery and engineering of more robust and productive halogenase biocatalysts.

  6. The Cholesterol-Lowering Effect of Alisol Acetates Based on HMG-CoA Reductase and Its Molecular Mechanism

    Directory of Open Access Journals (Sweden)

    Fei Xu

    2016-01-01

    Full Text Available This study measured the impact of alisol B 23-acetate and alisol A 24-acetate, the main active ingredients of the traditional Chinese medicine Alismatis rhizoma, on total cholesterol (TC, triglyceride (TG, high density lipoprotein-cholesterol (HDL-C, and low density lipoprotein-cholesterol (LDL-C levels of hyperlipidemic mice. The binding of alisol B 23-acetate and alisol A 24-acetate to the key enzyme involved in the metabolism of TC, 3-hydroxy-3-methylglutary-coenzyme A (HMG-CoA reductase, was studied using the reagent kit method and the western blotting technique combined with a molecular simulation technique. According to the results, alisol acetates significantly lower the TC, TG, and LDL-C concentrations of hyperlipidemic mice, while raising HDL-C concentrations. Alisol acetates lower HMG-CoA reductase activity in a dose-dependent fashion, both in vivo and in vitro. Neither of these alisol acetates significantly lower the protein expression of HMG-CoA. This suggests that alisol acetates lower the TC level via inhibiting the activity of HMG-CoA reductase by its prototype drug, which may exhibit an inhibition effect via directly and competitively binding to HMG-CoA. The side chain of the alisol acetate was the steering group via molecular simulation.

  7. Low activity of superoxide dismutase and high activity of glutathione reductase in erythrocytes from centenarians

    DEFF Research Database (Denmark)

    Andersen, Helle Raun; Jeune, B; Nybo, H

    1998-01-01

    aged between 60 and 79 years. MEASUREMENTS: enzyme activities of superoxide dismutase (CuZn-SOD), glutathione peroxidase, catalase and glutathione reductase (GR) in erythrocytes. Functional capacity among the centenarians was evaluated by Katz' index of activities of daily living, the Physical...

  8. Peroxo-Type Intermediates in Class I Ribonucleotide Reductase and Related Binuclear Non-Heme Iron Enzymes

    DEFF Research Database (Denmark)

    Kepp, Kasper Planeta; Bell, Caleb B.; Clay, MIchael D.

    2009-01-01

    We have performed a systematic study of chemically possible peroxo-type intermediates occurring in the non-heme di-iron enzyme class la ribonucleotide reductase, using spectroscopically calibrated computational chemistry. Density functional computations of equilibrium structures, Fe-O and O-O str...

  9. Knockdown of BAG3 sensitizes bladder cancer cells to treatment with the BH3 mimetic ABT-737.

    Science.gov (United States)

    Mani, Jens; Antonietti, Patrick; Rakel, Stefanie; Blaheta, Roman; Bartsch, Georg; Haferkamp, Axel; Kögel, Donat

    2016-02-01

    BAG3 is overexpressed in several malignancies and mediates a non-canonical, selective form of (macro)autophagy. By stabilizing pro-survival Bcl-2 proteins in complex with HSP70, BAG3 can also exert an apoptosis-antagonizing function. ABT-737 is a high affinity Bcl-2 inhibitor that fails to target Mcl-1. This failure may confer resistance in various cancers. Urothelial cancer cells were treated with the BH3 mimetics ABT-737 and (-)-gossypol, a pan-Bcl-2 inhibitor which inhibits also Mcl-1. To clarify the importance of the core autophagy regulator ATG5 and BAG3 in ABT-737 treatment, cell lines carrying a stable lentiviral knockdown of ATG5 and BAG3 were created. The synergistic effect of ABT-737 and pharmaceutical inhibition of BAG3 with the HSF1 inhibitor KRIBB11 or sorafenib was also evaluated. Total cell death and apoptosis were quantified by FACS analysis of propidium iodide, annexin. Target protein analysis was conducted by Western blotting. Knockdown of BAG3 significantly downregulated Mcl-1 protein levels and sensitized urothelial cancer cells to apoptotic cell death induced by ABT-737, while inhibition of bulk autophagy through depletion of ATG5 had no discernible effect on cell death. Similar to knockdown of BAG3, pharmacological targeting of the BAG3/Mcl-1 pathway with KRIBB11 was capable to sensitize both cell lines to treatment with ABT-737. Our results show that BAG3, but not bulk autophagy has a major role in the response of bladder cancer cells to BH3 mimetics. They also suggest that BAG3 is a suitable target for combined therapies aimed at synergistically inducing apoptosis in bladder cancer.

  10. [Knockdown of PRDX6 in microglia reduces neuron viability after OGD/R injury].

    Science.gov (United States)

    Tan, Li; Zhao, Yong; Jiang, Beibei; Yang, Bo; Zhang, Hui

    2016-08-01

    Objective To observe the effects of peroxiredoxin 6 (PRDX6) knockdown in the microglia on neuron viability after oxygen-glucose deprivation and reoxygenation (OGD/R). Methods Microglia was treated with lentivirus PRDX6-siRNA and Ca(2+)-independent phospholipase A2 (iPLA2) inhibitor, 1-hexadecyl-3-(trifluoroethgl)-sn-glycerol-2 phosphomethanol (MJ33). Twenty-four hours later, it was co-cultured with primary neuron to establish the microglia-neuron co-culture OGD/R model. According to the different treatment of microglia, the cells were divided into normal group, OGD/R group, negative control-siRNA treated OGD/R group, PRDX6-siRNA treated OGD/R group and PRDX6-siRNA combined with MJ33 treated OGD/R group. Western blot analysis and real-time quantitative PCR were respectively performed to detect PRDX6 protein and mRNA levels after knockdown of PRDX6 in microglia. The iPLA2 activity was measured by ELISA. MTS and lactate dehydrogenase (LDH) assay were used to measure neuron viability and cell damage. The oxidative stress level of neuron was determined by measuring superoxide dismutase (SOD) and malonaldehyde (MDA) content. Results In PRDX6-siRNA group, neuron viability was inhibited and oxidative stress damage was aggravated compared with OGD/R group. In PRDX6-siRNA combined with MJ33 group, cell viability was promoted and oxidative stress damage was alleviated compared with PRDX6-siRNA group. Conclusion PRDX6 in microglia protects neuron against OGD/R-induced injury, and iPLA2 activity has an effect on PRDX6.

  11. Induction of the Nitrate Assimilation nirA Operon and Protein-Protein Interactions in the Maturation of Nitrate and Nitrite Reductases in the Cyanobacterium Anabaena sp. Strain PCC 7120.

    Science.gov (United States)

    Frías, José E; Flores, Enrique

    2015-07-01

    Nitrate is widely used as a nitrogen source by cyanobacteria, in which the nitrate assimilation structural genes frequently constitute the so-called nirA operon. This operon contains the genes encoding nitrite reductase (nirA), a nitrate/nitrite transporter (frequently an ABC-type transporter; nrtABCD), and nitrate reductase (narB). In the model filamentous cyanobacterium Anabaena sp. strain PCC 7120, which can fix N2 in specialized cells termed heterocysts, the nirA operon is expressed at high levels only in media containing nitrate or nitrite and lacking ammonium, a preferred nitrogen source. Here we examined the genes downstream of the nirA operon in Anabaena and found that a small open reading frame of unknown function, alr0613, can be cotranscribed with the operon. The next gene in the genome, alr0614 (narM), showed an expression pattern similar to that of the nirA operon, implying correlated expression of narM and the operon. A mutant of narM with an insertion mutation failed to produce nitrate reductase activity, consistent with the idea that NarM is required for the maturation of NarB. Both narM and narB mutants were impaired in the nitrate-dependent induction of the nirA operon, suggesting that nitrite is an inducer of the operon in Anabaena. It has previously been shown that the nitrite reductase protein NirA requires NirB, a protein likely involved in protein-protein interactions, to attain maximum activity. Bacterial two-hybrid analysis confirmed possible NirA-NirB and NarB-NarM interactions, suggesting that the development of both nitrite reductase and nitrate reductase activities in cyanobacteria involves physical interaction of the corresponding enzymes with their cognate partners, NirB and NarM, respectively. Nitrate is an important source of nitrogen for many microorganisms that is utilized through the nitrate assimilation system, which includes nitrate/nitrite membrane transporters and the nitrate and nitrite reductases. Many cyanobacteria

  12. Carbohydrate restriction and dietary cholesterol modulate the expression of HMG-CoA reductase and the LDL receptor in mononuclear cells from adult men

    Directory of Open Access Journals (Sweden)

    Volek Jeff S

    2007-11-01

    Full Text Available Abstract The liver is responsible for controlling cholesterol homeostasis in the body. HMG-CoA reductase and the LDL receptor (LDL-r are involved in this regulation and are also ubiquitously expressed in all major tissues. We have previously shown in guinea pigs that there is a correlation in gene expression of HMG-CoA reductase and the LDL-r between liver and mononuclear cells. The present study evaluated human mononuclear cells as a surrogate for hepatic expression of these genes. The purpose was to evaluate the effect of dietary carbohydrate restriction with low and high cholesterol content on HMG-CoA reductase and LDL-r mRNA expression in mononuclear cells. All subjects were counseled to consume a carbohydrate restricted diet with 10–15% energy from carbohydrate, 30–35% energy from protein and 55–60% energy from fat. Subjects were randomly assigned to either EGG (640 mg/d additional dietary cholesterol or SUB groups [equivalent amount of egg substitute (0 dietary cholesterol contributions per day] for 12 weeks. At the end of the intervention, there were no changes in plasma total or LDL cholesterol (LDL-C compared to baseline (P > 0.10 or differences in plasma total or LDL-C between groups. The mRNA abundance for HMG-CoA reductase and LDL-r were measured in mononuclear cells using real time PCR. The EGG group showed a significant decrease in HMG-CoA reductase mRNA (1.98 ± 1.26 to 1.32 ± 0.92 arbitrary units P

  13. Cdna cloning and expression analyses of the isoflavone reductase-like gene of dendrobium officinale

    International Nuclear Information System (INIS)

    Qian, X.; Xu, S.Z.

    2015-01-01

    The full length of the isoflavone reductase-like gene (IRL) cDNA of Dendrobium officinale was cloned by using reverse transcription (RT) PCR combined with cDNA library, the IRL function was identified by Bioinformatics and prokaryotic expression analyses, and the IRL expression levels in the organs and tissues of D. officinale plants with different ages were determined by using real-time quantitative PCR (RT-qPCR). The results indicated that the full length of the cDNA of D. officinale IRL, DoIRL, was 1238 bp (accession no. KJ661023). Its open reading frame (ORF) was 930 bp which encoded 309 amino acids with a predicted molecular mass of 34 kDa, the 5 untranslated region (UTR) was 61 bp and the 3 UTR containing a poly (A) tail was 247 bp. The deduced amino acid sequence of DoIRL, DoIRL, was forecast to contain a NAD(P)H-binding motif (GGTGYIG) in the N-terminal region, two conserved N-glycosylation sites, a conserved nitrogen metabolite repression regulator (NmrA) domain and a phenylcoumaran benzylic ether reductase (PCBER) domain, to hold the nearest phylogenetic relationship with the PCBER of Striga asiatica, and to share both 73% identity with the isoflavone reductases-like (IRLs) of Cucumis sativus and Striga asiatica. In Escherichia coli 'BL21' cells, the DoIRL cDNA expression produced a protein band holding the predicted molecular mass of 34 kDa. DoIRL expressed in all organs and tissues of D. officinale plants with different ages at comparatively low levels, and the expression level in the leaves of the two-year-old plants was the highest. (author)

  14. Caracemide, a site-specific irreversible inhibitor of protein R1 of Escherichia coli ribonucleotide reductase

    DEFF Research Database (Denmark)

    Larsen, I. K.; Cornett, Claus; Karlsson, M.

    1992-01-01

    The anticancer drug caracemide, N-acetyl-N,O-di(methylcarbamoyl)hydroxylamine, and one of its degradation products, N-acetyl-O-methylcarbamoyl-hydroxylamine, were found to inhibit the enzyme ribonucleotide reductase of Escherichia coli by specific interaction with its larger component protein R1....

  15. Structure and expression of human dihydropteridine reductase

    International Nuclear Information System (INIS)

    Lockyer, J.; Cook, R.G.; Milstien, S.; Kaufman, S.; Woo, S.L.C.; Ledley, F.D.

    1987-01-01

    Dihydropteridine reductase catalyzes the NADH-mediated reduction of quinonoid dihydrobiopterin and is an essential component of the pterindependent aromatic amino acid hydroxylating systems. A cDNA for human DHPR was isolated from a human liver cDNA library in the vector λgt11 using a monospecific antibody against sheep DHPR. The nucleic acid sequence and amino acid sequence of human DHPR were determined from a full-length clone. A 112 amino acid sequence of sheep DHPR was obtained by sequencing purified sheep DHPR. This sequence is highly homologous to the predicted amino acid sequence of the human protein. Gene transfer of the recombinant human DHPR into COS cells leads to expression of DHPR enzymatic activity. These results indicate that the cDNA clone identified by antibody screening is an authentic and full-length cDNA for human DHPR

  16. Monodehydroascorbate reductase mediates TNT toxicity in plants.

    Science.gov (United States)

    Johnston, Emily J; Rylott, Elizabeth L; Beynon, Emily; Lorenz, Astrid; Chechik, Victor; Bruce, Neil C

    2015-09-04

    The explosive 2,4,6-trinitrotoluene (TNT) is a highly toxic and persistent environmental pollutant. Due to the scale of affected areas, one of the most cost-effective and environmentally friendly means of removing explosives pollution could be the use of plants. However, mechanisms of TNT phytotoxicity have been elusive. Here, we reveal that phytotoxicity is caused by reduction of TNT in the mitochondria, forming a nitro radical that reacts with atmospheric oxygen, generating reactive superoxide. The reaction is catalyzed by monodehydroascorbate reductase 6 (MDHAR6), with Arabidopsis deficient in MDHAR6 displaying enhanced TNT tolerance. This discovery will contribute toward the remediation of contaminated sites. Moreover, in an environment of increasing herbicide resistance, with a shortage in new herbicide classes, our findings reveal MDHAR6 as a valuable plant-specific target. Copyright © 2015, American Association for the Advancement of Science.

  17. Sertoli cell specific knockdown of RAR-related orphan receptor (ROR) alpha at puberty reduces sperm count in rats.

    Science.gov (United States)

    Mandal, Kamal; Sarkar, Rajesh K; Sen Sharma, Souvik; Jain, Ayushi; Majumdar, Subeer S

    2018-01-30

    Globally, there is an alarming decline in sperm count. Very often hormonal supplementation fails to restore normal sperm count. Sertoli cells (Sc) present within seminiferous tubules provide appropriate niche and factors required for the differentiation of germ cells (Gc) into mature sperm (spermatogenesis). Functionally compromised Sc may be one of the reasons for failure of hormones to facilitate normal spermatogenesis. Although role of secretory proteins and signaling molecules of Sc has been studied well, role of transcription factors regulating sperm count has not been addressed appropriately. Retinoic acid receptor-related orphan receptor (ROR)-alpha is one of such transcription factors reported in testis but its role in testicular function is not yet known. In a separate study, we found abundant ROR-alpha binding sites on promoter regions of several genes upregulated in pubertal rat Sc as compared to infant Sc. Immunostaining studies also revealed presence of ROR alpha in nucleus of pubertal Sc. We generated a transgenic knockdown rat model expressing shRNA targeted to ROR-alpha under Sc specific promoter, which is transcriptionally active only at and after puberty. ROR-alpha knockdown animals were found to have abnormal association of Sc and Gc, including Gc sloughing and restricted release of sperm. The knockdown animals displayed compromised spermatogenesis leading to significant reduction in sperm count. This is the first report describing the Sc specific role of ROR-alpha in maintaining quantitatively normal sperm output. Identification of various such molecules can generate avenues to limit or reverse an alarmingly declining sperm count witnessed globally in men. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Knockdown of the fat mass and obesity gene disrupts cellular energy balance in a cell-type specific manner.

    Directory of Open Access Journals (Sweden)

    Ryan T Pitman

    Full Text Available Recent studies suggest that FTO variants strongly correlate with obesity and mainly influence energy intake with little effect on the basal metabolic rate. We suggest that FTO influences eating behavior by modulating intracellular energy levels and downstream signaling mechanisms which control energy intake and metabolism. Since FTO plays a particularly important role in adipocytes and in hypothalamic neurons, SH-SY5Y neuronal cells and 3T3-L1 adipocytes were used to understand how siRNA mediated knockdown of FTO expression alters cellular energy homeostasis. Cellular energy status was evaluated by measuring ATP levels using a luminescence assay and uptake of fluorescent glucose. FTO siRNA in SH-SY5Y cells mediated mRNA knockdown (-82%, increased ATP concentrations by up to 46% (P = 0.013 compared to controls, and decreased phosphorylation of AMPk and Akt in SH-SY5Y by -52% and -46% respectively as seen by immunoblotting. In contrast, FTO siRNA in 3T3-L1 cells decreased ATP concentration by -93% (p<0.0005, and increased AMPk and Akt phosphorylation by 204% and 70%, respectively suggesting that FTO mediates control of energy levels in a cell-type specific manner. Furthermore, glucose uptake was decreased in both SH-SY5Y (-51% p = 0.015 and 3T3-L1 cells (-30%, p = 0.0002. We also show that FTO knockdown decreases NPY mRNA expression in SH-SY5Y cells (-21% through upregulation of pSTAT3 (118%. These results provide important evidence that FTO-variant linked obesity may be associated with altered metabolic functions through activation of downstream metabolic mediators including AMPk.

  19. 14CO2-fixation and nitrate reductase activity in vivo in relation to hybrid vigour in maize

    International Nuclear Information System (INIS)

    Balasubramanian, V.; Shanthakumari, P.; Sinha, S.K.

    1977-01-01

    Dry matter accumulation in maize shoots, leaf area, 14 CO 2 -fixation and nitrate reductase activity in vivo were measured in the field grown heterotic hybrid CM 400x CM 300 and its inbred parents CM 300 and CM 400 from seedling to maturity. Rates of dry matter accumulation and leaf area development were higher in the hybrid during the initial vegetative phase than in the inbreds. The hybrid had more absolute level of 14 CO 2 -fixation and nitrate reductase activity, although the rates of these processes on unit weight basis were not higher than those of inbreds. It is concluded that the rapid development of leaf area in hybrids during the early stages of vegetative growth is probably important for hybrid vigour. (author)

  20. Role of a novel dual flavin reductase (NR1) and an associated histidine triad protein (DCS-1) in menadione-induced cytotoxicity

    International Nuclear Information System (INIS)

    Kwasnicka-Crawford, Dorota A.; Vincent, Steven R.

    2005-01-01

    Microsomal cytochrome P450 reductase catalyzes the one-electron transfer from NADPH via FAD and FMN to various electron acceptors, such as cytochrome P450s or to some anti-cancer quinone drugs. This results in generation of free radicals and toxic oxygen metabolites, which can contribute to the cytotoxicity of these compounds. Recently, a cytosolic NADPH-dependent flavin reductase, NR1, has been described which is highly homologous to the microsomal cytochrome P450 reductase. In this study, we show that over-expression of NR1 in human embryonic kidney cells enhances the cytotoxic action of the model quinone, menadione. Furthermore, we show that a novel human histidine triad protein DCS-1, which is expressed together with NR1 in many tissues, can significantly reduce menadione-induced cytotoxicity in these cells. We also show that DCS-1 binds NF1 and directly modulates its activity. These results suggest that NR1 may play a role in carcinogenicity and cell death associated with one-electron reductions

  1. The knockdown of OsVIT2 and MIT affects iron localization in rice seed.

    Science.gov (United States)

    Bashir, Khurram; Takahashi, Ryuichi; Akhtar, Shamim; Ishimaru, Yasuhiro; Nakanishi, Hiromi; Nishizawa, Naoko K

    2013-11-20

    The mechanism of iron (Fe) uptake in plants has been extensively characterized, but little is known about how Fe transport to different subcellular compartments affects Fe localization in rice seed. Here, we discuss the characterization of a rice vacuolar Fe transporter 2 (OsVIT2) T-DNA insertion line (osvit2) and report that the knockdown of OsVIT2 and mitochondrial Fe transporter (MIT) expression affects seed Fe localization. osvit2 plants accumulated less Fe in their shoots when grown under normal or excess Fe conditions, while the accumulation of Fe was comparable to that in wild-type (WT) plants under Fe-deficient conditions. The accumulation of zinc, copper, and manganese also changed significantly in the shoots of osvit2 plants. The growth of osvit2 plants was also slow compared to that of WT plants. The concentration of Fe increased in osvit2 polished seeds. Previously, we reported that the expression of OsVIT2 was higher in MIT knockdown (mit-2) plants, and in this study, the accumulation of Fe in mit-2 seeds decreased significantly. These results suggest that vacuolar Fe trafficking is important for plant Fe homeostasis and distribution, especially in plants grown in the presence of excess Fe. Moreover, changes in the expression of OsVIT2 and MIT affect the concentration and localization of metals in brown rice as well as in polished rice seeds.

  2. Quantification of Functionalised Gold Nanoparticle-Targeted Knockdown of Gene Expression in HeLa Cells

    Science.gov (United States)

    Jiwaji, Meesbah; Sandison, Mairi E.; Reboud, Julien; Stevenson, Ross; Daly, Rónán; Barkess, Gráinne; Faulds, Karen; Kolch, Walter; Graham, Duncan; Girolami, Mark A.; Cooper, Jonathan M.; Pitt, Andrew R.

    2014-01-01

    Introduction Gene therapy continues to grow as an important area of research, primarily because of its potential in the treatment of disease. One significant area where there is a need for better understanding is in improving the efficiency of oligonucleotide delivery to the cell and indeed, following delivery, the characterization of the effects on the cell. Methods In this report, we compare different transfection reagents as delivery vehicles for gold nanoparticles functionalized with DNA oligonucleotides, and quantify their relative transfection efficiencies. The inhibitory properties of small interfering RNA (siRNA), single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) sequences targeted to human metallothionein hMT-IIa are also quantified in HeLa cells. Techniques used in this study include fluorescence and confocal microscopy, qPCR and Western analysis. Findings We show that the use of transfection reagents does significantly increase nanoparticle transfection efficiencies. Furthermore, siRNA, ssRNA and ssDNA sequences all have comparable inhibitory properties to ssDNA sequences immobilized onto gold nanoparticles. We also show that functionalized gold nanoparticles can co-localize with autophagosomes and illustrate other factors that can affect data collection and interpretation when performing studies with functionalized nanoparticles. Conclusions The desired outcome for biological knockdown studies is the efficient reduction of a specific target; which we demonstrate by using ssDNA inhibitory sequences targeted to human metallothionein IIa gene transcripts that result in the knockdown of both the mRNA transcript and the target protein. PMID:24926959

  3. [Effect of UV-radiation on the level of ascorbic acid, SH-groups, and activity of glutathione reductase in the eye lens].

    Science.gov (United States)

    Byshneva, L N; Senchuk, V V

    2002-01-01

    The effect of UV radiation in vitro on the level of ascorbate, SH-groups and glutathione reductase activity in the soluble fraction of bovine eye lens was studied. UV-Irradiation increased NADPH-oxidoreductase activity, the level of ascorbate oxidation and decreased the content of SH-groups and activity of glutathione reductase. Significant activation of the NADPH-oxidoreductase activity in the presence of ascorbate and Cu2+ was observed after UV-irradiation. It is suggested that ascorbate may play an important role in the UV-induced lens pathology.

  4. P450 reductase and cytochrome b5 interactions with cytochrome P450: Effects on house fly CYP6A1 catalysis

    OpenAIRE

    Murataliev, Marat B.; Guzov, Victor M.; Walker, F. Ann; Feyereisen, René

    2008-01-01

    The interactions of protein components of the xenobiotic-metabolizing cytochrome P450 system, CYP6A1, P450 reductase, and cytochrome b5 from the house fly (Musca domestica) have been characterized. CYP6A1 activity is determined by the concentration of the CYP6A1-P450 reductase complex, regardless of which protein is present in excess. Both holo- and apo-b5 stimulated CYP6A1 heptachlor epoxidase and steroid hydroxylase activities and influenced the regioselectivity of testosterone hydroxylatio...

  5. Identification and cloning of an NADPH-dependent hydroxycinnamoyl-CoA double bond reductase involved in dihydrochalcone formation in Malus×domestica Borkh.

    Science.gov (United States)

    Ibdah, Mwafaq; Berim, Anna; Martens, Stefan; Valderrama, Andrea Lorena Herrera; Palmieri, Luisa; Lewinsohn, Efraim; Gang, David R

    2014-11-01

    The apple tree (Malus sp.) is an agriculturally and economically important source of food and beverages. Many of the health beneficial properties of apples are due to (poly)phenolic metabolites that they contain, including various dihydrochalcones. Although many of the genes and enzymes involved in polyphenol biosynthesis are known in many plant species, the specific reactions that lead to the biosynthesis of the dihydrochalcone precursor, p-dihydrocoumaroyl-CoA (3), are unknown. To identify genes involved in the synthesis of these metabolites, existing genome databases of the Rosaceae were screened for apple genes with significant sequence similarity to Arabidopsis alkenal double bond reductases. Herein described are the isolation and characterization of a Malus hydroxycinnamoyl-CoA double bond reductase, which catalyzed the NADPH-dependent reduction of p-coumaroyl-CoA and feruloyl-CoA to p-dihydrocoumaroyl-CoA and dihydroferuloyl-CoA, respectively. Its apparent Km values for p-coumaroyl-CoA, feruloyl-CoA and NADPH were 96.6, 92.9 and 101.3μM, respectively. The Malus double bond reductase preferred feruloyl-CoA to p-coumaroyl-CoA as a substrate by a factor of 2.1 when comparing catalytic efficiencies in vitro. Expression analysis of the hydroxycinnamoyl-CoA double bond reductase gene revealed that its transcript levels showed significant variation in tissues of different developmental stages, but was expressed when expected for involvement in dihydrochalcone formation. Thus, the hydroxycinnamoyl-CoA double bond reductase appears to be responsible for the reduction of the α,β-unsaturated double bond of p-coumaroyl-CoA, the first step of dihydrochalcone biosynthesis in apple tissues, and may be involved in the production of these compounds. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Knockdown of miR-27a sensitizes colorectal cancer stem cells to TRAIL by promoting the formation of Apaf-1-caspase-9 complex.

    Science.gov (United States)

    Zhang, Rui; Xu, Jian; Zhao, Jian; Bai, Jinghui

    2017-07-11

    MicroRNAs have been proved to participate in multiple biological processes in cancers. For developing resistance to cytotoxic drug, cancer cells, especially the cancer stem cells, usually change their microRNA expression profile to survive in hostile environments. In the present study, we found that expression of microRNA-27a was increased in colorectal cancer stem cells. High level of microRNA-27a was indicated to induce the resistance to TNF-related apoptosis-inducing ligand (TRAIL). Knockdown of microRNA-27a resensitized colorectal cancer stem cells to TRAIL-induced cell death. Mechanically, the gene of Apaf-1, which is associated with the mitochondrial apoptosis, was demonstrated to be the target of microRNA-27a in colorectal cancer stem cells. Knockdown of microRNA-27a increased the expression level of Apaf-1, thus enhancing the formation of Apaf-1-caspase-9 complex and subsequently promoting the TRAIL-induced apoptosis in colorectal cancer stem cells. These findings suggested that knockdown of microRNA-27a in colorectal cancer stem cells by the specific antioligonucleotides was potential to reverse the chemoresistance to TRAIL. It may represent a novel therapeutic strategy for treating the colorectal cancer more effectively.

  7. Direct enzyme assay evidence confirms aldehyde reductase function of Ydr541cp and Ygl039wp from Saccharomyces cerevisiae.

    Science.gov (United States)

    Moon, Jaewoong; Liu, Z Lewis

    2015-04-01

    The aldehyde reductase gene ARI1 is a recently characterized member of an intermediate subfamily within the short-chain dehydrogenase/reductase (SDR) superfamily that clarified mechanisms of in situ detoxification of 2-furaldehyde and 5-hydroxymethyl-2-furaldehyde by Saccharomyces cerevisiae. Uncharacterized open reading frames (ORFs) are common among tolerant candidate genes identified for lignocellulose-to-advanced biofuels conversion. This study presents partially purified proteins of two ORFs, YDR541C and YGL039W, and direct enzyme assay evidence against aldehyde-inhibitory compounds commonly encountered during lignocellulosic biomass fermentation processes. Each of the partially purified proteins encoded by these ORFs showed a molecular mass of approximately 38 kDa, similar to Ari1p, a protein encoded by aldehyde reductase gene. Both proteins demonstrated strong aldehyde reduction activities toward 14 aldehyde substrates, with high levels of reduction activity for Ydr541cp toward both aromatic and aliphatic aldehydes. While Ydr541cp was observed to have a significantly higher specific enzyme activity at 20 U/mg using co-factor NADPH, Ygl039wp displayed a NADH preference at 25 U/mg in reduction of butylaldehyde. Amino acid sequence analysis identified a characteristic catalytic triad, Ser, Tyr and Lys; a conserved catalytic motif of Tyr-X-X-X-Lys; and a cofactor-binding sequence motif, Gly-X-X-Gly-X-X-Ala, near the N-terminus that are shared by Ydr541cp, Ygl039wp, Yol151wp/GRE2 and Ari1p. Findings of aldehyde reductase genes contribute to the yeast gene annotation and aids development of the next-generation biocatalyst for advanced biofuels production. Copyright © 2015 John Wiley & Sons, Ltd.

  8. Crystallization and preliminary X-ray diffraction analysis of salutaridine reductase from the opium poppy Papaver somniferum

    International Nuclear Information System (INIS)

    Higashi, Yasuhiro; Smith, Thomas J.; Jez, Joseph M.; Kutchan, Toni M.

    2010-01-01

    Recombinant P. somniferum salutaridine reductase (SalR) was purified and crystallized with NADPH using the hanging-drop vapor-diffusion method. Crystals of the SalR–NADPH complex diffracted X-rays to a resolution of 1.9 Å. The opium poppy Papaver somniferum is the source of the narcotic analgesics morphine and codeine. Salutaridine reductase (SalR; EC 1.1.1.248) reduces the C-7 keto group of salutaridine to the C-7 (S)-hydroxyl group of salutaridinol in the biosynthetic pathway that leads to morphine in the opium poppy plant. P. somniferum SalR was overproduced in Escherichia coli and purified using cobalt-affinity and size-exclusion chromatography. Hexagonal crystals belonging to space group P6 4 22 or P6 2 22 were obtained using ammonium sulfate as precipitant and diffracted to a resolution of 1.9 Å

  9. Structure of conjugated polyketone reductase from Candida parapsilosis IFO 0708 reveals conformational changes for substrate recognition upon NADPH binding.

    Science.gov (United States)

    Qin, Hui-Min; Yamamura, Akihiro; Miyakawa, Takuya; Kataoka, Michihiko; Nagai, Takahiro; Kitamura, Nahoko; Urano, Nobuyuki; Maruoka, Shintaro; Ohtsuka, Jun; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2014-01-01

    Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708, identified as a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ketopantoyl lactone reductase, belongs to the aldo-keto reductase superfamily. This enzyme reduces ketopantoyl lactone to D-pantoyl lactone in a strictly stereospecific manner. To elucidate the structural basis of the substrate specificity, we determined the crystal structures of the apo CPR-C2 and CPR-C2/NADPH complex at 1.70 and 1.80 Å resolutions, respectively. CPR-C2 adopted a triose-phosphate isomerase barrel fold at the core of the structure. Binding with the cofactor NADPH induced conformational changes in which Thr27 and Lys28 moved 15 and 5.0 Å, respectively, in the close vicinity of the adenosine 2'-phosphate group of NADPH to form hydrogen bonds. Based on the comparison of the CPR-C2/NADPH structure with 3-α-hydroxysteroid dehydrogenase and mutation analyses, we constructed substrate binding models with ketopantoyl lactone, which provided insight into the substrate specificity by the cofactor-induced structure. The results will be useful for the rational design of CPR-C2 mutants targeted for use in the industrial manufacture of ketopantoyl lactone.

  10. Knockdown of TWIST1 enhances arsenic trioxide- and ionizing radiation-induced cell death in lung cancer cells by promoting mitochondrial dysfunction

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Sung-Keum; Kim, Jae-Hee; Choi, Ha-Na [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of); Choe, Tae-Boo [Department of Microbiological Engineering, Kon-Kuk University, Gwangjin-gu, Seoul (Korea, Republic of); Hong, Seok-Il [Department of Laboratory Medicine, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of); Yi, Jae-Youn [Laboratory of Modulation of Radiobiological Responses, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of); Hwang, Sang-Gu [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of); Lee, Hyun-Gyu [Department of Microbiology and Immunology, College of Medicine, Yonsei University, 250 Seongsan-no, Seodaemun-gu, Seoul (Korea, Republic of); Lee, Yun-Han, E-mail: yhlee87@yuhs.ac [Department of Radiation Oncology, College of Medicine, Yonsei University, 250 Seongsan-no, Seodaemun-gu, Seoul (Korea, Republic of); Park, In-Chul, E-mail: parkic@kcch.re.kr [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of)

    2014-07-11

    Highlights: • Knockdown of TWIST1 enhanced ATO- and IR-induced cell death in NSCLCs. • Intracellular ROS levels were increased in cells treated with TWIST1 siRNA. • TWIST1 siRNA induced MMP loss and mitochondrial fragmentation. • TWIST1 siRNA upregulated the fission-related proteins FIS1 and DRP1. - Abstract: TWIST1 is implicated in the process of epithelial mesenchymal transition, metastasis, stemness, and drug resistance in cancer cells, and therefore is a potential target for cancer therapy. In the present study, we found that knockdown of TWIST1 by small interfering RNA (siRNA) enhanced arsenic trioxide (ATO)- and ionizing radiation (IR)-induced cell death in non-small-cell lung cancer cells. Interestingly, intracellular reactive oxygen species levels were increased in cells treated with TWIST1 siRNA and further increased by co-treatment with ATO or IR. Pretreatment of lung cancer cells with the antioxidant N-acetyl-cysteine markedly suppressed the cell death induced by combined treatment with TWIST1 siRNA and ATO or IR. Moreover, treatment of cells with TWIST1 siRNA induced mitochondrial membrane depolarization and significantly increased mitochondrial fragmentation (fission) and upregulated the fission-related proteins FIS1 and DRP1. Collectively, our results demonstrate that siRNA-mediated TWIST1 knockdown induces mitochondrial dysfunction and enhances IR- and ATO-induced cell death in lung cancer cells.

  11. A scalable double-barcode sequencing platform for characterization of dynamic protein-protein interactions.

    Science.gov (United States)

    Schlecht, Ulrich; Liu, Zhimin; Blundell, Jamie R; St Onge, Robert P; Levy, Sasha F

    2017-05-25

    Several large-scale efforts have systematically catalogued protein-protein interactions (PPIs) of a cell in a single environment. However, little is known about how the protein interactome changes across environmental perturbations. Current technologies, which assay one PPI at a time, are too low throughput to make it practical to study protein interactome dynamics. Here, we develop a highly parallel protein-protein interaction sequencing (PPiSeq) platform that uses a novel double barcoding system in conjunction with the dihydrofolate reductase protein-fragment complementation assay in Saccharomyces cerevisiae. PPiSeq detects PPIs at a rate that is on par with current assays and, in contrast with current methods, quantitatively scores PPIs with enough accuracy and sensitivity to detect changes across environments. Both PPI scoring and the bulk of strain construction can be performed with cell pools, making the assay scalable and easily reproduced across environments. PPiSeq is therefore a powerful new tool for large-scale investigations of dynamic PPIs.

  12. Synthesis of milligram quantities of proteins using a reconstituted in vitro protein synthesis system.

    Science.gov (United States)

    Kazuta, Yasuaki; Matsuura, Tomoaki; Ichihashi, Norikazu; Yomo, Tetsuya

    2014-11-01

    In this study, the amount of protein synthesized using an in vitro protein synthesis system composed of only highly purified components (the PURE system) was optimized. By varying the concentrations of each system component, we determined the component concentrations that result in the synthesis of 0.38 mg/mL green fluorescent protein (GFP) in batch mode and 3.8 mg/mL GFP in dialysis mode. In dialysis mode, protein concentrations of 4.3 and 4.4 mg/mL were synthesized for dihydrofolate reductase and β-galactosidase, respectively. Using the optimized system, the synthesized protein represented 30% (w/w) of the total protein, which is comparable to the level of overexpressed protein in Escherichia coli cells. This optimized reconstituted in vitro protein synthesis system may potentially be useful for various applications, including in vitro directed evolution of proteins, artificial cell assembly, and protein structural studies. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  13. Prevalence of Plasmodium falciparum resistance markers to sulfadoxine-pyrimethamine among pregnant women receiving intermittent preventive treatment for malaria in Uganda

    DEFF Research Database (Denmark)

    Mbonye, Anthony K; Birungi, Josephine; Yanow, Stephanie K

    2015-01-01

    The aim of this study was to assess the prevalence of mutations in Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes among pregnant women using sulfadoxine-pyrimethamine (SP) as an intermittent preventive treatment (IPTp). A molecular epidemiological...... in the Pfdhfr and Pfdhps genes that are associated with SP resistance. The prevalence of the single-nucleotide mutations in Pfdhfr at codons 51I, 59R, and 108N and in Pfdhps at codons 437G and 540E was high (>98%), reaching 100% fixation after one dose of SP, while the prevalence of 581G was 3.3% at baseline...... and anemia. However, women infected with P. falciparum had 1.3-g/dl-lower hemoglobin levels (P = 0.001) and delivered babies with a 400-g-lower birth weight (P = 0.001) compared to nonparasitemic women. Despite this, 44 women who were P. falciparum positive at baseline became negative after one or two doses...

  14. Quantifying the limits of transition state theory in enzymatic catalysis.

    Science.gov (United States)

    Zinovjev, Kirill; Tuñón, Iñaki

    2017-11-21

    While being one of the most popular reaction rate theories, the applicability of transition state theory to the study of enzymatic reactions has been often challenged. The complex dynamic nature of the protein environment raised the question about the validity of the nonrecrossing hypothesis, a cornerstone in this theory. We present a computational strategy to quantify the error associated to transition state theory from the number of recrossings observed at the equicommittor, which is the best possible dividing surface. Application of a direct multidimensional transition state optimization to the hydride transfer step in human dihydrofolate reductase shows that both the participation of the protein degrees of freedom in the reaction coordinate and the error associated to the nonrecrossing hypothesis are small. Thus, the use of transition state theory, even with simplified reaction coordinates, provides a good theoretical framework for the study of enzymatic catalysis. Copyright © 2017 the Author(s). Published by PNAS.

  15. Radiation-induced gene amplification in rodent and human cells

    International Nuclear Information System (INIS)

    Luecke-Huhle, C.; Gloss, B.; Herrlich, P.

    1990-01-01

    Ionizing and UV radiations induce amplification of SV40 DNA sequences integrated in the genome of Chinese hamster cells and increase amplification of the dihydrofolate reductase (DHFR) gene during methotrexate selection in human skin fibroblasts of a patient with ataxia telangiectasia. Various types of external (60-Co-γ-rays, 241-Am-α-particles, UV) or internal radiation (caused by the decay of 125 I incorporated into DNA in form of I-UdR) were applied. By cell fusion experiments it could be shown that SV40 gene amplification is mediated by one or several diffusible trans-acting factors induced or activated in a dose dependent manner by all types of radiation. One of these factors binds to a 10 bp sequence within the minimal origin of replication of SV40. In vivo competition with an excess of a synthetic oligonucleotide comprising this sequence blocks radiation-induced amplification. (author) 25 refs.; 8 figs

  16. Synergistic Enhancement of Enzyme Performance and Resilience via Orthogonal Peptide-Protein Chemistry Enabled Multilayer Construction.

    Science.gov (United States)

    Zhang, Xue-Jian; Wang, Xiao-Wei; Sun, Jiaxing; Su, Chao; Yang, Shuguang; Zhang, Wen-Bin

    2018-05-16

    Protein immobilization is critical to utilize their unique functions in diverse applications. Herein, we report that orthogonal peptide-protein chemistry enabled multilayer construction can facilitate the incorporation of various folded structural domains, including calmodulin in different states, affibody and dihydrofolate reductase (DHFR). An extended conformation is found to be the most advantageous for steady film growth. The resulting protein thin films exhibit sensitive and selective responsive behaviors to bio-signals (Ca2+, TFP, NADPH, etc.) and fully maintain the catalytic activity of DHFR. The approach is applicable to different substrates such as hydrophobic gold and hydrophilic silica microparticles. The DHFR enzyme can be immobilized onto silica microparticles with tunable amounts. The multi-layer set-up exhibits a synergistic enhancement of DHFR activity with increasing number of bilayers and also makes the embedded DHFR more resilient to lyophilization. Therefore, this is a convenient and versatile method for protein immobilization with potential benefits of synergistic enhancement in enzyme performance and resilience.

  17. A case of severe methylenetetrahydrofolate reductase deficiency presenting as neonatal encephalopathy, seizures, microcephaly and central hypoventilation

    NARCIS (Netherlands)

    Balasubramaniam, S.; Salomons, G.S.; Blom, H.J.

    2013-01-01

    Methylenetetrahydrofolate reductase (MTHFR) is a key regulatory enzyme in the remethylation of homocysteine to methionine. S-adenosylmethionine, formed from methionine and adenosine triphosphate, is the methyl donor in crucial reactions for brain development and function. MTHFR deficiency is the

  18. Acute sterol o-acyltransferase 2 (SOAT2 knockdown rapidly mobilizes hepatic cholesterol for fecal excretion.

    Directory of Open Access Journals (Sweden)

    Stephanie M Marshall

    Full Text Available The primary risk factor for atherosclerotic cardiovascular disease is LDL cholesterol, which can be reduced by increasing cholesterol excretion from the body. Fecal cholesterol excretion can be driven by a hepatobiliary as well as a non-biliary pathway known as transintestinal cholesterol efflux (TICE. We previously showed that chronic knockdown of the hepatic cholesterol esterifying enzyme sterol O-acyltransferase 2 (SOAT2 increased fecal cholesterol loss via TICE. To elucidate the initial events that stimulate TICE, C57Bl/6 mice were fed a high cholesterol diet to induce hepatic cholesterol accumulation and were then treated for 1 or 2 weeks with an antisense oligonucleotide targeting SOAT2. Within 2 weeks of hepatic SOAT2 knockdown (SOAT2HKD, the concentration of cholesteryl ester in the liver was reduced by 70% without a reciprocal increase in hepatic free cholesterol. The rapid mobilization of hepatic cholesterol stores resulted in a ∼ 2-fold increase in fecal neutral sterol loss but no change in biliary cholesterol concentration. Acute SOAT2HKD increased plasma cholesterol carried primarily in lipoproteins enriched in apoB and apoE. Collectively, our data suggest that acutely reducing SOAT2 causes hepatic cholesterol to be swiftly mobilized and packaged onto nascent lipoproteins that feed cholesterol into the TICE pathway for fecal excretion.

  19. Detoxification of hexavalent chromium by Leucobacter sp. uses a reductase with specificity for dihydrolipoamide.

    Science.gov (United States)

    Sarangi, Abhipsa; Krishnan, Chandraraj

    2016-02-01

    Leucobacter sp. belongs to the metal stressed community and possesses higher tolerance to metals including chromium and can detoxify toxic hexavalent chromium by reduction to less toxic trivalent chromium. But, the mechanism of reduction of hexavalent chromium by Leucobacter sp. has not been studied. Understanding the enzyme catalyzing reduction of chromium is important to improve the species for application in bioremediation. Hence, a soluble reductase catalyzing the reduction of hexavalent chromium was purified from a Leucobacter sp. and characterized. The pure chromate reductase was obtained from the cell-free extract through hydrophobic interaction and gel filtration column chromatographic methods. It was a monomeric enzyme and showed similar molecular weights in both gel filtration (∼68 KDa) and SDS-PAGE (64 KDa). It reduced Cr(VI) using both NADH and NADPH as the electron donor, but exhibited higher activity with NADH. The optimal activity was found at pH 5.5 and 30 °C. The K(m) and V(max) for Cr(VI) reduction with NADH were 46.57 μM and 0.37 μmol min(-1) (mg protein) (-1), respectively. The activity was inhibited by p-hydroxy mercury benzoate, Ag(2+) and Hg(2+) indicating the role of thiol groups in the catalysis. The spectrophotometric analysis of the purified enzyme showed the absence of bound flavin in the enzyme. The N-terminal amino acid sequence and LC/MS analysis of trypsin digested purified enzyme showed similarity to dihydrolipoyl dehydrogenase. The purified enzyme had dihydrolipoyl dehydrogenase activity with dihydrolipoamide as the substrate, which suggested that Leucobacter sp. uses reductase with multiple substrate specificity for reduction of Cr(VI) detoxification. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Expression, purification, crystallization and X-ray analysis of 3-quinuclidinone reductase from Agrobacterium tumefaciens

    International Nuclear Information System (INIS)

    Hou, Feng; Miyakawa, Takuya; Takeshita, Daijiro; Kataoka, Michihiko; Uzura, Atsuko; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2012-01-01

    The purification and crystallization of 3-quinuclidinone reductase from A. tumefaciens allowed the collection of a diffraction data set to 1.72 Å resolution. (R)-3-Quinuclidinol is a useful chiral building block for the synthesis of various pharmaceuticals and can be produced from 3-quinuclidinone by asymmetric reduction. A novel 3-quinuclidinone reductase from Agrobacterium tumefaciens (AtQR) catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol with NADH as a cofactor. Recombinant AtQR was overexpressed in Escherichia coli, purified and crystallized with NADH using the sitting-drop vapour-diffusion method at 293 K. Crystals were obtained using a reservoir solution containing PEG 3350 as a precipitant. X-ray diffraction data were collected to 1.72 Å resolution on beamline BL-5A at the Photon Factory. The crystal belonged to space group P2 1 , with unit-cell parameters a = 62.0, b = 126.4, c = 62.0 Å, β = 110.5°, and was suggested to contain four molecules in the asymmetric unit (V M = 2.08 Å 3 Da −1 )