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Sample records for active site substitution

  1. Loop substitution as a tool to identify active sites of interleukin-1 beta.

    PubMed

    Palla, E; Bensi, G; Solito, E; Buonamassa, D T; Fassina, G; Raugei, G; Spano, F; Galeotti, C; Mora, M; Domenighini, M

    1993-06-25

    By computer analysis of the amino acid sequence of human interleukin-1 beta (IL-1 beta) and of the human type I IL-1 receptor (IL-1RI), we have identified two hydropathically complementary peptides (Fassina, G., Roller, P. P., Olson, A. D., Thorgeirsson, S. S., and Omichinski, J. G. (1989) J. Biol. Chem. 264, 11252-11257) capable of binding to each other. The sequence of the IL-1 beta peptide corresponds to that of residues 88-99 (loop 7 of the crystal structure of mature IL-1 beta) of mature IL-1 beta, one of the exposed and highly charged regions of the molecule. The substitution of this loop with an amino acid sequence of the same length but different hydropathic profile generates a mutant with drastically reduced binding activity to IL-1RI. In contrast, the binding affinity to the type II IL-1R (IL-1RII) is the same as that of wild type IL-1 beta. The results show that 1) loop 7 is part of the binding site of IL-1 beta to IL-1RI, but not to IL-1RII. 2) The structure of the mutant protein is not grossly altered except locally at the position of the substituted loop. 3) The substitution of amino acids by site-directed mutagenesis of the loop 7 region generates mutants with binding affinity constants slightly lower than that of wild type IL-1 beta and not comparable to that of the loop substitution analogue. 4. All mutants analyzed, including the loop substitutions, are biologically active, confirming the structural integrity of the proteins. We propose a binding site in which the cooperation of several low energy bonds extended over a wide area results in a high affinity complex between IL-1 and the type I receptor. PMID:7685764

  2. Active-site Arg --> Lys substitutions alter reaction and substrate specificity of aspartate aminotransferase.

    PubMed

    Vacca, R A; Giannattasio, S; Graber, R; Sandmeier, E; Marra, E; Christen, P

    1997-08-29

    Arg386 and Arg292 of aspartate aminotransferase bind the alpha and the distal carboxylate group, respectively, of dicarboxylic substrates. Their substitution with lysine residues markedly decreased aminotransferase activity. The kcat values with L-aspartate and 2-oxoglutarate as substrates under steady-state conditions at 25 degrees C were 0.5, 2.0, and 0.03 s-1 for the R292K, R386K, and R292K/R386K mutations, respectively, kcat of the wild-type enzyme being 220 s-1. Longer dicarboxylic substrates did not compensate for the shorter side chain of the lysine residues. Consistent with the different roles of Arg292 and Arg386 in substrate binding, the effects of their substitution on the activity toward long chain monocarboxylic (norleucine/2-oxocaproic acid) and aromatic substrates diverged. Whereas the R292K mutation did not impair the aminotransferase activity toward these substrates, the effect of the R386K substitution was similar to that on the activity toward dicarboxylic substrates. All three mutant enzymes catalyzed as side reactions the beta-decarboxylation of L-aspartate and the racemization of amino acids at faster rates than the wild-type enzyme. The changes in reaction specificity were most pronounced in aspartate aminotransferase R292K, which decarboxylated L-aspartate to L-alanine 15 times faster (kcat = 0.002 s-1) than the wild-type enzyme. The rates of racemization of L-aspartate, L-glutamate, and L-alanine were 3, 5, and 2 times, respectively, faster than with the wild-type enzyme. Thus, Arg --> Lys substitutions in the active site of aspartate aminotransferase decrease aminotransferase activity but increase other pyridoxal 5'-phosphate-dependent catalytic activities. Apparently, the reaction specificity of pyridoxal 5'-phosphate-dependent enzymes is not only achieved by accelerating the specific reaction but also by preventing potential side reactions of the coenzyme substrate adduct. PMID:9268327

  3. Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination

    PubMed Central

    Rowley, Paul A.; Kachroo, Aashiq H.; Ma, Chien-Hui; Maciaszek, Anna D.; Guga, Piotr; Jayaram, Makkuni

    2015-01-01

    Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5′ to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions. PMID:25999343

  4. Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination.

    PubMed

    Rowley, Paul A; Kachroo, Aashiq H; Ma, Chien-Hui; Maciaszek, Anna D; Guga, Piotr; Jayaram, Makkuni

    2015-07-13

    Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5' to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions. PMID:25999343

  5. Asymmetric mutations in the tetrameric R67 dihydrofolate reductase reveal high tolerance to active-site substitutions

    PubMed Central

    Ebert, Maximilian C C J C; Morley, Krista L; Volpato, Jordan P; Schmitzer, Andreea R; Pelletier, Joelle N

    2015-01-01

    Type II R67 dihydrofolate reductase (DHFR) is a bacterial plasmid-encoded enzyme that is intrinsically resistant to the widely-administered antibiotic trimethoprim. R67 DHFR is genetically and structurally unrelated to E. coli chromosomal DHFR and has an unusual architecture, in that four identical protomers form a single symmetrical active site tunnel that allows only one substrate binding/catalytic event at any given time. As a result, substitution of an active-site residue has as many as four distinct consequences on catalysis, constituting an atypical model of enzyme evolution. Although we previously demonstrated that no single residue of the native active site is indispensable for function, library selection here revealed a strong bias toward maintenance of two native protomers per mutated tetramer. A variety of such “half-native” tetramers were shown to procure native-like catalytic activity, with similar KM values but kcat values 5- to 33-fold lower, illustrating a high tolerance for active-site substitutions. The selected variants showed a reduced thermal stability (Tm ∼12°C lower), which appears to result from looser association of the protomers, but generally showed a marked increase in resilience to heat denaturation, recovering activity to a significantly greater extent than the variant with no active-site substitutions. Our results suggest that the presence of two native protomers in the R67 DHFR tetramer is sufficient to provide native-like catalytic rate and thus ensure cellular proliferation. PMID:25401264

  6. Substitution of apolar residues in the active site of aspartate aminotransferase by histidine. Effects on reaction and substrate specificity.

    PubMed

    Vacca, R A; Christen, P; Malashkevich, V N; Jansonius, J N; Sandmeier, E

    1995-01-15

    In an attempt to change the reaction and substrate specificity of aspartate aminotransferase, several apolar active-site residues were substituted in turn with a histidine residue. Aspartate aminotransferase W140H (of Escherichia coli) racemizes alanine seven times faster (Kcat' = 2.2 x 10(-4) s-1) than the wild-type enzyme, while the aminotransferase activity toward L-alanine was sixfold decreased. X-ray crystallographic analysis showed that the structural changes brought about by the mutation are limited to the immediate environment of H140. In contrast to the tryptophan side chain in the wild-type structure, the imidazole ring of H140 does not form a stacking interaction with the coenzyme pyridine ring. The angle between the two ring planes is about 50 degrees. Pyridoxamine 5'-phosphate dissociates 50 times more rapidly from the W140H mutant than from the wild-type enzyme. A model of the structure of the quinonoid enzyme substrate intermediate indicates that H140 might assist in the reprotonation of C alpha of the amino acid substrate from the re side of the deprotonated coenzyme-substrate adduct in competition with si-side reprotonation by K258. In aspartate aminotransferase I17H (of chicken mitochondria), the substituted residue also lies on the re side of the coenzyme. This mutant enzyme slowly decarboxylates L-aspartate to L-alanine (Kcat' = 8 x 10(-5) s-1). No beta-decarboxylase activity is detectable in the wild-type enzyme. In aspartate aminotransferase V37H (of chicken mitochondria), the mutated residue lies besides the coenzyme in the plane of the pyridine ring; no change in reaction specificity was observed. All three mutations, i.e. W140-->H, I17-->H and V37--H, decreased the aminotransferase activity toward aromatic amino acids by 10-100-fold, while decreasing the activity toward dicarboxylic substrates only moderately to 20%, 20% and 60% of the activity of the wild-type enzymes, respectively. In all three mutant enzymes, the decrease in aspartate

  7. Substitution of conserved residues within the active site alters the cleavage religation equilibrium of DNA topoisomerase I.

    PubMed

    Colley, William C; van der Merwe, Marie; Vance, John R; Burgin, Alex B; Bjornsti, Mary-Ann

    2004-12-24

    Eukaryotic DNA topoisomerase I (Top1p) catalyzes the relaxation of supercoiled DNA and constitutes the cellular target of camptothecin (CPT). Mutation of conserved residues in close proximity to the active site tyrosine (Tyr(727) of yeast Top1p) alters the DNA cleavage religation equilibrium, inducing drug-independent cell lethality. Previous studies indicates that yeast Top1T722Ap and Top1N726Hp cytotoxicity results from elevated levels of covalent enzyme-DNA intermediates. Here we show that Top1T722Ap acts as a CPT mimetic by exhibiting reduced rates of DNA religation, whereas increased Top1N726Hp.DNA complexes result from elevated DNA binding and cleavage. We also report that the combination of the T722A and N726H mutations in a single protein potentiates the cytotoxic action of the enzyme beyond that induced by co-expression of the single mutants. Moreover, the addition of CPT to cells expressing the double top1T722A/N726H mutant did not enhance cell lethality. Thus, independent alterations in DNA cleavage and religation contribute to the lethal phenotype. The formation of distinct cytotoxic lesions was also evidenced by the different responses induced by low levels of these self-poisoning enzymes in isogenic strains defective for the Rad9 DNA damage checkpoint, processive DNA replication, or ubiquitin-mediated proteolysis. Substitution of Asn(726) with Phe or Tyr also produces self-poisoning enzymes, implicating stacking interactions in the increased kinetics of DNA cleavage by Top1N726Hp and Top1N726Fp. In contrast, replacing the amide side chain of Asn(726) with Gln renders Top1N726Qp resistant to CPT, suggesting that the orientation of the amide within the active site is critical for effective CPT binding. PMID:15489506

  8. Dual evaluation of some novel 2-amino-substituted coumarinylthiazoles as anti-inflammatory-antimicrobial agents and their docking studies with COX-1/COX-2 active sites.

    PubMed

    Chandak, Navneet; Kumar, Pawan; Kaushik, Pawan; Varshney, Parul; Sharma, Chetan; Kaushik, Dhirender; Jain, Sudha; Aneja, Kamal R; Sharma, Pawan K

    2014-08-01

    Synthesis of total eighteen 2-amino-substituted 4-coumarinylthiazoles including sixteen new compounds (3a-o and 5b) bearing the benzenesulfonamide moiety is described in the present report. All the synthesized target compounds were examined for their in vivo anti-inflammatory (AI) activity and in vitro antimicrobial activity. Results revealed that six compounds (3 d, 3 f, 3 g, 3 h, 3 j and 3 n) exhibited pronounced anti-inflammatory activity comparable to the standard drug indomethacin. AI results were further confirmed by the docking studies of the most active (3n) and the least active compound (3a) with COX-1 and COX-2 active sites. In addition, most of the compounds exhibited moderate antimicrobial activity against Gram-positive bacteria as well as fungal yeast, S. cervisiae. Comparison between 3 and 5 indicated that incorporation of additional substituted pyrazole nucleus into the scaffold significantly enhanced AI activity. PMID:23777557

  9. Influence of cation substitution and activator site exchange on the photoluminescence properties of Eu3+-doped quaternary pyrochlore oxides.

    PubMed

    Mahesh, S K; Rao, P Prabhakar; Thomas, Mariyam; Francis, T Linda; Koshy, Peter

    2013-12-01

    Stannate-based pyrochlore-type red phosphors CaGd(1-x)SnNbO7:xEu(3+), Ca(1-y)Sr(y)Gd(1-x)SnNbO7:xEu(3+), and Ca(0.8-x)Sr0.2GdSnNbO(7+δ): xEu(3+) were prepared via conventional solid-state method. Influence of cation substitution and activator site control on the photoluminescence properties of these phosphors are elucidated using powder X-ray diffraction, Rietveld analysis, Raman spectrum analysis, and photoluminescence excitation and emission spectra. The Eu(3+) luminescence in quaternary pyrochlore lattice exemplifies as a very good structural probe for the detection of short-range disorder in the lattice, which otherwise is not detected by normal powder X-ray diffraction technique. The Eu(3+) emission due to magnetic dipole transition ((5)D0-(7)F1 MD) is modified with the increase in europium concentration in the quaternary pyrochlore red phosphors. (5)D0-(7)F1 MD transition splitting is not observable for low Eu(3+) doping because of the short-range disorder in the pyrochlore lattice. Appearance of narrow peaks in Raman spectra confirms that short-range disorder in the crystal lattice disappears with progressive europium doping. By using Sr as a network modifier ion in place of Ca we were able to increase the f-f transition intensities and europium quenching concentration. The influence of effective positive charge of the central Eu(3+) ions when it replaces a metal ion having lower oxidation state such as Ca(2+) was also investigated. The relative intensities of A1g (∼500 cm(-1)) and F2g (∼330 cm(-1)) Raman vibrational modes get inverted when Eu(3+) ions replaces Ca(2+) ions instead of Gd(3+) as trivalent europium ions can attract the electron cloud of oxygen ions strongly in comparison with divalent calcium ions. The influence of positive charge effect of Eu(3+) in Ca0.7Sr0.2GdSnNbO7+δ:0.1Eu(3+) phosphor is greatly strengthened the charge transfer band and (7)F0-(5)L6 transition intensities than that of the Ca0.8Sr0.2Gd0.9SnNbO7:0.1Eu(3+) phosphor. Our

  10. Substitution of Tyr254 with Phe at the active site of flavocytochrome b2: consequences on catalysis of lactate dehydrogenation

    SciTech Connect

    Dubois, J.; Chapman, S.K.; Mathews, F.S.; Reid, G.A.; Lederer, F. )

    1990-07-10

    A role for Tyr254 in L-lactate dehydrogenation catalyzed by flavocytochrome b2 has recently been proposed on the basis of the known active-site structure and of studies that had suggested a mechanism involving the initial formation of a lactate carbanion. This role is now examined after replacement of Tyr254 with phenylalanine. The kcat is decreased about 40-fold, Km for lactate appears unchanged, and the mainly rate-limiting step is still alpha-hydrogen abstraction, as judged from the steady-state deuterium isotope effect. Modeling studies with lactate introduced into the active site indicate two possible substrate conformations with different hydrogen-bonding partners for the substrate hydroxyl. If the hydrogen bond is formed with Tyr254, as was initially postulated, the mechanism must involve removal by His373 of the C2 hydrogen, with carbanion formation. If, in the absence of the Tyr254 phenol group, the hydrogen bond is formed with His373 N3, the substrate is positioned in such a way that the reaction must proceed by hydride transfer. Therefore the mechanism of the Y254F enzyme was investigated so as to distinguish between the two mechanistic possibilities. 2-Hydroxy-3-butynoate behaves with the mutant as a suicide reagent, as with the wild-type enzyme. Similarly, the mutant protein also catalyzes the reduction and the dehydrohalogenation of bromopyruvate under transhydrogenation conditions.

  11. Active-Site Residues of Escherichia coli DNA Gyrase Required in Coupling ATP Hydrolysis to DNA Supercoiling and Amino Acid Substitutions Leading to Novobiocin Resistance

    PubMed Central

    Gross, Christian H.; Parsons, Jonathan D.; Grossman, Trudy H.; Charifson, Paul S.; Bellon, Steven; Jernee, James; Dwyer, Maureen; Chambers, Stephen P.; Markland, William; Botfield, Martyn; Raybuck, Scott A.

    2003-01-01

    DNA gyrase is a bacterial type II topoisomerase which couples the free energy of ATP hydrolysis to the introduction of negative supercoils into DNA. Amino acids in proximity to bound nonhydrolyzable ATP analog (AMP · PNP) or novobiocin in the gyrase B (GyrB) subunit crystal structures were examined for their roles in enzyme function and novobiocin resistance by site-directed mutagenesis. Purified Escherichia coli GyrB mutant proteins were complexed with the gyrase A subunit to form the functional A2B2 gyrase enzyme. Mutant proteins with alanine substitutions at residues E42, N46, E50, D73, R76, G77, and I78 had reduced or no detectable ATPase activity, indicating a role for these residues in ATP hydrolysis. Interestingly, GyrB proteins with P79A and K103A substitutions retained significant levels of ATPase activity yet demonstrated no DNA supercoiling activity, even with 40-fold more enzyme than the wild-type enzyme, suggesting that these amino acid side chains have a role in the coupling of the two activities. All enzymes relaxed supercoiled DNA to the same extent as the wild-type enzyme did, implying that only ATP-dependent reactions were affected. Mutant genes were examined in vivo for their abilities to complement a temperature-sensitive E. coli gyrB mutant, and the activities correlated well with the in vitro activities. We show that the known R136 novobiocin resistance mutations bestow a significant loss of inhibitor potency in the ATPase assay. Four new residues (D73, G77, I78, and T165) that, when changed to the appropriate amino acid, result in both significant levels of novobiocin resistance and maintain in vivo function were identified in E. coli. PMID:12604539

  12. Reversed DNA strand cleavage specificity in initiation of Cre-LoxP recombination induced by the His289Ala active-site substitution.

    PubMed

    Gelato, Kathy A; Martin, Shelley S; Baldwin, Enoch P

    2005-11-25

    During the first steps of site-specific recombination, Cre protein cleaves and religates a specific homologous pair of LoxP strands to form a Holliday junction (HJ) intermediate. The HJ is resolved into recombination products through exchange of the second homologous strand pair. CreH289A, containing a His to Ala substitution in the conserved R-H-R catalytic motif, has a 150-fold reduced recombination rate and accumulates HJs. However, to produce these HJs, CreH289A exchanges the opposite set of strands compared to wild-type Cre (CreWT). To investigate how CreH289A and CreWT impose strand exchange order, we characterized their reactivities and strand cleavage preferences toward LoxP duplex and HJ substrates containing 8bp spacer substitutions. Remarkably, CreH289A had different and often opposite strand exchange preferences compared to CreWT with nearly all substrates. CreH289N was much less perturbed, implying that overall recombination rate and strand exchange depend more on His289 hydrogen bonding capability than on its acid/base properties. LoxP substitutions immediately 5' (S1 nucleotide) or 3' (S1' nucleotide) of the scissile phosphate had large effects on substrate utilization and strand exchange order. S1' substitutions, designed to alter base-unstacking events concomitant with Cre-induced LoxP bending, caused HJ accumulation and dramatically inverted the cleavage preferences. That pre-formed HJs were resolved via either strand in vitro suggests that inhibition of the "conformational switch" isomerization required to trigger the second strand exchange accounts for the observed HJ accumulation. Rather than reflecting CreWT behavior, CreH289A accumulates HJs of opposite polarity through a combination of its unique cleavage specificity and an HJ isomerization defect. The overall implication is that cleavage specificity is mediated by sequence-dependent DNA deformations that influence the scissile phosphate positioning and reactivity. A role of His289 may be to

  13. Activity, selectivity, and stereochemical features in the copper-catalyzed hydrogenative ring-opening of alkyl-substituted cyclopropanes - Nature of active sites

    SciTech Connect

    Palinko, I.; Molnar, A.; Kiss, J.T.; Bartok, M. )

    1990-02-01

    The reactions of four alkyl-substituted cyclopropanes in a hydrogen atmosphere in a closed circulation system have been studied over silica-supported and support-free copper catalysts made by different methods. Both isomerization and direct hydrogen addition occur. The ring opens in both the sterically more hindered and less hindered directions. On the basis of the kinetic data, mechanistic considerations are discussed. The catalysts were studied by N{sub 2}O titration and TPR (temperature-programmed reduction). The properties of the reactions and the TPR data indicate that the active centers are atomic clusters for the direct hydrogenation and Cu(I) ions for the ring-opening from the sterically hindered direction through the formation of olefinic compounds.

  14. Active site substitution A82W improves the regioselectivity of steroid hydroxylation by cytochrome P450 BM3 mutants as rationalized by spin relaxation nuclear magnetic resonance studies.

    PubMed

    Rea, V; Kolkman, A J; Vottero, E; Stronks, E J; Ampt, K A M; Honing, M; Vermeulen, N P E; Wijmenga, S S; Commandeur, J N M

    2012-01-24

    Cytochrome P450 BM3 from Bacillus megaterium is a monooxygenase with great potential for biotechnological applications. In this paper, we present engineered drug-metabolizing P450 BM3 mutants as a novel tool for regioselective hydroxylation of steroids at position 16β. In particular, we show that by replacing alanine at position 82 with a tryptophan in P450 BM3 mutants M01 and M11, the selectivity toward 16β-hydroxylation for both testosterone and norethisterone was strongly increased. The A82W mutation led to a ≤42-fold increase in V(max) for 16β-hydroxylation of these steroids. Moreover, this mutation improves the coupling efficiency of the enzyme, which might be explained by a more efficient exclusion of water from the active site. The substrate affinity for testosterone increased at least 9-fold in M11 with tryptophan at position 82. A change in the orientation of testosterone in the M11 A82W mutant as compared to the orientation in M11 was observed by T(1) paramagnetic relaxation nuclear magnetic resonance. Testosterone is oriented in M11 with both the A- and D-ring protons closest to the heme iron. Substituting alanine at position 82 with tryptophan results in increased A-ring proton-iron distances, consistent with the relative decrease in the level of A-ring hydroxylation at position 2β. PMID:22208729

  15. Reversed DNA Strand Cleavage Specificity in Initiation of Cre–LoxP Recombination Induced by the His289Ala Active-site Substitution

    PubMed Central

    Gelato, Kathy A.; Martin, Shelley S.; Baldwin, Enoch P.

    2010-01-01

    During the first steps of site-specific recombination, Cre protein cleaves and religates a specific homologous pair of LoxP strands to form a Holliday junction (HJ) intermediate. The HJ is resolved into recombination products through exchange of the second homologous strand pair. CreH289A, containing a His to Ala substitution in the conserved R-H-R catalytic motif, has a 150-fold reduced recombination rate and accumulates HJs. However, to produce these HJs, CreH289A exchanges the opposite set of strands compared to wild-type Cre (CreWT). To investigate how CreH289A and CreWT impose strand exchange order, we characterized their reactivities and strand cleavage preferences toward LoxP duplex and HJ substrates containing 8 bp spacer substitutions. Remarkably, CreH289A had different and often opposite strand exchange preferences compared to CreWT with nearly all substrates. CreH289N was much less perturbed, implying that overall recombination rate and strand exchange depend more on His289 hydrogen bonding capability than on its acid/base properties. LoxP substitutions immediately 5′(S1 nucleotide) or 3′(S1′nucleotide) of the scissile phosphate had large effects on substrate utilization and strand exchange order. S1′substitutions, designed to alter base-unstacking events concomitant with Cre-induced LoxP bending, caused HJ accumulation and dramatically inverted the cleavage preferences. That pre-formed HJs were resolved via either strand in vitro suggests that inhibition of the “conformational switch” isomerization required to trigger the second strand exchange accounts for the observed HJ accumulation. Rather than reflecting CreWT behavior, CreH289A accumulates HJs of opposite polarity through a combination of its unique cleavage specificity and an HJ isomerization defect. The overall implication is that cleavage specificity is mediated by sequence-dependent DNA deformations that influence the scissile phosphate positioning and reactivity. A role of His289

  16. SufE D74R Substitution Alters Active Site Loop Dynamics To Further Enhance SufE Interaction with the SufS Cysteine Desulfurase

    PubMed Central

    Dai, Yuyuan; Kim, Dokyong; Dong, Guangchao; Busenlehner, Laura S.; Frantom, Patrick A.; Outten, F. Wayne

    2015-01-01

    Many essential metalloproteins require iron–sulfur (Fe–S) cluster cofactors for their function. In vivo persulfide formation from L-cysteine is a key step in the biogenesis of Fe–S clusters in most organisms. In Escherichia coli, the SufS cysteine desulfurase mobilizes persulfide from L-cysteine via a PLP-dependent ping-pong reaction. SufS requires the SufE partner protein to transfer the persulfide to the SufB Fe–S cluster scaffold. Without SufE, the SufS enzyme fails to efficiently turn over and remains locked in the persulfide-bound state. Coordinated protein–protein interactions mediate sulfur transfer from SufS to SufE. Multiple studies have suggested that SufE must undergo a conformational change to extend its active site Cys loop during sulfur transfer from SufS. To test this putative model, we mutated SufE Asp74 to Arg (D74R) to increase the dynamics of the SufE Cys51 loop. Amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) analysis of SufE D74R revealed an increase in solvent accessibility and dynamics in the loop containing the active site Cys51 used to accept persulfide from SufS. Our results indicate that the mutant protein has a stronger binding affinity for SufS than that of wild-type SufE. In addition, SufE D74R can still enhance SufS desulfurase activity and did not show saturation at higher SufE D74R concentrations, unlike wild-type SufE. These results show that dynamic changes may shift SufE to a sulfur-acceptor state that interacts more strongly with SufS. PMID:26171726

  17. New insights into the mechanism of nickel insertion into carbon monoxide dehydrogenase: analysis of Rhodospirillum rubrum carbon monoxide dehydrogenase variants with substituted ligands to the [Fe3S4] portion of the active-site C-cluster.

    PubMed

    Jeon, Won Bae; Singer, Steven W; Ludden, Paul W; Rubio, Luis M

    2005-12-01

    Carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum catalyzes the oxidation of CO to CO2. A unique [NiFe4S4] cluster, known as the C-cluster, constitutes the active site of the enzyme. When grown in Ni-deficient medium R. rubrum accumulates a Ni-deficient apo form of CODH that is readily activated by Ni. It has been previously shown that activation of apo-CODH by Ni is a two-step process involving the rapid formation of an inactive apo-CODH*Ni complex prior to conversion to the active holo-CODH. We have generated CODH variants with substitutions in cysteine residues involved in the coordination of the [Fe3S4] portion of the C-cluster. Analysis of the variants suggests that the cysteine residues at positions 338, 451, and 481 are important for CO oxidation activity catalyzed by CODH but not for Ni binding to the C-cluster. C451S CODH is the only new variant that retains residual CO oxidation activity. Comparison of the kinetics and pH dependence of Ni activation of the apo forms of wild-type, C451S, and C531A CODH allowed us to develop a model for Ni insertion into the C-cluster of CODH in which Ni reversibly binds to the C-cluster and subsequently coordinates Cys531 in the rate-determining step. PMID:16283394

  18. Base substitution mutations induced by metabolically activated aflatoxin B1.

    PubMed

    Foster, P L; Eisenstadt, E; Miller, J H

    1983-05-01

    We have determined the base substitutions generated by metabolically activated aflatoxin B1 in the lacI gene of a uvrB- strain of Escherichia coli. By monitoring over 70 different nonsense mutation sites, we show that activated aflatoxin B1 specifically induced GxC leads to TxA transversions. One possible pathway leading to this base change involves depurination at guanine residues. We consider this mechanism of mutagenesis in the light of our other findings that the carcinogens benzo[a]pyrene diol epoxide and N-acetoxyacetylaminofluorene also specifically induce GxC leads to TxA transversions. PMID:6405385

  19. Modeling of Substitutional Site Preference in Ordered Intermetallic Alloys

    NASA Technical Reports Server (NTRS)

    Bozzolo, Guillermo; Noebe, Ronald D.; Honecy, Frank

    1998-01-01

    We investigate the site substitution scheme of specific alloying elements in ordered compounds and the dependence of site occupancy on compound stoichiometry, alloy concentration. This basic knowledge, and the interactions with other alloying additions are necessary in order to predict and understand the effect of various alloying schemes on the physical properties of a material, its response to various temperature treatments, and the resulting mechanical properties. Many theoretical methods can provide useful but limited insight in this area, since most techniques suffer from constraints in the type of elements and the crystallographic structures that can be modeled. With this in mind, the Bozzolo-Ferrante-Smith (BFS) method for alloys was designed to overcome these limitations, with the intent of providing an useful tool for the theoretical prediction of fundamental properties and structure of complex systems. After a brief description of the BFS method, its use for the determination of site substitution schemes for individual as well as collective alloying additions to intermetallic systems is described, including results for the concentration dependence of the lattice parameter. Focusing on B2 NiAl, FeAl and CoAl alloys, the energetics of Si, Ti, V, Cr, Fe, Co, Ni, Cu, Zr, Nb, Mo, Ru, Hf, Ta and W alloying additions are surveyed. The effect of single additions as well as the result of two simultaneous additions, discussing the interaction between additions and their influence on site preference schemes is considered. Finally, the BFS analysis is extended to ternary L1(sub 2) (Heusler phase) alloys. A comparison between experimental and theoretical results for the limited number of cases for which experimental data is available is also included.

  20. Removal of the Side Chain at the Active-Site Serine by a Glycine Substitution Increases the Stability of a Wide Range of Serine β-Lactamases by Relieving Steric Strain.

    PubMed

    Stojanoski, Vlatko; Adamski, Carolyn J; Hu, Liya; Mehta, Shrenik C; Sankaran, Banumathi; Zwart, Peter; Prasad, B V Venkataram; Palzkill, Timothy

    2016-05-01

    Serine β-lactamases are bacterial enzymes that hydrolyze β-lactam antibiotics. They utilize an active-site serine residue as a nucleophile, forming an acyl-enzyme intermediate during hydrolysis. In this study, thermal denaturation experiments as well as X-ray crystallography were performed to test the effect of substitution of the catalytic serine with glycine on protein stability in serine β-lactamases. Six different enzymes comprising representatives from each of the three classes of serine β-lactamases were examined, including TEM-1, CTX-M-14, and KPC-2 of class A, P99 of class C, and OXA-48 and OXA-163 of class D. For each enzyme, the wild type and a serine-to-glycine mutant were evaluated for stability. The glycine mutants all exhibited enhanced thermostability compared to that of the wild type. In contrast, alanine substitutions of the catalytic serine in TEM-1, OXA-48, and OXA-163 did not alter stability, suggesting removal of the Cβ atom is key to the stability increase associated with the glycine mutants. The X-ray crystal structures of P99 S64G, OXA-48 S70G and S70A, and OXA-163 S70G suggest that removal of the side chain of the catalytic serine releases steric strain to improve enzyme stability. Additionally, analysis of the torsion angles at the nucleophile position indicates that the glycine mutants exhibit improved distance and angular parameters of the intrahelical hydrogen bond network compared to those of the wild-type enzymes, which is also consistent with increased stability. The increased stability of the mutants indicates that the enzyme pays a price in stability for the presence of a side chain at the catalytic serine position but that the cost is necessary in that removal of the serine drastically impairs function. These findings support the stability-function hypothesis, which states that active-site residues are optimized for substrate binding and catalysis but that the requirements for catalysis are often not consistent with the

  1. Paramagnetic 1H and 13C NMR studies on cobalt-substituted human carbonic anhydrase I carboxymethylated at active site histidine-200: molecular basis for the changes in catalytic properties induced by the modification.

    PubMed

    Khalifah, R G; Rogers, J I; Harmon, P; Morely, P J; Carroll, S B

    1984-07-01

    Using bromo[1-13C]acetate to modify N tau of His-200 of human carbonic anhydrase isozyme I leads to the introduction of a useful 13C NMR probe into the active site. To complement our previous diamagnetic NMR studies with this probe, we have now succeeded in directly observing the paramagnetically perturbed resonance of the carboxylate in the cobalt-substituted modified enzyme above pH 8. In the pH range 8-10, the resonance undergoes a pH-dependent slow-exchange process, with the more alkaline form having a much smaller pseudocontact shift and a narrower line width. Below pH 8, the resonance apparently undergoes a very large paramagnetic downfield shift that was estimated by extrapolation. An ionization of approximate pK of 6 appears to control this process. Paramagnetic spin-relaxation studies on the resonance under conditions where it was directly observed yielded distance measurements between the carboxylate carbon and the active site cobalt ion. In inhibitor complexes, this distance was in the range of 5-7 A. In the absence of inhibitors, the distance was approximately 3.0-3.2 A at pH 7.9, consistent with the coordination of the carboxylate to the metal. However, at pH 10, the distance was increased to 4.8 A. These distance determinations were aided by relaxation measurements of a paramagnetically shifted proton resonance at 60-65 ppm downfield assigned by others to a proton of a ligand histidine of metal and confirmed by us to be 5.2 +/- 0.1 A from the metal. Our findings provide a molecular basis for the observed changes in catalytic properties that accompany the carboxymethylation. PMID:6432037

  2. Arylmethyl substituted derivatives of Fosmidomycin: synthesis and antimalarial activity.

    PubMed

    Schlüter, Katrin; Walter, Rolf D; Bergmann, Bärbel; Kurz, Thomas

    2006-12-01

    The phosphonohydroxamic acid Fosmidomycin is a drug candidate for the treatment of Malaria, currently in phase II trials in combination with Clindamycin. In order to obtain compounds of higher lipophilicity, we recently synthesized alpha-phenyl substituted Fosmidomycin derivatives which display high antimalarial activity. We now report the synthesis and in vitro antimalarial activity of arylmethyl substituted bis(pivaloyloxymethyl) ester prodrugs of Fosmidomycin and its acetyl analogue FR900098. The 3,4-dichlorobenzyl substituted derivative of Fosmidomycin proved to be about twice as active as the respective Fosmidomycin prodrug, however, less active than the corresponding FR900098 prodrug. Electron donating substituents as well as voluminous substituents led to a significant reduction of activity. PMID:17055117

  3. Functional analysis of the BRI1 receptor kinase by Thr-for-Ser substitution in a regulatory autophosphorylation site

    PubMed Central

    Oh, Man-Ho; Bender, Kyle W.; Kim, Sang Y.; Wu, Xia; Lee, Seulki; Nou, Ill-Sup; Zielinski, Raymond E.; Clouse, Steven D.; Huber, Steven C.

    2015-01-01

    BRI1 becomes highly phosphorylated in vivo upon perception of the ligand, brassinolide, as a result of autophosphorylation and transphosphorylation by its co-receptor kinase, BAK1. Important autophosphorylation sites include those involved in activation of kinase activity and those that are inhibitory, such as Ser-891. The inhibitory sites are autophosphorylated after kinase activation has been achieved and are postulated to contribute to deactivation of the kinase. The function of phosphosites is usually tested by substituting a non-phosphorylatable residue or an acidic residue that can act as a phosphomimetic. What has typically not been examined is substitution of a Thr for a Ser phosphosite (or vice versa) but given that Thr and Ser are not equivalent amino acids this type of substitution may represent a new approach to engineer regulatory phosphorylation. In the present study with BRI1, we substituted Thr at the Ser-891 phosphosite to generate the S891T directed mutant. The recombinant Flag-BRI1 (S891T) cytoplasmic domain protein (the S891T protein) was catalytically active and phosphorylation occurred at the engineered Thr-891 site. However, the S891T recombinant protein autophosphorylated more slowly than the wild-type protein during expression in E. coli. As a result, activation of peptide kinase activity (measured in vitro) was delayed as was transphosphorylation of bacterial proteins in situ. Stable transgenic expression of BRI1 (S891T)-Flag in Arabidopsis bri1-5 plants did not fully rescue the brassinosteroid (BR) phenotype indicating that BR signaling was constrained. Our working model is that restricted signaling in the S891T plants occurs as a result of the reduced rate of activation of the mutant BRI1 kinase by autophosphorylation. These results provide the platform for future studies to critically test this new model in vivo and establish Ser-Thr substitutions at phosphosites as an interesting approach to consider with other protein kinases. PMID

  4. Amorphous Semiconductor Nanowires Created by Site-Specific Heteroatom Substitution with Significantly Enhanced Photoelectrochemical Performance.

    PubMed

    He, Ting; Zu, Lianhai; Zhang, Yan; Mao, Chengliang; Xu, Xiaoxiang; Yang, Jinhu; Yang, Shihe

    2016-08-23

    Semiconductor nanowires that have been extensively studied are typically in a crystalline phase. Much less studied are amorphous semiconductor nanowires due to the difficulty for their synthesis, despite a set of characteristics desirable for photoelectric devices, such as higher surface area, higher surface activity, and higher light harvesting. In this work of combined experiment and computation, taking Zn2GeO4 (ZGO) as an example, we propose a site-specific heteroatom substitution strategy through a solution-phase ions-alternative-deposition route to prepare amorphous/crystalline Si-incorporated ZGO nanowires with tunable band structures. The substitution of Si atoms for the Zn or Ge atoms distorts the bonding network to a different extent, leading to the formation of amorphous Zn1.7Si0.3GeO4 (ZSGO) or crystalline Zn2(GeO4)0.88(SiO4)0.12 (ZGSO) nanowires, respectively, with different bandgaps. The amorphous ZSGO nanowire arrays exhibit significantly enhanced performance in photoelectrochemical water splitting, such as higher and more stable photocurrent, and faster photoresponse and recovery, relative to crystalline ZGSO and ZGO nanowires in this work, as well as ZGO photocatalysts reported previously. The remarkable performance highlights the advantages of the ZSGO amorphous nanowires for photoelectric devices, such as higher light harvesting capability, faster charge separation, lower charge recombination, and higher surface catalytic activity. PMID:27494205

  5. New potent inhibitors of tyrosinase: novel clues to binding of 1,3,4-thiadiazole-2(3H)-thiones, 1,3,4-oxadiazole-2(3H)-thiones, 4-amino-1,2,4-triazole-5(4H)-thiones, and substituted hydrazides to the dicopper active site.

    PubMed

    Ghani, Usman; Ullah, Nisar

    2010-06-01

    A series of 1,3,4-thiadiazole-2(3H)-thiones, 1,3,4-oxadiazole-2(3H)-thiones, 4-amino-1,2,4-triazole-5(4H)-thiones, and substituted hydrazides were tailored and synthesized as new potent inhibitors of tyrosinase. The rationale for inhibitor design was based on the active site structural evidence from the crystal structures of bacterial tyrosinase and potato catechol oxidase enzymes. Kinetic and active site binding studies suggested mono-dentate binding of thiadiazole, oxadiazole, and triazole rings to the active site dicopper center of tyrosinase including hydrophobicity contributing to the potent inhibition. Kinetic plots showed mixed-type of inhibition by all 25 compounds. Substitutions at C3 of the triazole ring and C5 of the thiadiazole/oxadiazole rings were found to be playing a major role in the high binding affinity to tyrosinase. The current work may help develop new potent tyrosinase inhibitors against hyperpigmentation including potential insecticides. PMID:20452224

  6. Synthesis and herbicidal activity of phenyl-substituted benzoylpyrazoles.

    PubMed

    Siddall, Thomas L; Ouse, David G; Benko, Zoltan L; Garvin, Gail M; Jackson, Johnny L; McQuiston, Jeffrey M; Ricks, Michael J; Thibault, Thomas D; Turner, James A; Vanheertum, John C; Weimer, Monte R

    2002-12-01

    A novel series of substituted 3-phenyl benzoylpyrazoles were prepared and tested as potential grass herbicides. The targeted materials were prepared by three newly developed synthetic routes, which allowed a comprehensive study of the SAR (structure-activity relationships) of this series. The best combination of grass weed activity (Avena fatua L, Setaria viridis (L) Beauv and Alopecurus myosuroides Huds) and wheat selectivity was obtained with an alkoxy group in the 4-position of the phenyl ring. Activity was further enhanced by the presence of tert-butyl on the pyrazole and a methyl group at the C-2 position of the benzoyl moiety. The alkoxy-substituted 3-phenylbenzoylpyrazoles are a novel class of herbicides with potential utility for control of important grass weeds in cereals. PMID:12476990

  7. Detecting Gradients of Asymmetry in Site-Specific Substitutions in Mitochondrial Genomes

    PubMed Central

    KRISHNAN, NEERAJA M.; SELIGMANN, HERVÈ; RAINA, SAMEER Z.; POLLOCK, DAVID D.

    2010-01-01

    During mitochondrial replication, spontaneous mutations occur and accumulate asymmetrically during the time spent single stranded by the heavy strand (DssH). The predominant mutations appear to be deaminations from adenine to hypoxanthine (A → H, which leads to an A → G substitution) and cytosine to thymine (C → T). Previous findings indicated that C → T substitutions accumulate rapidly and then saturate at high DssH, suggesting protection or repair, whereas A → G accumulates linearly with DssH. We describe here the implementation of a simple hidden Markov model (HMM) of among-site rate correlations to provide an almost continuous profile of the asymmetry in substitution response for any particular substitution type. We implement this model using a phylogeny-based Bayesian Markov chain Monte Carlo (MCMC) approach. We compare and contrast the relative asymmetries in all 12 possible substitution types, and find that the observed transition substitution responses determined using our new method agree quite well with previous predictions of a saturating curve for C → T transition substitutions and a linear accumulation of A → G transitions. The patterns seen in transversion substitutions show much lower among-site variation, and are nonlinear and more complex than those seen in transitions. We also find that, after accounting for the principal linear effect, some of the residual variation in A → G/G → A response ratios is explained by the average predicted nucleic acid secondary structure propensity at a site, possibly due to protection from mutation when secondary structure forms. PMID:15585129

  8. Phase stability and site substitution in Nb-Al compounds

    SciTech Connect

    Sluiter, M.; Takahashi, M.; Kawazoe, Y.

    1995-07-01

    The site preferences of Ta, Mo, and W in A15 Nb{sub 3}Al, the phase stability of off-stoichiometric A15 Nb{sub 3}Al, and the atomic order of the Nb{sub 2}Al D8{sub b} ({sigma}) phase are investigated by performing electronic structure calculations based on the local density approximation. Mo, W, and Ta ale found to have a preference for the Nb sublattice. Ta has the strongest site preference. It was shown that some recently proposed ground states for off-stoichiometric A15 phases are unlikely to occur in the Nb-Al system. Moreover, it was shown that the atomic order in the D8{sub b} Nb{sub 2}Al phase contributes significantly to its stability.

  9. Effect of Heating on DPPH Radical Scavenging Activity of Meat Substitute

    PubMed Central

    Song, Hyeun Sung; Bae, Jun Kyu; Park, Inshik

    2013-01-01

    This study was conducted to evaluate the increase of DPPH radical scavenging activity of meat substitute by heating. The meat substitute showed higher DPPH radical scavenging activity than those of other foods rich in protein such as beef, pork, chicken, and soybean curd. The DPPH radical scavenging activity of meat substitute was dependent upon concentration, heating temperature and heating time of meat substitute. The DPPH radical scavenging activity of meat substitute was enhanced with increasing heating temperature and time. The increase of DPPH radical scavenging activity was only applied to meat substitute without showing any activation in other foods rich in protein such as beef, pork, chicken, and soybean curd. PMID:24471114

  10. Anti-arthritic and immunosuppressive activities of substituted triterpenoidal candidates.

    PubMed

    Alanazi, Amer M; Al-Omar, Mohamed A; Abdulla, Mohamed M; Amr, Abd El-Galil E

    2013-07-01

    We herein report the anti-arthritic and immunosuppressive activities of some synthesized substituted terpenoidal structure. Forty-four triterpenoid derivatives 1-21 containing a carboxylic, ester, amide and ketone groups attached to a triterpene moiety were conveniently synthesized and screened for their anti-arthritic and immunosuppressive activities. Synthetic triterpenoidal structures linked to a different function groups seem to be a promising approach in the search for novel leads for potent anti-arthritic and immunosuppressive agents. The detailed synthetic pathways of obtained compounds and anti-arthritic and immunosuppressive activities were reported. PMID:23603083

  11. Modeling of Ternary Element Site Substitution in NiAl

    NASA Technical Reports Server (NTRS)

    Bozzolo, Guillermo; Noebe, Ronald D.; Honecy, Frank

    2000-01-01

    It is well recognized that ternary alloying additions can have a dramatic impact on the behavior of ordered intermetallic alloys such as nickel aluminides. Properties as diverse as yield strength, fracture strength, fracture mode, cyclic oxidation resistance, creep strength, and thermal and electrical diffusivity can change by orders of magnitude when a few percent or less of a ternary element is added. Yet our understanding of the resulting point defect structures and the simple site preferences of ternary alloying additions is poor because these are extremely difficult characteristics to determine. This disconnection between the understanding of the structure and properties in ordered alloys is at least in part responsible for the limited development and commercialization of these materials. Theoretical methods have provided useful but limited insight in this area, since most techniques suffer from constraints in the type of elements and the crystallographic structures that can be modeled. In an effort to overcome these limitations, the Bozzolo-Ferrante-Smith (BFS) method for alloys was designed. After a brief description of this approximate quantum mechanical approach, we use BFS to investigate the energetics of Si, Ti, V, Cr, Fe, Co, Cu, Zr, Nb, Mo, Ru, Hf, Ta and W additions to B2-ordered, stoichiometric NiAl. In addition to determining the site preference for these alloying additions over a range of compositions, we include results for the concentration dependence of the lattice parameter. In this introductory paper, we performed our analyses in the absence of constitutional and thermal vacancies for alloys of the form Ni50(Al,X)50. Where data exist, a comparison between experimental, theoretical, and BFS results is also included.

  12. A novel method for protein-protein interaction site prediction using phylogenetic substitution models

    PubMed Central

    La, David; Kihara, Daisuke

    2011-01-01

    Protein-protein binding events mediate many critical biological functions in the cell. Typically, functionally important sites in proteins can be well identified by considering sequence conservation. However, protein-protein interaction sites exhibit higher sequence variation than other functional regions, such as catalytic sites of enzymes. Consequently, the mutational behavior leading to weak sequence conservation poses significant challenges to the protein-protein interaction site prediction. Here, we present a phylogenetic framework to capture critical sequence variations that favor the selection of residues essential for protein-protein binding. Through the comprehensive analysis of diverse protein families, we show that protein binding interfaces exhibit distinct amino acid substitution as compared with other surface residues. Based on this analysis, we have developed a novel method, BindML, which utilizes the substitution models to predict protein-protein binding sites of protein with unknown interacting partners. BindML estimates the likelihood that a phylogenetic tree of a local surface region in a query protein structure follows the substitution patterns of protein binding interface and non-binding surfaces. BindML is shown to perform well compared to alternative methods for protein binding interface prediction. The methodology developed in this study is very versatile in the sense that it can be generally applied for predicting other types of functional sites, such as DNA, RNA, and membrane binding sites in proteins. PMID:21989996

  13. Anticancer Activity of Methyl-Substituted Oxaliplatin Analogs†

    PubMed Central

    Jungwirth, Ute; Xanthos, Dimitris N.; Gojo, Johannes; Bytzek, Anna K.; Körner, Wilfried; Heffeter, Petra; Abramkin, Sergey A.; Jakupec, Michael A.; Hartinger, Christian G.; Windberger, Ursula; Galanski, Markus; Keppler, Bernhard K.; Berger, Walter

    2012-01-01

    Oxaliplatin is successfully used in systemic cancer therapy. However, resistance development and severe adverse effects are limiting factors for curative cancer treatment with oxaliplatin. The purpose of this study was to comparatively investigate in vitro and in vivo anticancer properties as well as the adverse effects of two methyl-substituted enantiomerically pure oxaliplatin analogs [[(1R,2R,4R)-4-methyl-1,2-cyclohexanediamine] oxalatoplatinum(II) (KP1537), and [(1R,2R,4S)-4-methyl-1,2-cyclohexanediamine]oxalatoplatinum(II) (KP1691)] and to evaluate the impact of stereoisomerism. Although the novel oxaliplatin analogs demonstrated in multiple aspects activities comparable with those of the parental compound, several key differences were discovered. The analogs were characterized by reduced vulnerability to resistance mechanisms such as p53 mutations, reduced dependence on immunogenic cell death induction, and distinctly attenuated adverse effects including weight loss and cold hyperalgesia. Stereoisomerism of the substituted methyl group had a complex and in some aspects even contradictory impact on drug accumulation and anticancer activity both in vitro and in vivo. To summarize, methyl-substituted oxaliplatin analogs harbor improved therapeutic characteristics including significantly reduced adverse effects. Hence, they might be promising metal-based anticancer drug candidates for further (pre)clinical evaluation. PMID:22331606

  14. Allegations of diversion and substitution of crude oil. Bayou Choctaw Storage Site, Strategic Petroleum Reserve

    SciTech Connect

    Not Available

    1984-03-30

    Investigation did not substantiate allegations that crude oil destined for the Strategic Petroleum Reserve storage site at Bayou Choctaw was diverted to private use and some other material substituted in its place. However, recommendations are made for handling intermediate transport and storage systems for crude oil to tighten security aspects. (PSB)

  15. Active site specificity of plasmepsin II.

    PubMed Central

    Westling, J.; Cipullo, P.; Hung, S. H.; Saft, H.; Dame, J. B.; Dunn, B. M.

    1999-01-01

    Members of the aspartic proteinase family of enzymes have very similar three-dimensional structures and catalytic mechanisms. Each, however, has unique substrate specificity. These distinctions arise from variations in amino acid residues that line the active site subsites and interact with the side chains of the amino acids of the peptides that bind to the active site. To understand the unique binding preferences of plasmepsin II, an enzyme of the aspartic proteinase class from the malaria parasite, Plasmodium falciparum, chromogenic octapeptides having systematic substitutions at various positions in the sequence were analyzed. This enabled the design of new, improved substrates for this enzyme (Lys-Pro-Ile-Leu-Phe*Nph-Ala/Glu-Leu-Lys, where * indicates the cleavage point). Additionally, the crystal structure of plasmepsin II was analyzed to explain the binding characteristics. Specific amino acids (Met13, Ser77, and Ile287) that were suspected of contributing to active site binding and specificity were chosen for site-directed mutagenesis experiments. The Met13Glu and Ile287Glu single mutants and the Met13Glu/Ile287Glu double mutant gain the ability to cleave substrates containing Lys residues. PMID:10548045

  16. Redox Potentials, Laccase Oxidation, and Antilarval Activities of Substituted Phenols

    PubMed Central

    Prasain, Keshar; Nguyen, Thi D. T.; Gorman, Maureen J.; Barrigan, Lydia M.; Peng, Zeyu; Kanost, Michael R.; Syed, Lateef U.; Li, Jun; Zhu, Kun Yan; Hua, Duy H.

    2012-01-01

    Laccases are copper-containing oxidases that are involved in sclerotization of the cuticle of mosquitoes and other insects. Oxidation of exogenous compounds by insect laccases may have the potential to produce reactive species toxic to insects. We investigated two classes of substituted phenolic compounds, halogenated di- and trihydroxybenzenes and substituted di-tert-butylphenols, on redox potential, oxidation by laccase and effects on mosquito larval growth. An inverse correlation between the oxidation potentials and laccase activity of halogenated hydroxybenzenes was found. Substituted di-tert-butylphenols however were found to impact mosquito larval growth and survival. In particular, 2,4-di-tert-butyl-6-(3-methyl-2-butenyl)phenol (15) caused greater than 98% mortality of Anopheles gambiae larvae in a concentration of 180 nM, whereas 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-methylpropanal oxime (13) and 6,8-di-tert-butyl-2,2-dimethyl-3,4-dihydro-2H-chromene (33) caused 93% and 92% mortalities in concentrations of 3.4 and 3.7 μM, respectively. Larvae treated with di-tert-butylphenolic compounds died just before pupation. PMID:22300888

  17. Site preference and magnetic properties of Ga/In-substituted strontium hexaferrite: An ab initio study

    NASA Astrophysics Data System (ADS)

    Dixit, Vivek; Nandadasa, Chandani N.; Kim, Seong-Gon; Kim, Sungho; Park, Jihoon; Hong, Yang-Ki

    2015-11-01

    The first-principles density functional theory has been used to study Ga/In-substituted strontium hexaferrite (SrFe12O19). Based on the calculation of the substitution energy of Ga and In in SrFe12O19 and the formation probability analysis, we conclude that in SrFe12-xGaxO19 the substituted Ga atoms prefer to occupy the 12k, 2a, and 4f1 sites, while In atoms in SrFe12-xInxO19 occupy the 12k, 4f2, and 4f1 sites. We used the site occupation probabilities to calculate the magnetic properties of the substituted SrFe12O19. It was found that as the fraction of Ga atoms in SrFe12-xGaxO19 increases, the saturation magnetization (Ms) as well as magnetic anisotropy energy (MAE) decrease, while the anisotropy field (Ha) increases. In the case of SrFe12-xInxO19, Ms, MAE, and Ha decrease with an increase of the concentration of In atoms.

  18. Site preference and magnetic properties of Ga/In-substituted strontium hexaferrite: An ab initio study

    SciTech Connect

    Dixit, Vivek; Nandadasa, Chandani N.; Kim, Seong-Gon; Kim, Sungho; Park, Jihoon; Hong, Yang-Ki

    2015-11-28

    The first-principles density functional theory has been used to study Ga/In-substituted strontium hexaferrite (SrFe{sub 12}O{sub 19}). Based on the calculation of the substitution energy of Ga and In in SrFe{sub 12}O{sub 19} and the formation probability analysis, we conclude that in SrFe{sub 12−x}Ga{sub x}O{sub 19} the substituted Ga atoms prefer to occupy the 12k, 2a, and 4f{sub 1} sites, while In atoms in SrFe{sub 12−x}In{sub x}O{sub 19} occupy the 12k, 4f{sub 2}, and 4f{sub 1} sites. We used the site occupation probabilities to calculate the magnetic properties of the substituted SrFe{sub 12}O{sub 19}. It was found that as the fraction of Ga atoms in SrFe{sub 12−x}Ga{sub x}O{sub 19} increases, the saturation magnetization (M{sub s}) as well as magnetic anisotropy energy (MAE) decrease, while the anisotropy field (H{sub a}) increases. In the case of SrFe{sub 12−x}In{sub x}O{sub 19}, M{sub s}, MAE, and H{sub a} decrease with an increase of the concentration of In atoms.

  19. Correlating valence state, site preference and co-substitution to the magnetoelastic properties of cobalt ferrite

    NASA Astrophysics Data System (ADS)

    Nlebedim, Cajetan; Jiles, David

    2015-03-01

    Understanding how to influence the physics of magnetism, especially the relationship between magnetic susceptibility and stress, can be very useful in designing non-contact stress and torque sensors using magnetoelastic materials. This is particularly important considering that materials rarely occur in states desirable for direct applications. In this work we show that the magnetoelastic properties of cobalt ferrite are strongly dependent on the valence states and site preferences of substituted cations. It was found that co-substitution of magnetic and non-magnetic cations, is key to achieving simultaneous improvement in magnetostriction amplitude and strain sensitivity to applied magnetic field. Nevertheless, Curie temperature decreased, irrespective of the valence state, site preference or co-substitution. This presentation will show why tetravalent Ge resulted in superior magnetostrictive properties compared to other tetravalent, trivalent and divalent cations substituted into the crystal lattice of cobalt ferrite. This work was supported by the U.S. DOE, Office of Science, Basic Energy Sciences, Materials Science and Engineering Division. The research was performed at Ames Laboratory, operated for the USDoE by Iowa State University (Contract #DE-AC02-07CH11358).

  20. Syntheses and biological activities of 13-substituted avermectin aglycons.

    PubMed

    Mrozik, H; Linn, B O; Eskola, P; Lusi, A; Matzuk, A; Preiser, F A; Ostlind, D A; Schaeffer, J M; Fisher, M H

    1989-02-01

    The reactions of sulfonate esters of the allylic/homoallylic 13-alcohol of 5-O-(tert-butyldimethylsilyl)-22,23-dihydroavermectin B1a aglycon (1a) were investigated. Nucleophilic substitution gave 13 beta-chloro and 13 beta-iodo derivatives, while solvolytic reaction conditions yielded 13 alpha-methoxy, 13 alpha-fluoro, and 13 alpha-chloro products. A mixture of 13 alpha- and 13 beta-fluorides was obtained upon reaction with DAST. The 13 beta-iodide gave, upon elimination with lutidine, the 8(9),10(11),12(13),14(15)-tetraene. The 13 beta-alcohol and the rearranged 15-ol 13(14)-ene and 15-amino 13(14)-ene derivatives were obtained by substitution via the allylic carbonium ion. MEM ethers 11 and 12 of the two epimeric 13-ols were prepared by alkylation with MEM chloride. In contrast, methylation of 1a with MeI and Ag2O in CH2Cl2 occurred exclusively at the tertiary 7-hydroxy group and not at the secondary 13 alpha-ol. Oxidation of the allylic alcohol 1a proceeded under Swern conditions but not with MnO2 to the 13-oxo aglycon, which was reduced by NaBH4 exclusively to the natural 13 alpha-ol, while reductive amination with NaCNBH3-NH4OAc gave the 13 alpha-amine. The methoxime derivative was obtained in the form of the two geometric isomers. Anthelmintic activities against the sheep nematode Trichostrongylus colubriformis, miticidal activities against the two-spotted spider mite (Tetranychus urticae), and insecticidal activities against the southern armyworm (Spodoptera eridania) as well as the binding constants to a free living nematode (Caenorhabditis elegans) derived receptor assay were obtained and compared to avermectin B1a, 22,23-dihydroavermectin B1a, and the 13-deoxy-22,23-dihydroavermectin B1 aglycon related to the milbemycins. None of the newly prepared derivatives exceeded the potency of the three reference compounds. Lipophilic 13-substituents such as halogen, alkoxy, and methoxime retained high biological activities in all assays, while the more polar

  1. Cytotoxic activities of several geranyl-substituted flavanones.

    PubMed

    Smejkal, Karel; Svacinová, Jana; Slapetová, Tereza; Schneiderová, Kristýna; Dall'acqua, Stefano; Innocenti, Gabbriella; Závalová, Veronika; Kollár, Peter; Chudík, Stanislav; Marek, Radek; Julínek, Ondrej; Urbanová, Marie; Kartal, Murat; Csöllei, Marek; Dolezal, Karel

    2010-04-23

    Nine geranylated flavanones isolated from the fruits of Paulownia tomentosa (4-12) and two from the roots of Morus alba (13 and 14) were examined for cytotoxicity to selected human cancer cell lines and normal human fibroblasts. Cytotoxicity was determined in vitro using a calcein AM cytotoxicity assay. Cytotoxicity for the THP-1 monocytic leukemia cell line was tested using erythrosin B cell staining. The geranylated compounds tested were compared with the known simple flavanone standards taxifolin (1), naringenin (2), and hesperetin (3) and with the standard anticancer drugs olomoucine II, diaziquone, and oxaliplatin and the antineoplastic compound camptothecin, and showed different levels of cytotoxicity. The effects of structural changes on cytotoxic activity, including geranyl substitution of the flavanone skeleton and the oxidation pattern of ring B of the flavanones, are discussed. PMID:20192247

  2. Synthesis, antimicrobial, and antiproliferative activities of substituted phenylfuranylnicotinamidines

    PubMed Central

    Youssef, Magdy M; Arafa, Reem K; Ismail, Mohamed A

    2016-01-01

    This research work deals with the design and synthesis of a series of substituted phenylfuranylnicotinamidines 4a–i. Facile preparation of the target compounds was achieved by Suzuki coupling-based synthesis of the nitrile precursors 3a–i, followed by their conversion to the corresponding nicotinamidines 4a–i utilizing LiN(TMS)2. The antimicrobial activities of the newly synthesized nicotinamidine derivatives were evaluated against the Gram-negative bacterial strains Escherichia coli and Pseudomonas aeruginosa as well as the Gram-positive bacterial strains Staphylococcus aureus and Bacillus megaterium. The minimum inhibitory concentration values of nicotinamidines against all tested microorganisms were in the range of 10–20 μM. In specific, compounds 4a and 4b showed excellent minimum inhibitory concentration values of 10 μM against Staphylococcus aureus bacterial strain and were similar to ampicillin as an antibacterial reference. On the other hand, selected nicotinamidine derivatives were biologically screened for their cytotoxic activities against a panel of 60 cell lines representing nine types of human cancer at a single high dose at National Cancer Institute, Bethesda, MD, USA. Nicotinamidines showing promising activities were further assessed in a five-dose screening assay to determine their compound concentration causing 50% growth inhibition of tested cell (GI50), compound concentration causing 100% growth inhibition of tested cell (TGI), and compound concentration causing 50% lethality of tested cell (LC50) values. Structure-activity relationship studies demonstrated that the activity of members of this series can be modulated from cytostatic to cytotoxic based on the substitution pattern/nature on the terminal phenyl ring. The most active compound was found to be 4e displaying a submicromolar GI50 value of 0.83 μM, with TGI and LC50 values of 2.51 and 100 μM, respectively. Finally, the possible underlying mechanism of action of this series of

  3. Synthesis, antimicrobial, and antiproliferative activities of substituted phenylfuranylnicotinamidines.

    PubMed

    Youssef, Magdy M; Arafa, Reem K; Ismail, Mohamed A

    2016-01-01

    This research work deals with the design and synthesis of a series of substituted phenylfuranylnicotinamidines 4a-i. Facile preparation of the target compounds was achieved by Suzuki coupling-based synthesis of the nitrile precursors 3a-i, followed by their conversion to the corresponding nicotinamidines 4a-i utilizing LiN(TMS)2. The antimicrobial activities of the newly synthesized nicotinamidine derivatives were evaluated against the Gram-negative bacterial strains Escherichia coli and Pseudomonas aeruginosa as well as the Gram-positive bacterial strains Staphylococcus aureus and Bacillus megaterium. The minimum inhibitory concentration values of nicotinamidines against all tested microorganisms were in the range of 10-20 μM. In specific, compounds 4a and 4b showed excellent minimum inhibitory concentration values of 10 μM against Staphylococcus aureus bacterial strain and were similar to ampicillin as an antibacterial reference. On the other hand, selected nicotinamidine derivatives were biologically screened for their cytotoxic activities against a panel of 60 cell lines representing nine types of human cancer at a single high dose at National Cancer Institute, Bethesda, MD, USA. Nicotinamidines showing promising activities were further assessed in a five-dose screening assay to determine their compound concentration causing 50% growth inhibition of tested cell (GI50), compound concentration causing 100% growth inhibition of tested cell (TGI), and compound concentration causing 50% lethality of tested cell (LC50) values. Structure-activity relationship studies demonstrated that the activity of members of this series can be modulated from cytostatic to cytotoxic based on the substitution pattern/nature on the terminal phenyl ring. The most active compound was found to be 4e displaying a submicromolar GI50 value of 0.83 μM, with TGI and LC50 values of 2.51 and 100 μM, respectively. Finally, the possible underlying mechanism of action of this series of

  4. Sodium channel activation mechanisms. Insights from deuterium oxide substitution

    SciTech Connect

    Alicata, D.A.; Rayner, M.D.; Starkus, J.G. )

    1990-04-01

    Schauf and Bullock, using Myxicola giant axons, demonstrated that solvent substitution with deuterium oxide (D2O) significantly affects both sodium channel activation and inactivation kinetics without corresponding changes in gating current or tail current rates. They concluded that (a) no significant component of gating current derives from the final channel opening step, and (b) channels must deactivate (during tail currents) by a different pathway from that used in channel opening. By contrast, Oxford found in squid axons that when a depolarizing pulse is interrupted by a brief (approximately 100 microseconds) return to holding potential, subsequent reactivation (secondary activation) is very rapid and shows almost monoexponential kinetics. Increasing the interpulse interval resulted in secondary activation rate returning towards control, sigmoid (primary activation) kinetics. He concluded that channels open and close (deactivate) via the same pathway. We have repeated both sets of observations in crayfish axons, confirming the results obtained in both previous studies, despite the apparently contradictory conclusions reached by these authors. On the other hand, we find that secondary activation after a brief interpulse interval (50 microseconds) is insensitive to D2O, although reactivation after longer interpulse intervals (approximately 400 microseconds) returns towards a D2O sensitivity similar to that of primary activation. We conclude that D2O-sensitive primary activation and D2O-insensitive tail current deactivation involve separate pathways. However, D2O-insensitive secondary activation involves reversal of the D2O-insensitive deactivation step. These conclusions are consistent with parallel gate models, provided that one gating particle has a substantially reduced effective valence.

  5. LAT-1 activity of meta-substituted phenylalanine and tyrosine analogs.

    PubMed

    Augustyn, Evan; Finke, Karissa; Zur, Arik A; Hansen, Logan; Heeren, Nathan; Chien, Huan-Chieh; Lin, Lawrence; Giacomini, Kathleen M; Colas, Claire; Schlessinger, Avner; Thomas, Allen A

    2016-06-01

    The transporter protein Large-neutral Amino Acid Transporter 1 (LAT-1, SLC7A5) is responsible for transporting amino acids such as tyrosine and phenylalanine as well as thyroid hormones, and it has been exploited as a drug delivery mechanism. Recently its role in cancer has become increasingly appreciated, as it has been found to be up-regulated in many different tumor types, and its expression levels have been correlated with prognosis. Substitution at the meta position of aromatic amino acids has been reported to increase affinity for LAT-1; however, the SAR for this position has not previously been explored. Guided by newly refined computational models of the binding site, we hypothesized that groups capable of filling a hydrophobic pocket would increase binding to LAT-1, resulting in improved substrates relative to parent amino acid. Tyrosine and phenylalanine analogs substituted at the meta position with halogens, alkyl and aryl groups were synthesized and tested in cis-inhibition and trans-stimulation cell assays to determine activity. Contrary to our initial hypothesis we found that lipophilicity was correlated with diminished substrate activity and increased inhibition of the transporter. The synthesis and SAR of meta-substituted phenylalanine and tyrosine analogs is described. PMID:27106710

  6. N-substituted 2-isonicotinoylhydrazinecarboxamides--new antimycobacterial active molecules.

    PubMed

    Rychtarčíková, Zuzana; Krátký, Martin; Gazvoda, Martin; Komlóová, Markéta; Polanc, Slovenko; Kočevar, Marijan; Stolaříková, Jiřina; Vinšová, Jarmila

    2014-01-01

    This report presents a new modification of the isoniazid (INH) structure linked with different anilines via a carbonyl group obtained by two synthetic procedures and with N-substituted 5-(pyridine-4-yl)-1,3,4-oxadiazole-2-amines prepared by their cyclisation. All synthesised derivatives were characterised by IR, NMR, MS and elemental analyses and were evaluated in vitro for their antimycobacterial activity against Mycobacterium tuberculosis H37Rv, Mycobacterium avium 330/88, Mycobacterium kansasii 235/80 and one clinical isolated strain of M. kansasii 6509/96. 2-Isonicotinoyl-N-(4-octylphenyl)hydrazinecarboxamide displayed an in vitro efficacy comparable to that of INH for M. tuberculosis with minimum inhibitory concentrations (MICs) of 1-2 μM. Among the halogenated derivatives, the best anti-tuberculosis activity was found for 2-isonicotinoyl-N-(2,4,6-trichlorophenyl)hydrazinecarboxamide (MIC=4 μM). In silico modelling on the enoyl-acyl carrier protein reductase InhA confirmed that longer alkyl substituents are advantageous for the interactions and affinity to InhA. Most of the hydrazinecarboxamides, especially those derived from 4-alkylanilines, exhibited significant activity against INH-resistant nontuberculous mycobacteria. PMID:24686575

  7. Salt site performance assessment activities

    SciTech Connect

    Kircher, J.F.; Gupta, S.K.

    1983-01-01

    During this year the first selection of the tools (codes) for performance assessments of potential salt sites have been tentatively selected and documented; the emphasis has shifted from code development to applications. During this period prior to detailed characterization of a salt site, the focus is on bounding calculations, sensitivity and with the data available. The development and application of improved methods for sensitivity and uncertainty analysis is a focus for the coming years activities and the subject of a following paper in these proceedings. Although the assessments to date are preliminary and based on admittedly scant data, the results indicate that suitable salt sites can be identified and repository subsystems designed which will meet the established criteria for protecting the health and safety of the public. 36 references, 5 figures, 2 tables.

  8. Local salt substitutes “Obu-otoyo” activate acetylcholinesterase and butyrylcholinesterase and induce lipid peroxidation in rat brain

    PubMed Central

    Oboh, Ganiyu; Ademiluyi, Adedayo O.

    2015-01-01

    Evidence has shown that ingestion of heavy metals can lead to neurodegenerative diseases. This study aimed to investigate the neurotoxic potential of salt substitutes (Obu-Otoyo); salt A (made by burning palm kernel shaft then soaked in water overnight and the extract from the resulting residue is used as the salt substitute) and salt B (an unrefined salt mined from a local site at Ilobu town, Osun-State, Nigeria) by assessing their effect on some key enzymes linked with neurodegenerative disease [acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities] as well as on malondialdehyde (MDA) content of the rat brain. Salt substitutes were fed to normal rats as dietary inclusion at doses of 0.5 and 1.0% for 30 days. Thereafter, the effect of the salt substitutes on AChE and BChE activities as well as on MDA level in the rat brain was determined. The results revealed that the salt substitutes caused a significant (p<0.05) increase in both AChE and BChE activity and also induced lipid peroxidation in the brain of rats in vivo as well as under in vitro condition in a dose-dependent manner. The effect of the salt substitutes on AChE and BChE activities could be attributed to the presence of some toxic heavy metals. Therefore, the ability of the salt substitutes to induce lipid peroxidation and activate AChE and BChE activities could provide some possible mechanism for their neurotoxic effect. PMID:27486373

  9. Mutational Biases Drive Elevated Rates of Substitution at Regulatory Sites across Cancer Types.

    PubMed

    Kaiser, Vera B; Taylor, Martin S; Semple, Colin A

    2016-08-01

    Disruption of gene regulation is known to play major roles in carcinogenesis and tumour progression. Here, we comprehensively characterize the mutational profiles of diverse transcription factor binding sites (TFBSs) across 1,574 completely sequenced cancer genomes encompassing 11 tumour types. We assess the relative rates and impact of the mutational burden at the binding sites of 81 transcription factors (TFs), by comparing the abundance and patterns of single base substitutions within putatively functional binding sites to control sites with matched sequence composition. There is a strong (1.43-fold) and significant excess of mutations at functional binding sites across TFs, and the mutations that accumulate in cancers are typically more disruptive than variants tolerated in extant human populations at the same sites. CTCF binding sites suffer an exceptionally high mutational load in cancer (3.31-fold excess) relative to control sites, and we demonstrate for the first time that this effect is seen in essentially all cancer types with sufficient data. The sub-set of CTCF sites involved in higher order chromatin structures has the highest mutational burden, suggesting a widespread breakdown of chromatin organization. However, we find no evidence for selection driving these distinctive patterns of mutation. The mutational load at CTCF-binding sites is substantially determined by replication timing and the mutational signature of the tumor in question, suggesting that selectively neutral processes underlie the unusual mutation patterns. Pervasive hyper-mutation within transcription factor binding sites rewires the regulatory landscape of the cancer genome, but it is dominated by mutational processes rather than selection. PMID:27490693

  10. Mutational Biases Drive Elevated Rates of Substitution at Regulatory Sites across Cancer Types

    PubMed Central

    Semple, Colin A.

    2016-01-01

    Disruption of gene regulation is known to play major roles in carcinogenesis and tumour progression. Here, we comprehensively characterize the mutational profiles of diverse transcription factor binding sites (TFBSs) across 1,574 completely sequenced cancer genomes encompassing 11 tumour types. We assess the relative rates and impact of the mutational burden at the binding sites of 81 transcription factors (TFs), by comparing the abundance and patterns of single base substitutions within putatively functional binding sites to control sites with matched sequence composition. There is a strong (1.43-fold) and significant excess of mutations at functional binding sites across TFs, and the mutations that accumulate in cancers are typically more disruptive than variants tolerated in extant human populations at the same sites. CTCF binding sites suffer an exceptionally high mutational load in cancer (3.31-fold excess) relative to control sites, and we demonstrate for the first time that this effect is seen in essentially all cancer types with sufficient data. The sub-set of CTCF sites involved in higher order chromatin structures has the highest mutational burden, suggesting a widespread breakdown of chromatin organization. However, we find no evidence for selection driving these distinctive patterns of mutation. The mutational load at CTCF-binding sites is substantially determined by replication timing and the mutational signature of the tumor in question, suggesting that selectively neutral processes underlie the unusual mutation patterns. Pervasive hyper-mutation within transcription factor binding sites rewires the regulatory landscape of the cancer genome, but it is dominated by mutational processes rather than selection. PMID:27490693

  11. A DFT+U study of A-site and B-site substitution in BaFeO3-δ.

    PubMed

    Baiyee, Zarah Medina; Chen, Chi; Ciucci, Francesco

    2015-09-28

    BaFeO3-δ (BFO)-based perovskites have emerged as cheap and effective oxygen electrocatalysts for oxygen reduction reaction at high temperatures. The BFO cubic phase facilitates a high oxygen deficiency and is commonly stabilised by partial substitution. Understanding the electronic mechanisms of substitution and oxygen deficiency is key to rational material design, and can be realised through DFT analysis. In this work an in-depth first principle DFT+U study is undertaken to determine site distinctive characteristics for 12.5%, Y, La and Ce substitutions in BFO. In particular, it is shown that B-site doped structures exhibit a lower energy cost for oxygen vacancy formation relative to A site doping and pristine BFO. This is attributed to the stabilisation of holes in the oxygen sub-lattice and increased covalency of the Fe-O bonds of the FeO6 octahedra in B-site-substituted BFO. Charge analysis shows that A-site substitution amounts to donor doping and consequently impedes the accommodation of other donors (i.e. oxygen vacancies). However, A-site substitution may also exhibit a higher electronic conductivity due to less lattice distortion for oxygen deficiency compared to B-site doped structures. Furthermore, analysis of the local structural effects provides physical insight into stoichiometric expansions observed for this material. PMID:26295283

  12. Substitution of Val72 residue alters the enantioselectivity and activity of Penicillium expansum lipase.

    PubMed

    Tang, Lianghua; Su, Min; Zhu, Ling; Chi, Liying; Zhang, Junling; Zhou, Qiong

    2013-01-01

    Error-prone PCR was used to create more active or enantioselective variants of Penicillium expansum lipase (PEL). A variant with a valine to glycine substitution at residue 72 in the lid structure exhibited higher activity and enantioselectivity than those of wild-type PEL. Site-directed saturation mutagenesis was used to explore the sequence-function relationship and the substitution of Val72 of P. expansum lipase changed both catalytic activity and enantioselectivity greatly. The variant V72A, displayed a highest enantioselectivity enhanced to about twofold for the resolution of (R, S)-naproxen (E value increased from 104 to 200.7 for wild-type PEL and V72A variant, respectively). In comparison to PEL, the variant V72A showed a remarkable increase in specific activity towards p-nitrophenyl palmitate (11- and 4-fold increase at 25 and 35 °C, respectively) whereas it had a decreased thermostability. The results suggest that the enantioselective variant V72A could be used for the production of pharmaceutical drugs such as enantiomerically pure (S)-naproxen and the residue Val 72 of P. expansum lipase plays a significant role in the enantioselectivity and activity of this enantioselective lipase. PMID:22972595

  13. Site occupancy and magnetic properties of Al-substituted M-type strontium hexaferrite

    SciTech Connect

    Dixit, Vivek; Nandadasa, Chandani N.; Kim, Seong-Gon; Kim, Sungho; Park, Jihoon; Hong, Yang-Ki; Liyanage, Laalitha S. I.; Moitra, Amitava

    2015-06-28

    We use first-principles total-energy calculations based on density functional theory to study the site occupancy and magnetic properties of Al-substituted M-type strontium hexaferrite SrFe{sub 12−x}Al{sub x}O{sub 19} with x = 0.5 and x = 1.0. We find that the non-magnetic Al{sup 3+} ions preferentially replace Fe{sup 3+} ions at two of the majority spin sites, 2a and 12k, eliminating their positive contribution to the total magnetization causing the saturation magnetization M{sub s} to be reduced as Al concentration x is increased. Our formation probability analysis further provides the explanation for increased magnetic anisotropy field when the fraction of Al is increased. Although Al{sup 3+} ions preferentially occupy the 2a sites at a low temperature, the occupation probability of the 12k site increases with the rise of the temperature. At a typical annealing temperature (>700 °C) Al{sup 3+} ions are much more likely to occupy the 12k site than the 2a site. Although this causes the magnetocrystalline anisotropy K{sub 1} to be reduced slightly, the reduction in M{sub s} is much more significant. Their combined effect causes the anisotropy field H{sub a} to increase as the fraction of Al is increased, consistent with recent experimental measurements.

  14. Site occupancy and magnetic properties of Al-substituted M-type strontium hexaferrite

    NASA Astrophysics Data System (ADS)

    Dixit, Vivek; Nandadasa, Chandani N.; Kim, Seong-Gon; Kim, Sungho; Park, Jihoon; Hong, Yang-Ki; Liyanage, Laalitha S. I.; Moitra, Amitava

    2015-06-01

    We use first-principles total-energy calculations based on density functional theory to study the site occupancy and magnetic properties of Al-substituted M-type strontium hexaferrite SrFe12-xAlxO19 with x = 0.5 and x = 1.0. We find that the non-magnetic Al3+ ions preferentially replace Fe3+ ions at two of the majority spin sites, 2a and 12k, eliminating their positive contribution to the total magnetization causing the saturation magnetization Ms to be reduced as Al concentration x is increased. Our formation probability analysis further provides the explanation for increased magnetic anisotropy field when the fraction of Al is increased. Although Al3+ ions preferentially occupy the 2a sites at a low temperature, the occupation probability of the 12k site increases with the rise of the temperature. At a typical annealing temperature (>700 °C) Al3+ ions are much more likely to occupy the 12k site than the 2a site. Although this causes the magnetocrystalline anisotropy K1 to be reduced slightly, the reduction in Ms is much more significant. Their combined effect causes the anisotropy field Ha to increase as the fraction of Al is increased, consistent with recent experimental measurements.

  15. 5-N-Substituted-2-(substituted benzenesulphonyl) glutamines as antitumor agents. Part II: synthesis, biological activity and QSAR study.

    PubMed

    Samanta, Soma; Srikanth, K; Banerjee, Suchandra; Debnath, Bikash; Gayen, Shovanlal; Jha, Tarun

    2004-03-15

    Cancer is a major killer disease throughout human history. Thus, cancer becomes a major point of interest in life science. It was proved that cancer is a nitrogen trap and tumor cells are avid glutamine consumers. The non-essential amino acid glutamine, which is a glutamic acid derivative, supplies its amide nitrogen to tumor cells in the biosynthesis of purine and pyrimidine bases of nucleic acids as well as takes part in protein synthesis. Based on these and in continuation of our composite programme of development of new potential anticancer agents through rational drug design, 17 new 5-N-Substituted-2-(substituted benzenesulphonyl) glutamines were selected for synthesis. These compounds as well as 36 earlier synthesized glutamine analogues were screened for antitumor activity using percentage inhibition of tumor cell count as the activity parameter. QSAR study was performed with 53 compounds in order to design leads with increased effectiveness for antitumor activity using both physicochemical and topological parameters. QSAR study showed that steric effect on the aromatic ring is conducive to the activity. n-butyl substitution on aliphatic side chain and atom no 12 is important for antitumor activity of glutamine analogues. PMID:15018914

  16. The Identity of the Nucleophile Substitution may Influence Metal Interactions with the Cleavage Site of the Minimal Hammerhead Ribozyme

    PubMed Central

    Osborne, Edith M.; Ward, W. Luke; Ruehle, Max Z.; DeRose, Victoria J.

    2010-01-01

    Potential metal interactions with the cleavage site of a minimal hammerhead ribozyme (mHHRz) were probed using 31P NMR-detected Cd2+ titration studies of HHRz constructs containing a phosphorothioate (PS) modification at the cleavage site. The mHHRz nucleophile position was replaced by either a 2′-F or a 2′-NH2 in order to block cleavage activity during the study. The 2′-F/PS cleavage site mHHRz construct, in which the 2′-F should closely imitate the atom size and electronegativity of a 2′OH, demonstrates low levels of metal ion association (<1 ppm 31P chemical shift changes). This observation indicates that having an atom size and electrostatic properties that are similar to the 2′-OH are not the governing factors in allowing metal interactions with the scissile phosphate of the mHHRz. With a 2′-NH2 substitution, a large upfield change in 31P NMR chemical shift of the phosphorothioate peak (Δ~3 ppm with 6 equivalents added Cd2+) indicates observable Cd2+ interactions with the substituted site. Since a 2′-NH2, but not a 2′-F, can serve as a metal ligand, these data suggest that a metal ion interaction with the HHRz cleavage site may include both the scissile phosphate and the 2′ nucleophile. Control samples in which the 2′-NH2/PS unit is placed either next to the mHHRz cleavage site (at U16.1), in a duplex, or in a amUPSU dinucleotide, show much weaker interactions with Cd2+. Results with these control samples indicate that simply the presence of a 2′-NH2/PS unit does not create a strong metal binding site, reinforcing the possibility that the 2′-NH2-moderated Cd-PS interaction is specific to the mHHRz cleavage site. Upfield chemical shifts of both 31P and H2′ 1H resonances in amUPSU are observed with addition of Cd2+, consistent with the predicted metal coordination to both 2′-NH2 and phosphorothioate ligands. These data suggest that metal ion association with the HHRz cleavage site may include an interaction with the 2

  17. Synthesis and Anticonvulsant Activities of (R)-N-(4′-Substituted) benzyl 2-Acetamido-3-methoxypropionamides

    PubMed Central

    Salomé, Christophe; Salomé-Grosjean, Elise; Park, Ki Duk; Morieux, Pierre; Swendiman, Robert; DeMarco, Erica; Stables, James P.; Kohn, Harold

    2010-01-01

    The structure-activity relationship (SAR) for the N-benzyl group in the clinical antiepileptic agent (R)-lacosamide ((R)-N-benzyl 2-acetamido-3-methoxypropionamide, (R)-3) has been explored. Forty-three compounds were prepared and then evaluated at the National Institute of Neurological Disorders and Stroke Anticonvulsant Screening Program for seizure protection in the maximal electroshock (MES) and subcutaneous Metrazol models. Comparing activities for two series of substituted aryl regioisomers (2′, 3′, 4′) showed that 4′-modified derivatives had the highest activity. Significantly, structural latitude existed at the 4′-site. The SAR indicated that non-bulky 4′-substituted (R)-3 derivatives exhibited superb activity, independent of their electronic properties. Activities in the MES test of several compounds were either comparable with or exceeded that of (R)-3, and surpassed the activities observed for the traditional antiepileptic agents phenytoin, phenobarbital, and valproate. PMID:20041718

  18. Using site-specific soil samples as a substitution for improved hydrological and nonpoint source predictions.

    PubMed

    Chen, Lei; Wang, Guobo; Zhong, Yucen; Zhao, Xin; Shen, Zhenyao

    2016-08-01

    Soil databases are one of the most important inputs for watershed models, and the quality of soil properties affects how well a model performs. The objectives of this study were to (1) quantify the sensitivity of model outputs to soil properties and to (2) use site-specific soil properties as a substitution for more accurate hydrological and nonpoint source (H/NPS) predictions. Soil samples were collected from a typical mountainous watershed in China, and the impacts of soil sample parameters on H/NPS predictions were quantified using the Soil and Water Assessment Tool (SWAT). The most sensitive parameters related to predicting flow, sediment, and total phosphorus (TP) mainly were the soil hydrological, the channel erosion processes, and the initial soil chemical environment, respectively. When the site-specific soil properties were used, the uncertainties (coefficient of variation) related to predicting the hydrology, sediment and TP decreased by 75∼80 %, 75∼84 %, and 46∼61 %, respectively. Based on changes in the Nash-Sutcliff coefficient, the model performance improved by 4.9 and 19.45 % for the hydrological and sediment model, accordingly. However, site-specific soil properties did not contribute to better TP predictions because of the high spatial variability of the soil P concentrations across the large watershed. Thus, although site-specific soil samples can be used to obtain more accurate H/NPS predictions, more sampling sites are required to apply this method in large watersheds. PMID:27146539

  19. C-H Bond Activation by Pd-substituted CeO2: Substituted Ions versus Reduced Species

    SciTech Connect

    Misch, Lauren M; Kurzman, Joshua A; Derk, Alan R; Kim, Young-Il; Seshadri, Ram; Metiu, Horia; McFarland, Eric W; Stucky, Galen D

    2012-02-07

    Substituted metal oxides containing ionic species have been attracting a great deal of attention because of their potential ability to reduce the usage of precious metals in heterogeneous catalysts. We investigate Pd-substituted CeO2 for C-H bond activation reactions including the partial oxidation and dry reforming of CH4. This catalyst has been previously studied for CO oxidation, NOx reduction, and the water-gas shift reaction. Pd-substituted CeO2, Ce1-xPdxO2-δ, was prepared as a powder with high surface area and a hollow sphere morphology using ultrasonic spray pyrolysis. The catalysts were extensively characterized using synchrotron X-ray diffraction and other techniques, confirming phase pure samples up to 10 mol % Pd substitution. Ce0.95Pd0.05O2-δ was found to be active for partial oxidation of CH4 around 500 °C and higher. Our studies, including postcatalytic synchrotron diffraction, suggest that the single-phase Ce1-xPdxO2-δ material is not the active species and that catalysis occurs instead over the reduced two-phase Pd0/CeO2. This observation has been further confirmed by verifying the activity of the reduced Pd0/CeO2 catalysts for ethylene hydrogenation, a reaction that is known to require Pd0.

  20. Securin can have a separase cleavage site by substitution mutations in the domain required for stabilization and inhibition of separase.

    PubMed

    Nagao, Koji; Yanagida, Mitsuhiro

    2006-03-01

    Securin-separase complex is required for sister chromatid separation. Securin degrades in an APC/cyclosome dependent manner. Separase is activated on the destruction of securin and cleaves cohesin. Fission yeast securin/Cut2 required for proper separase localization has the motifs for destruction and separase-binding at the N- and C-termini, respectively. We report here the third essential domain, which becomes toxic when the 76-amino acid fragment (81-156) in the middle is overproduced. The fragment inhibits separase, while separase is recruited normally and securin is destroyed. It may interfere with separase activation after destruction of securin. If the 127DIE129 stretch is substituted for AIA, the fragment toxicity and the full-length function are abolished. Interestingly, Cut2 is cleaved in a separase dependent manner if the cleavage consensus is introduced following the DIE sequence. This finding is consistent with the proposed model that the DIE region may mimic the cleavage site of separase and inhibit the activation of separase. Evidence for physical interaction between the fragment and separase is provided. A temperature sensitive mutation cut1-K73 isolated by its specific resistance to the fragment toxicity resides in the superhelical region of separase, suggesting that the catalytic site and the helical region in separase may cooperate for activation. PMID:16483313

  1. An Asp7Gly substitution in PPARG is associated with decreased transcriptional activation activity.

    PubMed

    Hua, Liushuai; Wang, Jing; Li, Mingxun; Sun, Xiaomei; Zhang, Liangzhi; Lei, Chuzhao; Lan, Xianyong; Fang, Xingtang; Zhao, Xin; Chen, Hong

    2014-01-01

    As the master regulator of adipogenesis, peroxisome proliferator-activated receptor gamma (PPARG) is required for the accumulation of adipose tissue and hence contributes to obesity. A previous study showed that the substitution of +20A>G in PPARG changed the 7(th) amino acid from Asp to Gly, creating a mutant referred to as PPARG Asp7Gly. In this study, association analysis indicated that PPARG Asp7Gly was associated with lower body height, body weight and heart girth in cattle (P<0.05). Overexpression of PPARG in NIH3T3-L1 cells showed that the Asp7Gly substitution may cause a decrease in its adipogenic ability and the mRNA levels of CIDEC (cell death-inducing DFFA-like effector c) and aP2, which are all transcriptionally activated by PPARG during adipocyte differentiation. A dual-luciferase reporter assay was used to analyze the promoter activity of CIDEC. The results confirmed that the mutant PPARG exhibited weaker transcriptional activation activity than the wild type (P<0.05). These findings likely explain the associations between the Asp7Gly substitution and the body measurements. Additionally, the Asp7Gly mutation may be used in molecular marker assisted selection (MAS) of cattle breeding in the future. PMID:24466299

  2. Red Shifted Absorbance of A-site Substituted Bismuth Titanate Pyrochlore: Characterization and Stability Analysis from First Principles

    NASA Astrophysics Data System (ADS)

    Mayfield, Cedric; Huda, Muhammad

    2015-03-01

    Transition metal inclusion has enhanced photocatalytic activity of bismuth titanate (Bi2Ti2O7) up to an impurity threshold concentration. Beyond the threshold, spectral absorbance is continually red shifted but increased photocurrent is not reciprocated. We investigated, from first principles, the origin of decreased photocurrent in modified Bi2Ti2O7 (BTO) by calculating the electronic structures of a representative set of doping configurations and by performing a phase stability analysis of the doping. We report our theoretical/computational strategy of analyzing free energy space and show an explicit dependence of pure phase synthesis on changes in free energy. Also, we present a probability distribution of the doping configurations based on formation enthalpy to better understand the nature of doping in BTO. We found that transition metal substitutions are favorable at the A-sites due to unchanging coordination with O ions. This work is supported by National Science Foundation, Award No. 1133672.

  3. Activation of c-Jun transcription factor by substitution of a charged residue in its N-terminal domain.

    PubMed Central

    Hoeffler, W K; Levinson, A D; Bauer, E A

    1994-01-01

    C-Jun is a cellular transcription factor that can control gene expression in response to treatment of cells with phorbol esters, growth factors, and expression of some oncogenes. The ability of c-Jun to catalyze the transcription of certain genes is controlled, in part, by changes in the phosphorylation state of specific amino acids in c-Jun. One of the major sites that is phosphorylated during signal response is Ser73. Here we show that substitution of a negatively charged aspartic acid residue at 73 constitutively increased transcriptional activity of c-Jun. The Asp73 substitution also enhanced its availability to bind to DNA in a whole cell extract without altering its intrinsic DNA binding activity since the intrinsic activity was unaltered for the c-Jun mutant proteins expressed in a bacterial system. The negatively charged Asp substitution may mimic the negative charge of a phosphorylated serine at 73. The substitution of an uncharged alanine at 73 resulted in lowered activities. The N-terminal end of c-Jun containing these substitutions was fused to the DNA-binding region of the bovine papilloma virus E2 protein, and was able to confer the same activation properties to the fusion protein at the heterologous E2 DNA-binding site. Ser73 lies in a region of c-Jun previously proposed to bind an uncharacterized inhibitor, perhaps related to a protein of approximately 17.5 kD that coprecipitates along with our c-Jun or the JunE2 fusion products. Images PMID:8165146

  4. Synthesis, cytotoxicity, and hemolytic activity of 6'-O-substituted dioscin derivatives.

    PubMed

    Li, Wei; Qiu, Zaozao; Wang, Yibing; Zhang, Yichun; Li, Ming; Yu, Jia; Zhang, Lihong; Zhu, Ziyan; Yu, Biao

    2007-12-28

    Dioscin derivatives (1-12) with a variety of substitutions at the 6'-OH of the chacotriosyl residue and the 3',6'-anhydrosaponin derivatives (26, 30, and 32) were synthesized. All these derivatives showed much lower cytotoxicity than that of the parent dioscin, while their hemolytic activities were partially retained depending on the various 6'-O-substitutions. PMID:17945208

  5. Synthesis and antitubercular activity of heterocycle substituted diphenyl ether derivatives

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Despite being an ancient disease, tuberculosis (TB) remains the leading single-agent infectious disease killer in the world. The emerging serious problem due to TB control and clinical management prompted us to synthesize novel series of heterocyclic substituted diphenyl ether derivatives and determ...

  6. Biphenyl-4-yl-acrylohydroxamic acids: Identification of a novel indolyl-substituted HDAC inhibitor with antitumor activity.

    PubMed

    Cincinelli, Raffaella; Zwick, Vincent; Musso, Loana; Zuco, Valentina; De Cesare, Michelandrea; Zunino, Franco; Simoes-Pires, Claudia; Nurisso, Alessandra; Giannini, Giuseppe; Cuendet, Muriel; Dallavalle, Sabrina

    2016-04-13

    Modification of the cap group of biphenylacrylohydroxamic acid-based HDAC inhibitors led to the identification of a new derivative (3) characterized by an indolyl-substituted 4-phenylcinnamic skeleton. Molecular docking was used to predict the optimal conformation in the class I HDACs active site. Compound 3 showed HDAC inhibitory activity and antiproliferative activity against a panel of tumor cell lines, in the low μM range. The compound was further tested in vitro for acetylation of histone H4 and other non-histone proteins, and in vivo in a colon carcinoma model, showing significant proapoptotic and antitumor activities. PMID:26890116

  7. Directed evolution of P-glycoprotein cysteines reveals site-specific, non-conservative substitutions that preserve multidrug resistance.

    PubMed

    Swartz, Douglas J; Mok, Leo; Botta, Sri K; Singh, Anukriti; Altenberg, Guillermo A; Urbatsch, Ina L

    2014-01-01

    Pgp (P-glycoprotein) is a prototype ABC (ATP-binding-cassette) transporter involved in multidrug resistance of cancer. We used directed evolution to replace six cytoplasmic Cys (cysteine) residues in Pgp with all 20 standard amino acids and selected for active mutants. From a pool of 75000 transformants for each block of three Cys, we identified multiple mutants that preserved drug resistance and yeast mating activity. The most frequent substitutions were glycine and serine for Cys427 (24 and 20%, respectively) and Cys1070 (37 and 25%) of the Walker A motifs in the NBDs (nucleotide-binding domains), Cys1223 in NBD2 (25 and 8%) and Cys638 in the linker region (24 and 16%), whereas close-by Cys669 tolerated glycine (16%) and alanine (14%), but not serine (absent). Cys1121 in NBD2 showed a clear preference for positively charged arginine (38%) suggesting a salt bridge with Glu269 in the ICL2 (intracellular loop 2) may stabilize domain interactions. In contrast, three Cys residues in transmembrane α-helices could be successfully replaced by alanine. The resulting CL (Cys-less) Pgp was fully active in yeast cells, and purified proteins displayed drug-stimulated ATPase activities indistinguishable from WT (wild-type) Pgp. Overall, directed evolution identified site-specific, non-conservative Cys substitutions that allowed building of a robust CL Pgp, an invaluable new tool for future functional and structural studies, and that may guide the construction of other CL proteins where alanine and serine have proven unsuccessful. PMID:24825346

  8. Chromium and Tantalum Site Substitution Patterns in Ni3Al (L1(sub 2))gamma(prime)- Precipitates

    NASA Technical Reports Server (NTRS)

    Booth-Morrison, Christopher; Mao, Zugang; Seidman, David N.; Noebe, Ronald D.

    2008-01-01

    The site substitution behavior of Cr and Ta in the Ni3Al (Ll2)-type gamma'-precipitates of a Ni-Al-Cr-Ta alloy is investigated by atom-probe tomography (APT) and first-principles calculations. Measurements of the gamma'-phase composition by APT suggest that Al, Cr, and Ta share the Al sublattice sites of the gamma'-precipitates. The calculated substitutional energies of the solute atoms at the Ni and Al sublattice sites indicate that Ta has a strong preference for the Al sites, while Cr has a weak Al site preference. Furthermore, Ta is shown to replace Cr at the Al sublattice sites of the gamma'-precipitates, altering the elemental phase partitioning behavior of the Ni-Al-Cr-Ta alloy.

  9. Highly fluorinated 2,2'-biphenols and related compounds: relationship between substitution pattern and herbicidal activity.

    PubMed

    Francke, Robert; Reingruber, Rüdiger; Schollmeyer, Dieter; Waldvogel, Siegfried R

    2013-05-22

    A broad range of halogenated 2,2'-biphenols was tested for applicability as crop protection agents. The activity of these compounds toward four typical pest plants was observed after application by spraying of diluted solutions. Despite their rather simple structure, it was found that the studied compounds reveal a surprisingly high herbicidal impact. To gain a better understanding of the structure-activity relationship, specific sites of the molecule were chemically modified and the core structures thus gradually changed. The influence of the substitution pattern on the herbicidal properties is discussed, and conclusions on the active site of the biphenol structure are drawn. It was observed that type and position of the halogen substituents have a significant influence on the activity of the core structure. The hydroxy functionalities play a crucial role for the effectiveness of the tested compounds. Because the blocking of the hydroxy moiety leads to dramatically deteriorated performances, the presence of these functionalities on the aromatic ring seems to be indispensable. PMID:23641939

  10. Substitutions at the cofactor phosphate-binding site of a clostridial alcohol dehydrogenase lead to unexpected changes in substrate specificity.

    PubMed

    Maddock, Danielle J; Patrick, Wayne M; Gerth, Monica L

    2015-08-01

    Changing the cofactor specificity of an enzyme from nicotinamide adenine dinucleotide 2'-phosphate (NADPH) to the more abundant NADH is a common strategy for increasing overall enzyme efficiency in microbial metabolic engineering. The aim of this study was to switch the cofactor specificity of the primary-secondary alcohol dehydrogenase from Clostridium autoethanogenum, a bacterium with considerable promise for the bio-manufacturing of fuels and other petrochemicals, from strictly NADPH-dependent to NADH-dependent. We used insights from a homology model to build a site-saturation library focussed on residue S199, the position deemed most likely to disrupt binding of the 2'-phosphate of NADPH. Although the CaADH(S199X) library did not yield any NADH-dependent enzymes, it did reveal that substitutions at the cofactor phosphate-binding site can cause unanticipated changes in the substrate specificity of the enzyme. Using consensus-guided site-directed mutagenesis, we were able to create an enzyme that was stringently NADH-dependent, albeit with a concomitant reduction in activity. This study highlights the role that distal residues play in substrate specificity and the complexity of enzyme-cofactor interactions. PMID:26034298

  11. Substitutions at the cofactor phosphate-binding site of a clostridial alcohol dehydrogenase lead to unexpected changes in substrate specificity

    PubMed Central

    Maddock, Danielle J.; Patrick, Wayne M.; Gerth, Monica L.

    2015-01-01

    Changing the cofactor specificity of an enzyme from nicotinamide adenine dinucleotide 2′-phosphate (NADPH) to the more abundant NADH is a common strategy for increasing overall enzyme efficiency in microbial metabolic engineering. The aim of this study was to switch the cofactor specificity of the primary–secondary alcohol dehydrogenase from Clostridium autoethanogenum, a bacterium with considerable promise for the bio-manufacturing of fuels and other petrochemicals, from strictly NADPH-dependent to NADH-dependent. We used insights from a homology model to build a site-saturation library focussed on residue S199, the position deemed most likely to disrupt binding of the 2′-phosphate of NADPH. Although the CaADH(S199X) library did not yield any NADH-dependent enzymes, it did reveal that substitutions at the cofactor phosphate-binding site can cause unanticipated changes in the substrate specificity of the enzyme. Using consensus-guided site-directed mutagenesis, we were able to create an enzyme that was stringently NADH-dependent, albeit with a concomitant reduction in activity. This study highlights the role that distal residues play in substrate specificity and the complexity of enzyme–cofactor interactions. PMID:26034298

  12. Inhibitory potential of chemical substitutions at bioinspired sites of β-D-galactopyranose on neoglycoprotein/cell surface binding of two classes of medically relevant lectins.

    PubMed

    Giguère, Denis; André, Sabine; Bonin, Marc-André; Bellefleur, Marc-André; Provencal, Alexandre; Cloutier, Philipe; Pucci, Bernard; Roy, René; Gabius, Hans-Joachim

    2011-05-15

    Galactose is the key contact site for plant AB-toxins and the human adhesion/growth-regulatory galectins. Natural anomeric extensions and 3'-substitutions enhance its reactivity, thus prompting us to test the potential of respective chemical substitutions of galactose in the quest to develop potent inhibitors. Biochemical screening of a respective glycoside library with 60 substances in a solid-phase assay was followed by examining the compounds' activity to protect cells from lectin binding. By testing 32 anomeric extensions, 18 compounds with additional 3'-substitution, three lactosides and two Lewis-type trisaccharides rather mild effects compared to the common haptenic inhibitor lactose were detected in both assays. When using trivalent glycoclusters marked enhancements with 6- to 8-fold increases were revealed for the toxin and three of four tested galectins. Since the most potent compound and also 3'-substituted thiogalactosides reduced cell growth of a human tumor line at millimolar concentrations, biocompatible substitutions and scaffolds will be required for further developments. The synthesis of suitable glycoclusters, presenting headgroups which exploit differences in ligand selection in interlectin comparison to reduce cross-reactivity, and the documented strategic combination of initial biochemical screening with cell assays are considered instrumental to advance inhibitor design. PMID:21524586

  13. Probing the promiscuous active site of myo-inositol dehydrogenase using synthetic substrates, homology modeling, and active site modification.

    PubMed

    Daniellou, Richard; Zheng, Hongyan; Langill, David M; Sanders, David A R; Palmer, David R J

    2007-06-26

    The active site of myo-inositol dehydrogenase (IDH, EC 1.1.1.18) from Bacillus subtilis recognizes a variety of mono- and disaccharides, as well as 1l-4-O-substituted inositol derivatives. It catalyzes the NAD+-dependent oxidation of the axial alcohol of these substrates with comparable kinetic constants. We have found that 4-O-p-toluenesulfonyl-myo-inositol does not act as a substrate for IDH, in contrast to structurally similar compounds such as those bearing substituted benzyl substituents in the same position. X-ray crystallographic analysis of 4-O-p-toluenesulfonyl-myo-inositol and 4-O-(2-naphthyl)methyl-myo-inositol, which is a substrate for IDH, shows a distinct difference in the preferred conformation of the aryl substituent. Conformational analysis of known substrates of IDH suggests that this conformational difference may account for the difference in reactivity of 4-O-p-toluenesulfonyl-myo-inositol in the presence of IDH. A sequence alignment of IDH with the homologous glucose-fructose oxidoreductase allowed the construction of an homology model of inositol dehydrogenase, to which NADH and 4-O-benzyl-scyllo-inosose were docked and the active site energy minimized. The active site model is consistent with all experimental results and suggests that a conserved tyrosine-glycine-tyrosine motif forms the hydrophobic pocket adjoining the site of inositol recognition. Y233F and Y235F retain activity, while Y233R and Y235R do not. A histidine-aspartate pair, H176 and D172, are proposed to act as a dyad in which H176 is the active site acid/base. The enzyme is inactivated by diethyl pyrocarbonate, and the mutants H176A and D172N show a marked loss of activity. Kinetic isotope effect experiments with D172N indicate that chemistry is rate-determining for this mutant. PMID:17539607

  14. Differential Active Site Loop Conformations Mediate Promiscuous Activities in the Lactonase SsoPox

    PubMed Central

    Elias, Mikael; Chabriere, Eric

    2013-01-01

    Enzymes are proficient catalysts that enable fast rates of Michaelis-complex formation, the chemical step and products release. These different steps may require different conformational states of the active site that have distinct binding properties. Moreover, the conformational flexibility of the active site mediates alternative, promiscuous functions. Here we focused on the lactonase SsoPox from Sulfolobus solfataricus. SsoPox is a native lactonase endowed with promiscuous phosphotriesterase activity. We identified a position in the active site loop (W263) that governs its flexibility, and thereby affects the substrate specificity of the enzyme. We isolated two different sets of substitutions at position 263 that induce two distinct conformational sampling of the active loop and characterized the structural and kinetic effects of these substitutions. These sets of mutations selectively and distinctly mediate the improvement of the promiscuous phosphotriesterase and oxo-lactonase activities of SsoPox by increasing active-site loop flexibility. These observations corroborate the idea that conformational diversity governs enzymatic promiscuity and is a key feature of protein evolvability. PMID:24086491

  15. Effect of Cr and Mn ions on the structure and magnetic properties of GaFeO{sub 3}: Role of the substitution site

    SciTech Connect

    Saha, Rana; Shireen, Ajmala; Shirodkar, Sharmila N.; Waghmare, Umesh V.; Sundaresan, A.; Rao, C.N.R.

    2011-09-15

    Effect of substitution of Cr and Mn in the Fe and Ga sites of GaFeO{sub 3} on the structural parameters and magnetic properties has been investigated by preparing GaFe{sub 1-x}Cr{sub x}(Mn{sub x})O{sub 3} and Ga{sub 1-x}Cr{sub x}(Mn{sub x})FeO{sub 3} starting with appropriate oxide precursors. It is shown that, starting with Cr or Mn substituted Ga{sub 2}O{sub 3}, one obtains Ga{sub 1-x}Cr{sub x}(Mn{sub x})FeO{sub 3}, while reaction of Cr or Mn substituted {alpha}-Fe{sub 2}O{sub 3} with Ga{sub 2}O{sub 3} yields GaFe{sub 1-x}Cr{sub x}(Mn{sub x})O{sub 3}. The structural parameters and magnetic properties vary significantly with the substitution site of Cr showing a large decrease in the unit cell parameters as well as the T{sub C} and other magnetic properties when the substitution is at the octahedral Fe (1, 2) site. Substitution of Cr at the octahedral Ga2 site results in marginal changes. Substitution of Mn in the Ga and Fe sites also show differences although the changes themselves are much smaller. First-principles calculations confirm such site-specificity and show how Cr substitution affects the properties differently when substituted at the Ga2 and Fe1 sites. - Graphical abstract: The important role of site-specific substitution of transition metal ions on the structure and magnetic properties of GaFeO{sub 3} has been investigated experimentally and theoretically. Highlights: > Substitution of transition metal ions to specific cation site. > Ionic radii of the cation dictate the disorder associated with the specific site. > Site-specificity explains magnetic ordering and structural parameters.

  16. Semisynthesis, Cytotoxic Activity, and Oral Availability of New Lipophilic 9-Substituted Camptothecin Derivatives

    PubMed Central

    2013-01-01

    Despite that 9-substituted camptothecins are promising candidates in cancer therapy, the limited accessibility to this position has reduced the studies of these derivatives to a few standard modifications. We report herein a novel semisynthetic route based on the Tscherniac–Einhorn reaction to synthesize new lipophilic camptothecin derivatives with amidomethyl and imidomethyl substitutions in position 9. Compounds were evaluated for their antiproliferative activity, topoisomerase I inhibition, and oral availability. Preliminary data demonstrated that bulky imidomethyl modification is an appropriate lipophilic substitution for an effective oral administration relative to topotecan. In addition, this general procedure paves the way for obtaining new camptothecin derivatives. PMID:24900725

  17. Semisynthesis, cytotoxic activity, and oral availability of new lipophilic 9-substituted camptothecin derivatives.

    PubMed

    Rodriguez-Berna, Guillermo; Cabañas, Maria Jose Díaz; Mangas-Sanjuán, Victor; Gonzalez-Alvarez, Marta; Gonzalez-Alvarez, Isabel; Abasolo, Ibane; Schwartz, Simó; Bermejo, Marival; Corma, Avelino

    2013-07-11

    Despite that 9-substituted camptothecins are promising candidates in cancer therapy, the limited accessibility to this position has reduced the studies of these derivatives to a few standard modifications. We report herein a novel semisynthetic route based on the Tscherniac-Einhorn reaction to synthesize new lipophilic camptothecin derivatives with amidomethyl and imidomethyl substitutions in position 9. Compounds were evaluated for their antiproliferative activity, topoisomerase I inhibition, and oral availability. Preliminary data demonstrated that bulky imidomethyl modification is an appropriate lipophilic substitution for an effective oral administration relative to topotecan. In addition, this general procedure paves the way for obtaining new camptothecin derivatives. PMID:24900725

  18. High-Temperature Decomposition of Brønsted Acid Sites in Gallium-Substituted Zeolites

    SciTech Connect

    K Al-majnouni; N Hould; W Lonergan; D Vlachos; R Lobo

    2011-12-31

    The dehydroxylation of Broensted acid sites (BAS) in Ga-substituted zeolites was investigated at temperatures up to 850 C using X-ray absorption spectroscopy (XAS), Fourier transform infrared spectroscopy (FTIR), and mass spectrometry-temperature programmed desorption (MS-TPD). X-ray absorption near-edge spectroscopy (XANES) revealed that the majority of gallium has tetrahedral coordination even after complete dehydroxylation. The interatomic gallium-oxygen distance and gallium coordination number determined by extended X-ray absorption fine structure (EXAFS) are consistent with gallium in tetrahedral coordination at low T (< 550 C). Upon heating Ga-Beta and Ga-ZSM5 to 850 C, analysis of the EXAFS showed that 70 and 80% of the gallium was still in tetrahedral coordination. The remainder of the gallium was found to be in octahedral coordination. No trigonal Ga atoms were observed. FTIR measurements carried out at similar temperatures show that the intensity of the OH vibration due to BAS has been eliminated. MS-TPD revealed that hydrogen in addition to water evolved from the samples during dehydroxylation. This shows that dehydrogenation in addition to dehydration is a mechanism that contributes to BAS decomposition. Dehydrogenation was further confirmed by exposing the sample to hydrogen to regenerate some of the BAS as monitored by FTIR and MS-TPD.

  19. Probing structural features of Alzheimer's amyloid-β pores in bilayers using site-specific amino acid substitutions.

    PubMed

    Capone, Ricardo; Jang, Hyunbum; Kotler, Samuel A; Kagan, Bruce L; Nussinov, Ruth; Lal, Ratnesh

    2012-01-24

    A current hypothesis for the pathology of Alzheimer's disease (AD) proposes that amyloid-β (Aβ) peptides induce uncontrolled, neurotoxic ion flux across cellular membranes. The mechanism of ion flux is not fully understood because no experiment-based Aβ channel structures at atomic resolution are currently available (only a few polymorphic states have been predicted by computational models). Structural models and experimental evidence lend support to the view that the Aβ channel is an assembly of loosely associated mobile β-sheet subunits. Here, using planar lipid bilayers and molecular dynamics (MD) simulations, we show that amino acid substitutions can be used to infer which residues are essential for channel structure. We created two Aβ(1-42) peptides with point mutations: F19P and F20C. The substitution of Phe19 with Pro inhibited channel conductance. MD simulation suggests a collapsed pore of F19P channels at the lower bilayer leaflet. The kinks at the Pro residues in the pore-lining β-strands induce blockage of the solvated pore by the N-termini of the chains. The cysteine mutant is capable of forming channels, and the conductance behavior of F20C channels is similar to that of the wild type. Overall, the mutational analysis of the channel activity performed in this work tests the proposition that the channels consist of a β-sheet rich organization, with the charged/polar central strand containing the mutation sites lining the pore, and the C-terminal strands facing the hydrophobic lipid tails. A detailed understanding of channel formation and its structure should aid studies of drug design aiming to control unregulated Aβ-dependent ion fluxes. PMID:22242635

  20. Serine substitution for cysteine residues in levansucrase selectively abolishes levan forming activity.

    PubMed

    Senthilkumar, Velusamy; Busby, Stephen J W; Gunasekaran, Paramasamy; Senthikumar, Velusamy; Bushby, Stephen J W

    2003-10-01

    Levansucrase is responsible for levan formation during sucrose fermentation of Zymomonas mobilis, and this decreases the efficiency of ethanol production. As thiol modifying agents decrease levan formation, a role for cysteine residues in levansucrase activity has been examined using derivatives of Z. mobilis levansucrase that carry serine substitutions of cysteine at positions 121, 151 or 244. These substitutions abolished the levan forming activity of levansucrase whilst only halving its activity in sucrose hydrolysis. Thus, polymerase and hydrolase activities of Z. mobilis levansucrase are separate and have different requirements for the enzyme's cysteine residues. PMID:14584923

  1. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1990-10-01

    DOE Order 5820.2A requires that low-level waste (LLW) disposal sites active on or after September 1988 and all transuranic (TRU) waste storage sites be monitored periodically to assure that radioactive contamination does not escape from the waste sites and pose a threat to the public or to the environment. This plan describes such a monitoring program for the active LLW disposal sites in SWSA 6 and the TRU waste storage sites in SWSA 5 North. 14 refs., 8 figs.

  2. [Synthesis of 2-methyl-5-substituted phenoxy-primaquine and antimalarials activity].

    PubMed

    Zhong, B H; Deng, R X; Zhong, J X; Wang, J

    1990-01-01

    In searching for efficient, safe and radically curative agent and causal prophylactics for malaria, seven 2-methyl-5-substituted phenoxy-6-methoxy-8-(1-methyl-4-aminobutylamino)-quinolines (II1-7) were synthesized and their antimalarial activities were compared with the corresponding 4-methyl substituted derivatives of primaquine. The starting material, 2-nitro-4-methoxy-5-bromo-acetanilide (III), was prepared from p-methoxy aniline through acetylation, bromination and nitration. III was then condensed with substituted phenols in the presence of potassium carbonate. The condensed products were subsequently hydrolyzed with dilute alcoholic hydrochloric acid to yield 2-nitro-4-methoxy-5-substituted phenoxy-aniline (V) which underwent Skraup's reaction with 2-butenal to provide the key intermediates 2-methyl-5-substituted phenoxy-6-methoxy-8-nitroquinolines (VI). These 8-nitroquinoline derivatives were reduced to 8-aminoquinoline derivatives (VII). The latter were condensed with 4-bromo-1-phthalimido-pentane and then hydrolyzed with hydrazine hydrate, the final products were obtained as oxalate or succinate. The structure of the target compounds and unknown intermediates were confirmed by elementary and spectral analysis. Primary biological evaluation showed that all compounds II1-7 were much less active than the 4-methyl substituted derivatives and slightly less active than primaquine in both causal prophylactic test against Plasmodium yoelii and suppressive antimalarial test against P. berhei. PMID:2239331

  3. Characterization of mutagenic activity in grain-based coffee-substitute blends and instant coffees

    SciTech Connect

    Johansson, M.A.E.; Knize, M.G.; Felton, J.S.; Jagerstad, M.

    1994-06-01

    Several grain-based coffee-substitute blends and instant coffees showed a mutagenic response in the Ames/Salmonella test using TA98, YG1024 and YG1O29 with metabolic activation. The beverage powders contained 150 to 500 TA98 and 1150 to 4050 YG1024 revertant colonies/gram, respectively. The mutagenic activity in the beverage powders was shown to be stable to heat and the products varied in resistance to acid nitrite treatment. Characterization of the mutagenic activity, using HPLC-and the Ames test of the collected fractions, showed the coffee-substitutes and instant coffees contain several mutagenic compounds, which are most likely aromatic amines.

  4. Synthesis and evaluation of antitubercular activity of fluorinated 5-aryl-4-(hetero)aryl substituted pyrimidines.

    PubMed

    Verbitskiy, Egor V; Baskakova, Svetlana A; Kravchenko, Marionella A; Skornyakov, Sergey N; Rusinov, Gennady L; Chupakhin, Oleg N; Charushin, Valery N

    2016-08-15

    Various 5-(fluoroaryl)-4-(hetero)aryl substituted pyrimidines have been synthesized based on the Suzuki cross-coupling and nucleophilic aromatic substitution of hydrogen (SN(H)) reactions starting from commercially available 5-bromopyrimidine and their antitubercular activity against Mycobacterium tuberculosis H37Rv has been explored. The outcome of the study disclose that, some of the compounds have showed promising activity in micromolar concentration against Mycobacterium tuberculosis H37Rv, Mycobacterium avium, Mycobacterium terrae, and multidrug-resistant strains isolated from tuberculosis patients in Ural region (Russia). The data concerning the 'structure-activity' relationship for fluorinated compounds have been discussed. PMID:27338658

  5. Site occupancy and magnetic study of Al 3+ and Cr 3+ co-substituted Y 3Fe 5O 12

    NASA Astrophysics Data System (ADS)

    Bouziane, K.; Yousif, A.; Widatallah, H. M.; Amighian, J.

    Single-phased polycrystalline Y 3Fe 5-2xAl xCr xO 12 garnet samples ( x=0, 0.2, 0.4 and 0.6) have been prepared by the conventional ceramic technique. Rietveld refinement of X-ray diffraction patterns of the samples shows them to crystallize in the Ia3d space group and the corresponding lattice constant to decrease with increasing Al 3+ and Cr 3+ contents ( x). Mössbauer results indicate that Cr 3+ substitutes for Fe 3+ at the octahedral sites whilst Al 3+ essentially replaces Fe 3+ at the tetrahedral sites. This result indicates that co-doping of Y 3Fe 5O 12 does not affect the preferential site occupancy for separate individual substitution of either Cr 3+ or Al 3+. The magnetization measurements reveal that the Curie temperature ( Tc) monotonically decreases with increasing x while the magnetic moment per unit formula decreases up to x=0.4 and then slightly increases for x=0.6. This reflects a progressive weakening of the ferrimagnetic exchange interaction between the Fe 3+ ions at octahedral and tetrahedral sites due to co-substitution. The magnetic moment was calculated using the cations distribution inferred from the Mössbauer data and the collinear ferrimagnetic model, and was found to agree reasonably with the experimentally measured value. The phenomenological amplitude crossover, characterized by the temperature T*, has also been observed in the doped YIG and briefly discussed.

  6. Improved lithium storage performance of lithium sodium titanate anode by titanium site substitution with aluminum

    NASA Astrophysics Data System (ADS)

    Wang, Pengfei; Li, Peng; Yi, Ting-Feng; Lin, Xiaoting; Zhu, Yan-Rong; Shao, Lianyi; Shui, Miao; Long, Nengbing; Shu, Jie

    2015-10-01

    Li2Na2Ti6O14 and its Ti-site substitution Li2Na2Ti5.9M0.1O14 (M = Al, Zr, V) are prepared by a solid-state reaction method and used as anode materials for lithium-ion batteries. It is found that metal doping can effectively enhance the electronic conductivity and ionic diffusion coefficient of Li2Na2Ti6O14. Especially for Li2Na2Ti5.9Al0.1O14, it reveals the highest electronic conductivity (1.02 × 10-9 S cm-1) and lithium ion diffusion coefficient (8.38 × 10-15 cm2 s-1) among all the samples. As a result, Li2Na2Ti5.9Al0.1O14 reveals the best electrochemical performance. It can deliver a charge specific capacity of 270.3 mAh g-1 at 50 mA g-1. Even cycled at 1000 mA g-1, it still can present a charge capacity of 180.7 mAh g-1. All these enhanced lithium storage capabilities of Li2Na2Ti5.9Al0.1O14 should be attributed to the increased electronic/ionic conductivities and the decreased charge transfer resistance induced by Al doping. Besides, in-situ X-ray diffraction observation also confirms that the structural change of Li2Na2Ti5.9Al0.1O14 is highly reversible process for lithium storage.

  7. Synthesis and antikinetoplastid activities of 3-substituted quinolinones derivatives.

    PubMed

    Audisio, Davide; Messaoudi, Samir; Cojean, Sandrine; Peyrat, Jean-François; Brion, Jean-Daniel; Bories, Christian; Huteau, Françoise; Loiseau, Philippe M; Alami, Mouad

    2012-06-01

    A new family of quinolinone derivatives has been synthesized and evaluated for their antikinetoplastid activities against Leishmania donovani and Trypanosoma brucei brucei. Results from these structure-activity relationship studies enabled identification of compounds 3a and 4g as the most active compounds against L. donovani promastigotes and amastigotes parasites (IC(50) values in a range of 2-11 μM). Additionally, compound 3b has emerged from this study as the most active compound in the series against T. b. brucei with a MEC value of 12 μM. These three compounds are worth of further in vivo evaluation. PMID:22472166

  8. Substitute sweeteners: diverse bacterial oligosaccharyltransferases with unique N-glycosylation site preferences.

    PubMed

    Ollis, Anne A; Chai, Yi; Natarajan, Aravind; Perregaux, Emily; Jaroentomeechai, Thapakorn; Guarino, Cassandra; Smith, Jessica; Zhang, Sheng; DeLisa, Matthew P

    2015-01-01

    The central enzyme in the Campylobacter jejuni asparagine-linked glycosylation pathway is the oligosaccharyltransferase (OST), PglB, which transfers preassembled glycans to specific asparagine residues in target proteins. While C. jejuni PglB (CjPglB) can transfer many diverse glycan structures, the acceptor sites that it recognizes are restricted predominantly to those having a negatively charged residue in the -2 position relative to the asparagine. Here, we investigated the acceptor-site preferences for 23 homologs with natural sequence variation compared to CjPglB. Using an ectopic trans-complementation assay for CjPglB function in glycosylation-competent Escherichia coli, we demonstrated in vivo activity for 16 of the candidate OSTs. Interestingly, the OSTs from Campylobacter coli, Campylobacter upsaliensis, Desulfovibrio desulfuricans, Desulfovibrio gigas, and Desulfovibrio vulgaris, exhibited significantly relaxed specificity towards the -2 position compared to CjPglB. These enzymes glycosylated minimal N-X-T motifs in multiple targets and each followed unique, as yet unknown, rules governing acceptor-site preferences. One notable example is D. gigas PglB, which was the only bacterial OST to glycosylate the Fc domain of human immunoglobulin G at its native 'QYNST' sequon. Overall, we find that a subset of bacterial OSTs follow their own rules for acceptor-site specificity, thereby expanding the glycoengineering toolbox with previously unavailable biocatalytic diversity. PMID:26482295

  9. Substitute sweeteners: diverse bacterial oligosaccharyltransferases with unique N-glycosylation site preferences

    PubMed Central

    Ollis, Anne A.; Chai, Yi; Natarajan, Aravind; Perregaux, Emily; Jaroentomeechai, Thapakorn; Guarino, Cassandra; Smith, Jessica; Zhang, Sheng; DeLisa, Matthew P.

    2015-01-01

    The central enzyme in the Campylobacter jejuni asparagine-linked glycosylation pathway is the oligosaccharyltransferase (OST), PglB, which transfers preassembled glycans to specific asparagine residues in target proteins. While C. jejuni PglB (CjPglB) can transfer many diverse glycan structures, the acceptor sites that it recognizes are restricted predominantly to those having a negatively charged residue in the −2 position relative to the asparagine. Here, we investigated the acceptor-site preferences for 23 homologs with natural sequence variation compared to CjPglB. Using an ectopic trans-complementation assay for CjPglB function in glycosylation-competent Escherichia coli, we demonstrated in vivo activity for 16 of the candidate OSTs. Interestingly, the OSTs from Campylobacter coli, Campylobacter upsaliensis, Desulfovibrio desulfuricans, Desulfovibrio gigas, and Desulfovibrio vulgaris, exhibited significantly relaxed specificity towards the −2 position compared to CjPglB. These enzymes glycosylated minimal N-X-T motifs in multiple targets and each followed unique, as yet unknown, rules governing acceptor-site preferences. One notable example is D. gigas PglB, which was the only bacterial OST to glycosylate the Fc domain of human immunoglobulin G at its native ‘QYNST’ sequon. Overall, we find that a subset of bacterial OSTs follow their own rules for acceptor-site specificity, thereby expanding the glycoengineering toolbox with previously unavailable biocatalytic diversity. PMID:26482295

  10. Multi-Site Prenylation of 4-Substituted Tryptophans by Dimethylallyltryptophan Synthase

    PubMed Central

    Rudolf, Jeffrey D.; Wang, Hong; Poulter, C. Dale

    2013-01-01

    The aromatic prenyltransferase dimethylallyltryptophan synthase in Claviceps purpurea catalyzes the normal prenylation of tryptophan at C4 of the indole nucleus in the first committed step of ergot alkaloid biosynthesis. 4-Methyltryptophan is a competitive inhibitor of the enzyme that has been used in kinetic studies. Upon investigation of background activity during incubations of 4-methyltryptophan with dimethylallyl diphosphate, we found that the analogue was an alternate substrate, which gave four products. The structures of three of these compounds were established by 1H NMR and 2D NMR studies and revealed that dimethylallyltryptophan synthase catalyzed both normal and reverse prenylation at C3 of the indole ring and normal prenylation of N1. Similarly, 4-methoxytryptophan was an alternate substrate, giving normal prenylation at C5 as the major product. 4-Aminotryptophan, another alternate substrate, gave normal prenylation at C5 and C7. The ability of dimethylallyltryptophan synthase to prenylate at five different sites on the indole nucleus, with normal and reverse prenylation at one of the sites, is consistent with a dissociative electrophilic alkylation of the indole ring where orientation of the substrates within the active site and substituent electronic effects determine the position and type of prenylation. These results suggest a common mechanism for prenylation of tryptophan by all of the members of the structurally related dimethylallyltryptophan synthase family. PMID:23301871

  11. Synthesis, anti-inflammatory, analgesic, 5-lipoxygenase (5-LOX) inhibition activities, and molecular docking study of 7-substituted coumarin derivatives.

    PubMed

    Srivastava, Pavan; Vyas, Vivek K; Variya, Bhavesh; Patel, Palak; Qureshi, Gulamnizami; Ghate, Manjunath

    2016-08-01

    In the present study, 7-subsituted coumarin derivatives were synthesized using various aromatic and heterocyclic amines, and evaluated in vivo for anti-inflammatory and analgesic activity, and for ulcerogenic risk. The most active compounds were evaluated in vitro for 5-lipoxygenase (5-LOX) inhibition. Docking study was performed to predict the binding affinity, and orientation at the active site of the enzyme. In vivo anti-inflammatory and analgesic activity, and in vitro 5-LOX enzyme inhibition study revealed that compound 33 and 35 are the most potent compounds in all the screening methods. In vitro kinetic study of 35 showed mixed or non-competitive type of inhibition with 5-LOX enzyme. Presence of OCH3 group in 35 and Cl in 33 at C6-position of benzothiazole ring were found very important substitutions for potent activity. PMID:27376460

  12. Toxicity of N-substituted aromatics to acetoclastic methanogenic activity in granular sludge.

    PubMed Central

    Donlon, B A; Razo-Flores, E; Field, J A; Lettinga, G

    1995-01-01

    N-substituted aromatics are important priority pollutants entering the environment primarily through anthropogenic activities associated with the industrial production of dyes, explosives, pesticides, and pharmaceuticals. Anaerobic treatment of wastewaters discharged by these industries could potentially be problematical as a result of the high toxicity of N-substituted aromatics. The objective of this study was to examine the structure-toxicity relationships of N-substituted aromatic compounds to acetoclastic methanogenic bacteria. The toxicity was assayed in serum flasks by measuring methane production in granular sludge. Unacclimated cultures were used to minimize the biotransformation of the toxic organic chemicals during the test. The nature and the degree of the aromatic substitution were observed to have a profound effect on the toxicity of the test compound. Nitroaromatic compounds were, on the average, over 500-fold more toxic than their corresponding aromatic amines. Considering the facile reduction of nitro groups by anaerobic microorganisms, a dramatic detoxification of nitroaromatics towards methanogens can be expected to occur during anaerobic wastewater treatment. While the toxicity exerted by the N-substituted aromatic compounds was closely correlated with compound apolarity (log P), it was observed that at any given log P, N-substituted phenols had a toxicity that was 2 orders of magnitude higher than that of chlorophenols and alkylphenols. This indicates that toxicity due to the chemical reactivity of nitroaromatics is much more important than partitioning effects in bacterial membranes. PMID:8526501

  13. Synthesis and antitumor activity of some substituted indazole derivatives.

    PubMed

    Abbassi, Najat; Rakib, El Mostapha; Chicha, Hakima; Bouissane, Latifa; Hannioui, Abdellah; Aiello, Cinzia; Gangemi, Rosaria; Castagnola, Patrizio; Rosano, Camillo; Viale, Maurizio

    2014-06-01

    Some new N-[6-indazolyl]arylsulfonamides and N-[alkoxy-6-indazolyl]arylsulfonamides were prepared by the reduction of 2-alkyl-6-nitroindazoles with SnCl2 in different alcohols, followed by coupling the corresponding amine with arylsulfonyl chlorides in pyridine. The newly synthesized compounds were evaluated for their antiproliferative and apoptotic activities against two human tumor cell lines: A2780 (ovarian carcinoma) and A549 (lung adenocarcinoma). Preliminary in vitro pharmacological studies revealed that N-(2-allyl-2H-indazol-6-yl)-4-methoxybenzenesulfonamide 4 and N-[7-ethoxy-2-(4-methyl-benzyl)-2H-indazol-6-yl]-4-methyl-benzenesulfonamide 9 exhibited significant antiproliferative activity against the A2780 and A549 cell lines with IC50 values in the range from 4.21 to 18.6 µM, and also that they trigger apoptosis in a dose-dependent manner. Furthermore, both active compounds were able to cause an arrest of cells in the G2/M phase of the cell cycle, typical but not exclusive of tubulin interacting agents, although only infrequent interactions with the microtubule network were observed by immunofluorescence microscopy, while docking analysis showed a possible different behavior between the two active compounds. PMID:24554280

  14. Educational Activity Sites for High School Students

    ERIC Educational Resources Information Center

    Troutner, Joanne

    2005-01-01

    Finding quality Internet resources for high school students is a continuing challenge. Several high-quality web sites are presented for educators and students. These sites offer activities to learn how an art conservator looks at paintings, create a newspaper, research and develop an end product, build geometry and physics skills, explore science…

  15. Putative mechanisms of antitumor activity of cyano-substituted heteroaryles in HeLa cells.

    PubMed

    Ester, Katja; Supek, Fran; Majsec, Kristina; Marjanović, Marko; Lembo, David; Donalisio, Manuela; Šmuc, Tomislav; Jarak, Ivana; Karminski-Zamola, Grace; Kralj, Marijeta

    2012-04-01

    Six recently synthesized cyano-substituted heteroaryles, which do not bind to DNA but are highly cytotoxic against the human tumor cell line HeLa, were analyzed for their antitumor mechanisms of action (MOA). They did not interfere with the expression of human papillomavirus oncogenes integrated in the HeLa cell genome, but they did induce strong G1 arrest and result in the activation of caspase-3 and apoptosis. A computational analysis was performed that compared the antiproliferative activities of our compounds in 13 different tumor cell lines with those of compounds listed in the National Cancer Institute database. The results indicate that interference with cytoskeletal function and inhibition of mitosis are the likely antitumor MOA. Furthermore, a second in silico investigation revealed that the tumor cells that are sensitive to the cyano-substituted compounds show differences in their expression of locomotion genes compared with that of insensitive cell lines, thus corroborating the involvement of the cytoskeleton. This MOA was also confirmed experimentally: the cyano-substituted heteroaryles disrupted the actin and the tubulin networks in HeLa cells and inhibited cellular migration. However, further analysis indicated that multiple MOA may exist that depend on the position of the cyano-group; while cyano-substituted naphthiophene reduced the expression of cytoskeletal proteins, cyano-substituted thieno-thiophene-carboxanilide inhibited the formation of cellular reactive oxygen species. PMID:21046426

  16. New p-Substituted Salicylaldehyde Phenylhydrazone Derivatives: Synthesis, Characterization, and Antioxidant Activities

    PubMed Central

    Yorur-Goreci, Cigdem; Altas-Kiymaz, Nilay; Peksel, Aysegul; Bilgin-Eran, Belkis; Sonmez, Mustafa

    2014-01-01

    Abstract A series of new p-nitrophenylhydrazone derivatives 3a–f were synthesized, characterized, and investigated for their antioxidant activities. These compounds have been synthesized by refluxing (p-nitrophenyl)hydrazine with 4-sub-stituted salicylaldehydes. The structures of the compounds were established by IR, 1H- and 13C-NMR, and MS data. The antioxidant activities (free radical-scavenging activity, reducing power, metal chelating activity, and total anti-oxidant activity) of the hydrazone compounds were evaluated. All of the compounds exhibited significant activities, while compound 3a, with the shortest chain, showed the highest antioxidant activity in all of the tests. PMID:26279974

  17. Synthesis and antibacterial activity of novel lincomycin derivatives. I. Enhancement of antibacterial activities by introduction of substituted azetidines.

    PubMed

    Kumura, Ko; Wakiyama, Yoshinari; Ueda, Kazutaka; Umemura, Eijiro; Watanabe, Takashi; Shitara, Eiki; Fushimi, Hideki; Yoshida, Takuji; Ajito, Keiichi

    2016-06-01

    The synthesis and antibacterial activity of (7S)-7-sulfur-azetidin-3-yl lincomycin derivatives are described. Modification was achieved by a simple reaction of (7R)-7-O-methanesulfonyllincomycin and the corresponding substituted azetidine-2-thiol. Several compounds first showed moderate antibacterial activity against Streptococcus pneumoniae and Streptococcus pyogenes with erm gene as lincomycin derivatives. PMID:26758495

  18. Synthesis and antimalarial activity of chain substituted pivaloyloxymethyl ester analogues of Fosmidomycin and FR900098.

    PubMed

    Kurz, Thomas; Schlüter, Katrin; Kaula, Uwe; Bergmann, Bärbel; Walter, Rolf D; Geffken, Detlef

    2006-08-01

    Fosmidomycin is a promising antimalarial drug candidate with a unique chemical structure and a novel mode of action. Chain substituted pivaloyloxymethyl ester derivatives of Fosmidomycin and its acetyl analogue FR900098 have been synthesized and their in vitro antimalarial activity versus the Chloroquine sensitive strain 3D7 of Plasmodium falciparum has been determined. PMID:16679022

  19. Anticancer, Anti-Inflammatory, and Analgesic Activities of Synthesized 2-(Substituted phenoxy) Acetamide Derivatives

    PubMed Central

    Pal, Dilipkumar; Hegde, Rahul Rama; Hashim, Syed Riaz

    2014-01-01

    The aphorism was to develop new chemical entities as potential anticancer, anti-inflammatory, and analgesic agents. The Leuckart synthetic pathway was utilized in development of novel series of 2-(substituted phenoxy)-N-(1-phenylethyl)acetamide derivatives. The compounds containing 1-phenylethylamine as basic moiety attached to substituted phenols were assessed for their anticancer activity against MCF-7 (breast cancer), SK-N-SH (neuroblastoma), anti-inflammatory activity, and analgesic activity. These investigations revealed that synthesized products 3a–j with halogens on the aromatic ring favors as the anticancer and anti-inflammatory activity. Among all, compound 3c N-(1-(4-chlorophenyl)ethyl)-2-(4-nitrophenoxy)acetamide exhibited anticancer, anti-inflammatory, and analgesic activities. In conclusion, 3c may have potential to be developed into a therapeutic agent. PMID:25197642

  20. Anticancer, anti-inflammatory, and analgesic activities of synthesized 2-(substituted phenoxy) acetamide derivatives.

    PubMed

    Rani, Priyanka; Pal, Dilipkumar; Hegde, Rahul Rama; Hashim, Syed Riaz

    2014-01-01

    The aphorism was to develop new chemical entities as potential anticancer, anti-inflammatory, and analgesic agents. The Leuckart synthetic pathway was utilized in development of novel series of 2-(substituted phenoxy)-N-(1-phenylethyl)acetamide derivatives. The compounds containing 1-phenylethylamine as basic moiety attached to substituted phenols were assessed for their anticancer activity against MCF-7 (breast cancer), SK-N-SH (neuroblastoma), anti-inflammatory activity, and analgesic activity. These investigations revealed that synthesized products 3a-j with halogens on the aromatic ring favors as the anticancer and anti-inflammatory activity. Among all, compound 3c N-(1-(4-chlorophenyl)ethyl)-2-(4-nitrophenoxy)acetamide exhibited anticancer, anti-inflammatory, and analgesic activities. In conclusion, 3c may have potential to be developed into a therapeutic agent. PMID:25197642

  1. Effect of Element Substitution at V site on Thermoelectric Properties of Aurivillius Phase Bi2VO5.5

    NASA Astrophysics Data System (ADS)

    Kohri, Hitoshi; Yagasaki, Takayoshi

    2016-06-01

    Thermoelectric oxides are suitable at the high temperature range because of chemical stability. Aurivillius compounds are bismuth layered oxides, and known as oxygen ion conductors. The Aurivillius compounds consist of Perovskite layers and Bi-O layers. It is expected that nano-layered structure shows high Seebeck coefficients due to the quantum confinement of carriers in Perovskite layers. It was reported that the Seebeck coefficient of hot pressed specimens for Aurivillius phase Bi2VO5.5 was a high value of -28.3 mVK-1 at 1010 K, and the electrical resistivity of one was also a high value of 0.033 Ωm at 1010 K. In this paper, the effect of element substitution at the V site on thermoelectric properties of Aurivillius phase Bi2VO5.5 was investigated. Bi2V1-x M x O5.5 (M = Cr, Mo, W x = 0, 0.05, 0.1, 0.2) were prepared by solid-state reaction. The electrical resistivity of Cr-substituted specimens were indicated at larger values than the ones for unsubstituted specimens over the measurement temperature range. The resistivity above 800 K was reduced by substitution of W or Mo. W as a substituted element was effective for reducing the thermal conductivity of Bi2VO5.5. The maximum value of the dimensionless figure of merit ZT was 0.05 at 799 K for Bi2V0.8Mo0.2O5.5 and at 902 K for Bi2V0.8W0.1O5.5. The maximum ZT of an unsubstituted sample was 0.02 at 993 K. From these results, it was found that tungsten or molybdenum substitution was effective to improve ZT for Aurivillius phase Bi2VO5.5.

  2. Synthesis, characterization and antiamoebic activity of chalcones bearing N-substituted ethanamine tail.

    PubMed

    Leeza Zaidi, Saadia; Mittal, Sonam; Rajala, Maitreyi S; Avecilla, Fernando; Husain, Mohammad; Azam, Amir

    2015-06-15

    A series of chalcones (4-21) possessing N-substituted ethanamine were synthesized by the aldol condensation reaction of 1-(4-(2-substituted ethoxy)phenyl)ethanones with different aldehydes preceded by the reaction of 2-chloro N-substituted ethanamine hydrochloride and 4-hydroxy acetophenone. The structure of all the synthesized compounds was elucidated by various spectral and X-ray diffraction studies. The compounds were screened against HM1: IMSS strain of Entamoeba histolytica and cytotoxicity was performed on A549 (non-small cell lung cancer cell line) cells by MTT assay. Out of eighteen compounds twelve showed better activity then the standard drug metronidazole. The compound 9, 14 and 19 showed good cell viability, hence were least toxic. PMID:26021707

  3. Effect of heterovalent substitution at Mn site on the magnetic and transport properties of La0.67Sr0.33MnO3

    NASA Astrophysics Data System (ADS)

    Priolkar, K. R.; Rawat, R.

    Magnetic and transport properties of Ti substituted La0.67Sr0.33MnO3 are drastically affected with a change in preparation conditions. Low temperature infra-red absorption measurements reveal that this is perhaps due to inhomogeniety in substitution of Ti on Mn sites. It is found that, in the high temperature annealed samples, the substitution of Ti supresses the double exchange interaction due to the formation of Mn-O- Ti chains. While in the low temperature annealed case substitution of Ti causes formation of isolated ferromagnetic clusters linked to each other by a variable range hopping polaron.

  4. Antiradical and cytoprotective activities of several C-geranyl-substituted flavanones from Paulownia tomentosa fruit.

    PubMed

    Zima, Ales; Hosek, Jan; Treml, Jakub; Muselík, Jan; Suchý, Pavel; Prazanová, Gabriela; Lopes, Ana; Zemlicka, Milan

    2010-09-01

    Antiradical and cytoprotective activities of several flavanones isolated from Paulownia tomentosa (Thunb.) Steud. (Scrophulariaceae) have been evaluated using different in vitro and in vivo methods. The capacity of flavanones to scavenge radicals was measured in vitro by means of DPPH and ABTS assays, the inhibition of hydroxyl radicals produced in Fenton reactions, FRAP, scavenging superoxide radicals using enzymatic and nonenzymatic assays and the inhibition of peroxynitrite-induced nitration of tyrosine. The in vivo testing involved measuring the cytoprotective effect of chosen flavanones against alloxan-induced diabetes in mice. The activity of tested compounds was expressed either as a Trolox® equivalent or was compared with rutin or morine as known antioxidant compounds. The highest activity in most tests was observed for diplacone and 3´-O-methyl-5´-hydroxydiplacone, and the structure vs. the antioxidant activity relationship of geranyl or prenyl-substituted flavonoids with different substitutions at the B and C ring was discussed. PMID:20877208

  5. A facile synthesis and antimicrobial activity evaluation of sydnonyl-substituted thiazolidine derivatives.

    PubMed

    Shih, Mei-Hsiu; Xu, Yu-Yuan; Yang, Yu-Sheng; Lin, Guan-Ling

    2015-01-01

    Some new sydnonyl-substituted thiazolidine derivatives were synthesized in high yields by the modified Knoevenagel condensation of 3-aryl-4-formylsydnones with thiazolidine-2,4-dione and 2-thioxo-thiazolidine-4-one, respectively. All the synthesized thiazolidine derivatives were screened by paper-disc method to identify their antimicrobial activities against three bacteria viz. Staphylococcus aureus, Proteus vulgaris and Escherichia coli, and two fungal cultures viz. Aspergillus niger and Penicillium citrinum. The reference drugs were Norfloxacin and Griseofulvin, respectively. The screening data indicated that the tested sydnonyl-substituted thiazolidine derivatives exhibited no obvious antibacterial activity compared with the standard drug Norfloxacin. However, thiazolidine derivatives displayed significant antifungal activities against Penicillium citrinum and Aspergillus niger. Notably, all of the tested compounds showed growth inhibitory activity 1.5-4.4 times higher than that of the standard drug Griseofulvin against the two fungi. PMID:25871371

  6. Low dielectric response in enzyme active site

    PubMed Central

    Mertz, Edward L.; Krishtalik, Lev I.

    2000-01-01

    The kinetics of charge transfer depend crucially on the dielectric reorganization of the medium. In enzymatic reactions that involve charge transfer, atomic dielectric response of the active site and of its surroundings determines the efficiency of the protein as a catalyst. We report direct spectroscopic measurements of the reorganization energy associated with the dielectric response in the active site of α-chymotrypsin. A chromophoric inhibitor of the enzyme is used as a spectroscopic probe. We find that water strongly affects the dielectric reorganization in the active site of the enzyme in solution. The reorganization energy of the protein matrix in the vicinity of the active site is similar to that of low-polarity solvents. Surprisingly, water exhibits an anomalously high dielectric response that cannot be described in terms of the dielectric continuum theory. As a result, sequestering the active site from the aqueous environment inside low-dielectric enzyme body dramatically reduces the dielectric reorganization. This reduction is particularly important for controlling the rate of enzymatic reactions. PMID:10681440

  7. Synthesis and antiproliferative activity of D-ring substituted steroidal benzamidothiazoles.

    PubMed

    Fan, Ning-Juan; He, Qiu-Rui; Duan, Min; Bai, Yu-Bin; Tang, Jiang-Jiang

    2016-08-01

    Using progesterone as the starting material, we synthesized a series of steroidal derivatives possessing a D-ring substituted benzamidothiazole. All of the final structures were reported and identified by NMR and HRMS spectrometry for the first time. The antiproliferative activity of the synthesized compounds against PC-3 (human prostate cancer cell line) and SKOV-3 (ovarian cancer cells) were investigated. The preliminary results showed that compounds 8b, 8d and 8g possessed moderate antiproliferative activities. PMID:27137356

  8. Calcium substitution at yttrium site in YBa2Cu3Oy

    NASA Astrophysics Data System (ADS)

    Chandrachood, M. R.; Mulla, I. S.; Gorwadkar, S. M.; Sinha, A. P. B.

    1990-01-01

    We have successfully synthesized single-phase superconducting Y1-xCaxBa2Cu3Oy for 0≤x≤0.4 using a coprecipitation technique. All these compounds are orthorhombic with lattice parameters a˜3.83 and c varying from 11.69 to 11.67 Å. The Tc remains unchanged around 90 K. It may be noted that in these compounds Ca substitutes for Y. On the other hand, it is known that in the compounds where Ca substitutes for Ba, the structure changes to tetragonal and Tc decreases rapidly. [See G. J. Baldha, R. B. Jotania, H. H. Joshi, H. N. Pandya, and R. G. Kulkarni, Solid State Commun. 71, 839 (1989)]. Furthermore, these compositions sinter much better than pure 1:2:3 and one gets high-density pellets (>95% of total density) at relatively lower temperature (<950 °C).

  9. Synthesis and In Vitro Antioxidant Activity of New 3-Substituted-2-Oxindole Derivatives

    PubMed Central

    Gupta, A. K.; Kalpana, S.; Malik, J. K.

    2012-01-01

    A series of new 1,3-dihydro-3-hydroxy-3-(2-phenyl-2-oxoethyl)-2H-indol-2-ones (1a-g) and 1,3-dihydro-3-(2-phenyl-2-oxoethylidene)-2H-indol-2-ones (2a-g) were synthesised by Knoevenagel condensation of substituted indole-2,3-diones (isatins) with various acetophenones. The synthesised compounds were characterised by their physical data, elemental, IR, 1H NMR, 13C NMR and mass spectral analyses and their in vitro antioxidant activity was determined by 2,2-diphenyl-1-picrylhydrazyl free radical scavenging assay. These compounds showed moderate to good antioxidant activities as compared with the standard, ascorbic acid. The antioxidant potential of 3-hydroxy-3-substituted oxindoles (1a-g) increased in a concentration-dependent manner from 10 to 500 μg/ml with 5-fluoro and 5-methyl analogues showing maximum activity. Of 3-aroyl methylene indol-2-ones (2a-g), majority of compounds with halogen substitution at position 5 of isatin ring exhibited good antioxidant activity within a concentration range of 5-100 μg/ml and the maximum activity was observed at 20 and 25 μg/ml concentrations. Thus, our study provides evidence that some newly synthesised isatin derivatives exhibit substantial antioxidant activity at low concentrations. PMID:23716882

  10. Systematic mutagenesis of the active site omega loop of TEM-1 beta-lactamase.

    PubMed Central

    Petrosino, J F; Palzkill, T

    1996-01-01

    Beta-Lactamase is a bacterial protein that provides resistance against beta-lactam antibiotics. TEM-1 beta-lactamase is the most prevalent plasmid-mediated beta-lactamase in gram-negative bacteria. Normally, this enzyme has high levels of hydrolytic activity for penicillins, but mutant beta-lactamases have evolved with activity toward a variety of beta-lactam antibiotics. It has been shown that active site substitutions are responsible for changes in the substrate specificity. Since mutant beta-lactamases pose a serious threat to antimicrobial therapy, the mechanisms by which mutations can alter the substrate specificity of TEM-1 beta-lactamase are of interest. Previously, screens of random libraries encompassing 31 of 55 active site amino acid positions enabled the identification of the residues responsible for maintaining the substrate specificity of TEM-1 beta-lactamase. In addition to substitutions found in clinical isolates, many other specificity-altering mutations were also identified. Interestingly, many nonspecific substitutions in the N-terminal half of the active site omega loop were found to increase ceftazidime hydrolytic activity and decrease ampicillin hydrolytic activity. To complete the active sight study, eight additional random libraries were constructed and screened for specificity-altering mutations. All additional substitutions found to alter the substrate specificity were located in the C-terminal half of the active site loop. These mutants, much like the N-terminal omega loop mutants, appear to be less stable than the wild-type enzyme. Further analysis of a 165-YYG-167 triple mutant, selected for high levels of ceftazidime hydrolytic activity, provides an example of the correlation which exists between enzyme instability and increased ceftazidime hydrolytic activity in the ceftazidime-selected omega loop mutants. PMID:8606154

  11. Molybdenum substitution for improving the charge compensation and activity of Li2MnO3.

    PubMed

    Ma, Jun; Zhou, Yong-Ning; Gao, Yurui; Kong, Qingyu; Wang, Zhaoxiang; Yang, Xiao-Qing; Chen, Liquan

    2014-07-01

    Lithium-rich layer-structured oxides xLi2 MnO3 ⋅ (1-x)LiMO2 (0substitution was performed in this work to provide another electron donor and to enhance the electrochemical activity of Li2 MnO3 -based cathode materials. X-ray diffraction and adsorption studies indicated that Mo(5+) substitution expands the unit cell in the crystal lattice and weakens the LiO and MnO bonds, as well as enhancing the activity of Li2 MnO3 by lowering its delithiation potential and suppressing the release of oxygen. In addition, the chemical environment of O(2-) ions in molybdenum-substituted Li2 MnO3 is more reversible than in the unsubstituted sample during cycling. Therefore molybdenum substitution is expected to improve the performances of the Li2 MnO3 -based lithium-rich cathode materials. PMID:24939463

  12. Mixture models of nucleotide sequence evolution that account for heterogeneity in the substitution process across sites and across lineages.

    PubMed

    Jayaswal, Vivek; Wong, Thomas K F; Robinson, John; Poladian, Leon; Jermiin, Lars S

    2014-09-01

    Molecular phylogenetic studies of homologous sequences of nucleotides often assume that the underlying evolutionary process was globally stationary, reversible, and homogeneous (SRH), and that a model of evolution with one or more site-specific and time-reversible rate matrices (e.g., the GTR rate matrix) is enough to accurately model the evolution of data over the whole tree. However, an increasing body of data suggests that evolution under these conditions is an exception, rather than the norm. To address this issue, several non-SRH models of molecular evolution have been proposed, but they either ignore heterogeneity in the substitution process across sites (HAS) or assume it can be modeled accurately using the distribution. As an alternative to these models of evolution, we introduce a family of mixture models that approximate HAS without the assumption of an underlying predefined statistical distribution. This family of mixture models is combined with non-SRH models of evolution that account for heterogeneity in the substitution process across lineages (HAL). We also present two algorithms for searching model space and identifying an optimal model of evolution that is less likely to over- or underparameterize the data. The performance of the two new algorithms was evaluated using alignments of nucleotides with 10 000 sites simulated under complex non-SRH conditions on a 25-tipped tree. The algorithms were found to be very successful, identifying the correct HAL model with a 75% success rate (the average success rate for assigning rate matrices to the tree's 48 edges was 99.25%) and, for the correct HAL model, identifying the correct HAS model with a 98% success rate. Finally, parameter estimates obtained under the correct HAL-HAS model were found to be accurate and precise. The merits of our new algorithms were illustrated with an analysis of 42 337 second codon sites extracted from a concatenation of 106 alignments of orthologous genes encoded by the nuclear

  13. Interactions between lac repressor protein and site-specific bromodeoxyuridine-substituted operator DNA. Ultraviolet footprinting and protein-DNA cross-link formation

    SciTech Connect

    Wick, K.L.; Matthews, K.S. )

    1991-04-05

    Specific contacts between the lac repressor and operator have been explored using 5-bromodeoxyuridine-substituted DNA. Substitution of BrdU for single thymidine positions in a synthetic 40-base pair operator provides substrate for ultraviolet irradiation; upon irradiation, strand scission occurs at the BrdU residues. When bound, lac repressor protein provides protection against UV-induced breakage depending on the nature of the sites and type of interaction. We have confirmed 13 unique sites of inducer-sensitive protection along the operator sequence using this method compared to complete substitution with BrdU; differences were observed at two positions for singly substituted versus completely substituted DNAs. The ability of these photosensitive DNAs to form short range cross-links to bound protein has been used to determine the efficiency with which cross-linked protein-DNA complexes are generated at each individual site of BrdU substitution. Five sites of high efficiency cross-linking to the repressor protein have been identified. At one site, cross-linking without protection from strand scission was observed; this result suggests an unusual mechanism of strand scission and/or cross-linking at this site. Comparison of the UV protection results and the cross-linking data show that these processes provide complementary tools for identifying and analyzing individual protein-DNA contacts.

  14. Synthesis and antimycobacterial activity of novel thiadiazolylhydrazones of 1-substituted indole-3-carboxaldehydes.

    PubMed

    Tehrani, Kamaleddin Haj Mohammad Ebrahim; Mashayekhi, Vida; Azerang, Parisa; Sardari, Soroush; Kobarfard, Farzad; Rostamizadeh, Kobra

    2014-02-01

    A series of novel thiocarbohydrazones of substituted indoles and their corresponding thiadiazole derivatives were prepared, and their structures were confirmed by different analytical and spectroscopic methods. The derivatives were prepared by a sequential synthetic strategy including substitution at N-1 position of indole ring by various aliphatic and benzylic substituents, followed by condensation with thiocarbohydrazide, and finally cyclization by triethyl orthoformate. The derivatives were tested for their antimycobacterial activity against Mycobacterium bovis BCG, and the results revealed that among the synthesized compounds, thiadiazole derivatives 4e, 4f, 4n, 4p, 4q, and 4t exhibited the highest activity with IC₅₀ value of 3.91 μg/mL. The results indicate that the thiadiazole moiety plays a vital role in exerting antimycobacterial activity. PMID:24103309

  15. Design, synthesis, and anticancer activity of C8-substituted-4'-thionucleosides as potential HSP90 inhibitors.

    PubMed

    Qu, Shuhao; Mulamoottil, Varughese A; Nayak, Akshata; Ryu, Seungyeon; Hou, Xiyan; Song, Jayoung; Yu, Jinha; Sahu, Pramod K; Zhao, Long Xuan; Choi, Sun; Lee, Sang Kook; Jeong, Lak Shin

    2016-08-15

    A series of C8-substituted-4'-thioadenosine analogs 3a-3g, 15, and 17 and their truncated derivatives 4a-4j, 23-25, and 27 have been successfully synthesized from d-ribose and d-mannose, respectively, employing Pummerer type or Vorbrüggen condensation reactions and the functionalization at the C8-position of nucleobase via Stille coupling or nucleophilic aromatic substitution reactions as key steps. All the synthesized compounds were assayed for their HSP90 inhibitory activity, but they were found to be inactive up to 100μM. However, the 8-iodo derivatives 15, 17, and 27 exhibited potent anticancer activity, indicating that different mechanism of action might be involved in their biological activity. PMID:27283788

  16. Substitution of valine for histidine 265 in carbon monoxide dehydrogenase from Rhodospirillum rubrum affects activity and spectroscopic states.

    PubMed

    Spangler, N J; Meyers, M R; Gierke, K L; Kerby, R L; Roberts, G P; Ludden, P W

    1998-02-13

    In carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum, histidine 265 was replaced with valine by site-directed mutagenesis of the cooS gene. The altered form of CODH (H265V) had a low nickel content and a dramatically reduced level of catalytic activity. Although treatment with NiCl2 and CoCl2 increased the activity of H265V CODH by severalfold, activity levels remained more than 1000-fold lower than that of wild-type CODH. Histidine 265 was not essential for the formation and stability of the Fe4S4 clusters. The Km and KD for CO as well as the KD for cyanide were relatively unchanged as a result of the amino acid substitution in CODH. The time-dependent reduction of the [Fe4S4]2+ clusters by CO occurred on a time scale of hours, suggesting that, as a consequence of the mutation, a rate-limiting step had been introduced prior to the transfer of electrons from CO to the cubanes in centers B and C. EPR spectra of H265V CODH lacked the gav = 1.86 and gav = 1.87 signals characteristic of reduced forms of the active site (center C) of wild-type CODH. This indicates that the electronic properties of center C have been modified possibly by the disruption or alteration of the ligand-mediated interaction between the nickel site and Fe4S4 chromophore. PMID:9461598

  17. Structural evolution of luciferase activity in Zophobas mealworm AMP/CoA-ligase (protoluciferase) through site-directed mutagenesis of the luciferin binding site.

    PubMed

    Prado, R A; Barbosa, J A; Ohmiya, Y; Viviani, V R

    2011-07-01

    The structural origin and evolution of bioluminescent activity of beetle luciferases from AMP/CoA ligases remains a mystery. Previously we cloned the luciferase-like enzyme from Zophobas morio mealworm, a reasonable protoluciferase model that could shine light on this mystery. Kinetic characterization and studies with D- and L-luciferin and their adenylates showed that stereoselectivity constitutes a critical feature for the origin of luciferase activity in AMP/CoA ligases. Comparison of the primary structures and modeling studies of this protoluciferase and the three main families of beetle luciferases showed that the carboxylic acid substrate binding site of this enzyme is smaller and more hydrophobic than the luciferin binding site of beetle luciferases, showing several substitutions of otherwise conserved residues. Thus, here we performed a site-directed mutagenesis survey of the carboxylic binding site motifs of the protoluciferase by replacing their residues by the respective conserved ones found in beetle luciferases in order to identify the structural determinants of luciferase/oxygenase activity. Although most of the substitutions had negative impact on the luminescence activity of the protoluciferase, only the substitution I327T improved the luminescence activity, resulting in a broad and 15 nm blue-shifted luminescence spectrum. Such substitution indicates the importance of the loop motif 322YGMSEI327 (341YGLTETT347 in Photinus pyralis luciferase) for luciferase activity, and indicates a possible route for the evolution of bioluminescence function of beetle luciferases. PMID:21505686

  18. Ferredoxin/ferredoxin-thioredoxin reductase complex: Complete NMR mapping of the interaction site on ferredoxin by gallium substitution.

    PubMed

    Xu, Xingfu; Kim, Sung-Kun; Schürmann, Peter; Hirasawa, Masakazu; Tripathy, Jatindra N; Smith, Jody; Knaff, David B; Ubbink, Marcellus

    2006-12-11

    The reduction of ferredoxin-thioredoxin reductase (FTR) by plant-type ferredoxin plays an important role in redox regulation in plants and cyanobacteria. Nuclear magnetic resonance (NMR) was used to map the binding sites on Synechocystis ferredoxin for FTR. A gallium-substituted structural analog of this [2Fe-2S] ferredoxin was obtained by reconstituting the apoprotein in a refolding buffer containing gallium. For the first time, the complete interaction interface of a [2Fe-2S] ferredoxin with a target enzyme has been mapped by NMR chemical shift perturbation with this diamagnetic structural analog. PMID:17134703

  19. Identification of active-site residues in protease 3C of hepatitis A virus by site-directed mutagenesis.

    PubMed Central

    Gosert, R; Dollenmaier, G; Weitz, M

    1997-01-01

    Picornavirus 3C proteases (3Cpro) are cysteine proteases related by amino acid sequence to trypsin-like serine proteases. Comparisons of 3Cpro of hepatitis A virus (HAV) to those of other picornaviruses have resulted in prediction of active-site residues: histidine at position 44 (H44), aspartic acid (D98), and cysteine (C172). To test whether these residues are key members of a putative catalytic triad, oligonucleotide-directed mutagenesis was targeted to 3Cpro in the context of natural polypeptide precursor P3. Autocatalytic processing of the polyprotein containing wild-type or variant 3Cpro was tested by in vivo expression of vaccinia virus-HAV chimeras in an animal cell-T7 hybrid system and by in vitro translation of corresponding RNAs. Comparison with proteins present in HAV-infected cells showed that both expression systems mimicked authentic polyprotein processing. Individual substitutions of H44 by tyrosine and of C172 by glycine or serine resulted in complete loss of the virus-specific proteolytic cascade. In contrast, a P3 polyprotein in which D98 was substituted by asparagine underwent only slightly delayed processing, while an additional substitution of valine (V47) by glycine within putative protein 3A caused a more pronounced loss of processing. Therefore, apparently H44 and C172 are active-site constituents whereas D98 is not. The results, furthermore, suggest that substitution of amino acid residues distant from polyprotein cleavage sites may reduce proteolytic activity, presumably by altering substrate conformation. PMID:9060667

  20. [Electrodermal activity in heroin addicts and patients with methadone and morphine substitution].

    PubMed

    Linzmayer, Leopold; Boeck, Gerda; Fischer, Gabriele

    2003-01-01

    In the present investigation we tried to answer the question whether differences between heroin-dependent patients (n = 26, age: M = 24.96, SD = 6.30 years), a methadone substitution group (n = 20, age: M = 30.92, SD = 8.21 years) and a morphine substitution group (n = 20, age: M = 33.25, SD = 4.59 years) and healthy normals (n = 31, age: M = 25.07, SD = 4.62 years) could be found by means of measurement of electrodermal activity (SC, SCR, habituation of the SCR). Concerning "basal" skin conductance reflecting sympathetic activity, no significant differences were obtained. The methadone substitution group showed slight shortened onset latencies (information processing). In the morphine substitution group as compared to the other groups a small increase of the amplitude was observed indicating a slight increase in cognitive emotional intensity of appraisal after presentation of an acoustic stimulus. This small changes could be mediated by adaptation processes of the vegetative nervous system to the opioid, which occur "below" of those neuronal networks connected directly with the emotional stimuli processing. Concerning the speed of habituation no significant differences between the groups could be obtained. This indicates that no psychovegetative attenuation could be observed. The morphine substitution group as compared to the other groups was characterized by a longer persistence and a small increase of the intensity of excitement. However these variables ranged within normal limits and did not reach the level of statistical significance. This could be mediated by the effects of the opioid on the vegetative nervous system. PMID:12658967

  1. Dual mode antibacterial activity of ion substituted calcium phosphate nanocarriers for bone infections.

    PubMed

    Sampath Kumar, T S; Madhumathi, K; Rubaiya, Y; Doble, Mukesh

    2015-01-01

    Nanotechnology has tremendous potential for the management of infectious diseases caused by multi-drug resistant bacteria, through the development of newer antibacterial materials and efficient modes of antibiotic delivery. Calcium phosphate (CaP) bioceramics are commonly used as bone substitutes due to their similarity to bone mineral and are widely researched upon for the treatment of bone infections associated with bone loss. CaPs can be used as local antibiotic delivery agents for bone infections and can be substituted with antibacterial ions in their crystal structure to have a wide spectrum, sustained antibacterial activity even against drug resistant bacteria. In the present work, a dual mode antibiotic delivery system with antibacterial ion substituted calcium deficient hydroxyapatite (CDHA) nanoparticles has been developed. Antibacterial ions such as zinc, silver, and strontium have been incorporated into CDHA at concentrations of 6, 0.25-0.75, and 2.5-7.5 at. %, respectively. The samples were found to be phase pure, acicular nanoparticles of length 40-50 nm and width 5-6 nm approximately. The loading and release profile of doxycycline, a commonly used antibiotic, was studied from the nanocarriers. The drug release was studied for 5 days and the release profile was influenced by the ion concentrations. The release of antibacterial ions was studied over a period of 21 days. The ion substituted CDHA samples were tested for antibacterial efficacy on Staphylococcus aureus and Escherichia coli by MIC/MBC studies and time-kill assay. AgCDHA and ZnCDHA showed high antibacterial activity against both bacteria, while SrCDHA was weakly active against S. aureus. Present study shows that the antibiotic release can provide the initial high antibacterial activity, and the sustained ion release can provide a long-term antibacterial activity. Such dual mode antibiotic and antibacterial ion release offers an efficient and potent way to treat an incumbent drug

  2. Pharmacophore modeling of substituted 1,2,4-Trioxanes for quantitative prediction of their antimalarial activity.

    PubMed

    Gupta, Amit K; Chakroborty, S; Srivastava, Kumkum; Puri, Sunil K; Saxena, Anil K

    2010-08-23

    A pharmacophore model has been developed for determining the essential structural requirements for antimalarial activity from the eight series of substituted 1,2,4-trioxanes. The best pharmacophore model possessing two aliphatic hydrophobic, one aromatic hydrophobic, one hydrogen-bond (H-bond) acceptor, and one H-bond acceptor (lipid) feature for antimalarial activity showed an excellent correlation coefficient for the training (r(2)(training) = 0.85) and a fair correlation coefficient for the test set (r(2)(test) = 0.51) molecules. The model predicts well to other known substituted 1,2,4-trioxanes including those which either are drugs or are undergoing clinical trials. In order to further validate this model, five substituted 1,2,4-trioxanes were synthesized from the generated focused library and screened for antimalarial activity. The observed activity of these molecules was consistent with the pharmacophore model, suggesting that the model may be useful in the design of potent antimalarial agents. PMID:20726605

  3. Design, synthesis and antimicrobial activity of 6-N-substituted chitosan derivatives.

    PubMed

    Hu, Linfeng; Meng, Xiangtao; Xing, Ronge; Liu, Song; Chen, Xiaolin; Qin, Yukun; Yu, Huahua; Li, Pengcheng

    2016-09-15

    Three novel 6-N-substituted chitosan derivatives were designed and synthesised and characterized by FTIR and NMR. The degree of substitution was calculated by elemental analysis results. The antimicrobial activities of the target compounds were evaluated by twofold serial broth dilution method and poisoned food technique. The antifungal activities of 6-aminoethylamino-6-deoxy chitosan (3), 6-butylamino-6-deoxy chitosan (4) and 6-pyridyl-6-deoxy chitosan (5) were significantly increased against Rhizoctonia cerealis, Fusarium oxysporum and Botrytis cinerea, and the inhibition rate ranged from 22.48% to 63.56% at the concentration of 0.2mg/mL. The compound 3 had better antibacterial activities than chitosan, and the minimum inhibition concentration of which ranged between 6.25 and 25mg/L against gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis and Bacillus anthracis) and gram-negative bacteria (Escherichia coli, Salmonella typhi). The antibacterial activities of 6-N-substituted chitosan tended to increase with the increase of the number of -NH2 group. PMID:27506558

  4. Synthesis and local anaesthetic activity of 2-substituted-N-(2-diethylaminoethyl)-acetamides.

    PubMed

    Jindal, Dharam Paul; Coumar, Mohane S; Singh, Babita; Ismail, Mohammed Muhiyiden Mohammed; Zambare, Girish Nilkanth; Bodhankar, Subhash Laxmanrao

    2003-01-01

    The synthesis of 2-substituted-N-(2-diethylaminoethyl)acetamide oxalates (6a, 6b) and the evaluation of their in vivo local anaesthetic activities are described. The compounds 6a and 6b were obtained starting from 4-acetamidophenol and 1-naphthol, respectively. The in vivo local anesthetic activity was evaluated by infiltration anaesthesia, sciatic nerve block and corneal anaesthesia models. N-(2-Diethylaminoethyl)-2-(naphthalen-1-yloxy)acetamide oxalate (6b) was found to have potency, onset and duration of action comparable to that of lidocaine (2) (lidocaine hydrochloride, CAS 6108-05-0). Procaine (1) (procaine hydrochloride, CAS 51-05-8) was also used for comparison. Dissociation constants (pKa) of compounds 5a and 5b (2-substituted-N-(2-diethylaminoethyl)acetamide) have been determined to be 8.9 and 8.6, respectively. PMID:12608012

  5. Methyl substitution of a rexinoid agonist improves potency and reveals site of lipid toxicity.

    PubMed

    Atigadda, Venkatram R; Xia, Gang; Desphande, Anil; Boerma, LeeAnn J; Lobo-Ruppert, Susan; Grubbs, Clinton J; Smith, Craig D; Brouillette, Wayne J; Muccio, Donald D

    2014-06-26

    (2E,4E,6Z,8E)-8-(3',4'-Dihydro-1'(2'H)-naphthalen-1'-ylidene)-3,7-dimethyl-2,4,6-octatrienoic acid, 9cUAB30, is a selective rexinoid that displays substantial chemopreventive capacity with little toxicity. 4-Methyl-UAB30, an analogue of 9cUAB30, is a potent RXR agonist but caused increased lipid biosynthesis unlike 9cUAB30. To evaluate how methyl substitution influenced potency and lipid biosynthesis, we synthesized four 9cUAB30 homologues with methyl substitutions at the 5-, 6-, 7-, or 8-position of the tetralone ring. The syntheses and biological evaluations of these new analogues are reported here along with the X-ray crystal structures of each homologue bound to the ligand binding domain of hRXRα. We demonstrate that each homologue of 9cUAB30 is a more potent agonist, but only the 7-methyl-9cUAB30 caused severe hyperlipidemia in rats. On the basis of the X-ray crystal structures of these new rexinoids and bexarotene (Targretin) bound to hRXRα-LBD, we reveal that each rexinoid, which induced hyperlipidemia, had methyl groups that interacted with helix 7 residues of the LBD. PMID:24801499

  6. Methyl Substitution of a Rexinoid Agonist Improves Potency and Reveals Site of Lipid Toxicity

    PubMed Central

    2015-01-01

    (2E,4E,6Z,8E)-8-(3′,4′-Dihydro-1′(2′H)-naphthalen-1′-ylidene)-3,7-dimethyl-2,4,6-octatrienoic acid, 9cUAB30, is a selective rexinoid that displays substantial chemopreventive capacity with little toxicity. 4-Methyl-UAB30, an analogue of 9cUAB30, is a potent RXR agonist but caused increased lipid biosynthesis unlike 9cUAB30. To evaluate how methyl substitution influenced potency and lipid biosynthesis, we synthesized four 9cUAB30 homologues with methyl substitutions at the 5-, 6-, 7-, or 8-position of the tetralone ring. The syntheses and biological evaluations of these new analogues are reported here along with the X-ray crystal structures of each homologue bound to the ligand binding domain of hRXRα. We demonstrate that each homologue of 9cUAB30 is a more potent agonist, but only the 7-methyl-9cUAB30 caused severe hyperlipidemia in rats. On the basis of the X-ray crystal structures of these new rexinoids and bexarotene (Targretin) bound to hRXRα-LBD, we reveal that each rexinoid, which induced hyperlipidemia, had methyl groups that interacted with helix 7 residues of the LBD. PMID:24801499

  7. Tuning of magnetic ordering by Y substitution onto Tb site in the nanocrystalline TbMnO3

    NASA Astrophysics Data System (ADS)

    Chakraborty, Keka R.; Shukla, Rakesh; Kaushik, S. D.; Mukadam, M. D.; Siruguri, V.; Tyagi, A. K.; Yusuf, S. M.

    2015-10-01

    We report the magnetic properties, of nano-crystalline powders Tb1-xYxMnO3 (x = 0, 0.1, 0.2, 0.3 and 0.4), as perceived by neutron diffraction, and elucidate the effect of Tb site substitution on the magnetic structure of TbMnO3. All samples crystallized in the orthorhombic structure conforming to space group Pnma, and exhibited an incommensurate collinear antiferromagnetic ordering of the Mn ions below ˜40 K. Furthermore, at T ≤ 20 K, all these samples showed a change in magnetic structure (of Mn moments) to a spiral ordering down to 2 K, the lowest measured temperature. For the samples with x = 0, 0.1, and 0.2, a short-ranged two dimensional (2D) ordering of Tb moments was also observed at 2 K. However, for the other samples (x = 0.3 and 0.4), no magnetic ordering of Tb moments was found down to 2 K. So with Y substitution, a crossover from 2D ordering to a disordering of Tb moments was observed. The moments at the Mn site were found to be lower than the full Mn3+ (4μB) moment for all the samples below 40 K. The magnetic properties of all the samples studied by us in nano form are more pronounced than those of the reported single crystals of same compositions [V. Yu. Ivanov et al., JETP Lett. 91, 392-397 (2010)].

  8. Anti-infective and herbicidal activity of N-substituted 2-aminobenzothiazoles.

    PubMed

    Fajkusova, Dagmar; Pesko, Matus; Keltosova, Stanislava; Guo, Jiahui; Oktabec, Zbynek; Vejsova, Marcela; Kollar, Peter; Coffey, Aidan; Csollei, Jozef; Kralova, Katarina; Jampilek, Josef

    2012-12-15

    In this study, a series of N-substituted 2-aminobenzothiazoles was prepared according to a recently developed method. Twelve compounds were tested for their activity related to the inhibition of photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. Primary in vitro screening of the discussed compounds was also performed against fungal, bacterial and mycobacterial species. The biological activities of some compounds were comparable or higher than the standards phenoxymethylpenicillin or pyrazinamide. The most effective compounds demonstrated insignificant toxicity against the human monocytic leukemia THP-1 cell line. For all compounds, the structure-activity relationships are discussed. PMID:23140987

  9. Chemical Synthesis and Biological Activities of Novel Pleuromutilin Derivatives with Substituted Amino Moiety

    PubMed Central

    Shang, Ruofeng; Wang, Shengyu; Xu, Ximing; Yi, Yunpeng; Guo, Wenzhu; YuLiu; Liang, Jianping

    2013-01-01

    Novel pleuromutilin derivatives designed based on the structure of valnemulin were synthesized and evaluated for their in vitro antibacterial activities. These pleuromutilin derivatives with substituted amino moiety exhibited excellent activities against methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, Escherichia coli, and Streptococcus agalactiae. Compound 5b showed the highest antibacterial activities and even exceeded tiamulin. Moreover, the docking experiments provided information about the binding model between the synthesized compounds and peptidyl transferase center (PTC) of 23S rRNA. PMID:24376551

  10. Active site electrostatics protect genome integrity by blocking abortive hydrolysis during DNA recombination

    PubMed Central

    Ma, Chien-Hui; Rowley, Paul A; Macieszak, Anna; Guga, Piotr; Jayaram, Makkuni

    2009-01-01

    Water, acting as a rogue nucleophile, can disrupt transesterification steps of important phosphoryl transfer reactions in DNA and RNA. We have unveiled this risk, and identified safeguards instituted against it, during strand cleavage and joining by the tyrosine site-specific recombinase Flp. Strand joining is threatened by a latent Flp endonuclease activity (type I) towards the 3′-phosphotyrosyl intermediate resulting from strand cleavage. This risk is not alleviated by phosphate electrostatics; neutralizing the negative charge on the scissile phosphate through methylphosphonate (MeP) substitution does not stimulate type I endonuclease. Rather, protection derives from the architecture of the recombination synapse and conformational dynamics within it. Strand cleavage is protected against water by active site electrostatics. Replacement of the catalytic Arg-308 of Flp by alanine, along with MeP substitution, elicits a second Flp endonuclease activity (type II) that directly targets the scissile phosphodiester bond in DNA. MeP substitution, combined with appropriate active site mutations, will be useful in revealing anti-hydrolytic mechanisms engendered by systems that mediate DNA relaxation, DNA transposition, site-specific recombination, telomere resolution, RNA splicing and retrohoming of mobile introns. PMID:19440204

  11. Effects of 2′-O-Methyl Nucleotide Substitution on EcoRI Endonuclease Cleavage Activities

    PubMed Central

    Zhao, Guojie; Zhao, Bin; Tong, Zhaoxue; Mu, Runqing; Guan, Yifu

    2013-01-01

    To investigate the effect of sugar pucker conformation on DNA-protein interactions, we used 2′-O-methyl nucleotide (2′-OMeN) to modify the EcoRI recognition sequence -TGAATTCT-, and monitored the enzymatic cleavage process using FRET method. The 2′-O-methyl nucleotide has a C3′-endo sugar pucker conformation different from the C2′-endo sugar pucker conformation of native DNA nucleotides. The initial reaction velocities were measured and the kinetic parameters, Km and Vmax were derived using Michaelis-Menten equation. Experimental results showed that 2′-OMeN substitutions for the EcoRI recognition sequence decreased the cleavage efficiency for A2, A3 and T4 substitutions significantly, and 2′-OMeN substitution for T5 residue inhibited the enzymatic activity completely. In contrast, substitutions for G1 and C6 could maintain the original activity. 2′-fluoro nucleic acid (2′-FNA) and locked nucleic acid (LNA) having similar C3′-endo sugar pucker conformation also demonstrated similar enzymatic results. This position-dependent enzymatic cleavage property might be attributed to the phosphate backbone distortion caused by the switch from C2′-endo to C3′-endo sugar pucker conformation, and was interpreted on the basis of the DNA-EcoRI structure. These 2′-modified nucleotides could behave as a regulatory element to modulate the enzymatic activity in vitro, and this property will have potential applications in genetic engineering and biomedicine. PMID:24194862

  12. Na-site substitution effects on the thermoelectric properties of NaCo2O4

    NASA Astrophysics Data System (ADS)

    Kawata, T.; Iguchi, Y.; Itoh, T.; Takahata, K.; Terasaki, I.

    1999-10-01

    The resistivity and thermopower of Na1+xCo2O4 and Na1.1-xCaxCo2O4 are measured and analyzed. In Na1+xCo2O4, whereas the resistivity increases with x, the thermopower is nearly independent of x. This suggests that the excess Na is unlikely to supply carriers, and decreases effective conduction paths in the sample. In Na1.1-xCaxCo2O4, the resistivity and the thermopower increase with x, and the Ca2+ substitution for Na+ reduces the majority carriers in NaCo2O4. This means that they are holes, which is consistent with the positive sign of the thermopower. Strong correlation in this compound is evidenced by the peculiar temperature dependence of the resistivity.

  13. Quantitative determination of occupation sites of trace Co substituted for multiple Fe sites in M-type hexagonal ferrite using statistical beam-rocking TEM-EDXS analysis.

    PubMed

    Ohtsuka, Masahiro; Muto, Shunsuke; Tatsumi, Kazuyoshi; Kobayashi, Yoshinori; Kawata, Tsunehiro

    2016-04-01

    The occupation sites and the occupancies of trace dopants in La/Co co-doped Sr-M-type ferrite, SrFe12O19, were quantitatively and precisely determined by beam-rocking energy-dispersive X-ray spectroscopy (EDXS) on the basis of electron-channeling effects. Because the Co atoms, in particular, should be partially substituted for the five crystallographically inequivalent sites, which could be key parameters in improving the magneto-crystalline anisotropy, it is difficult yet intriguing to discover their occupation sites and occupancies without using the methods of large-scale facilities, such as neutron diffraction and synchrotron radiation. In the present study, we tackled this problem by applying an extended statistical atom location by channeling enhanced microanalysis method, using conventional transmission electron microscopy, EDXS and dynamical electron elastic/inelastic scattering theories. The results show that the key occupation sites of Co were the 2a, 4f1 and 12k sites. The quantified occupancies of Co were consistent with those of the previous study, which involved a combination of neutron diffraction and extended X-ray absorption fine structure analysis, as well as energetics considerations based on by first-principles calculations. PMID:26520786

  14. Local structure and site occupancy of Cd and Hg substitutions in CeTIn5 (T=Co, Rh, Ir)

    SciTech Connect

    Bauer, Eric D; Ronning, Filip; Thompson, J D; Sarrao, J L; Booth, C H; Bianchi, A D; Cho, J Y; Chan, J Y; Capan, C; Fisk, Z

    2009-01-01

    The CeTIn{sub 5} superconductors (T = Co, Rh, or Ir) have generated great interest due to their relatively high transition temperatures, non-Fermi liquid behavior, and their proximity to antiferromagnetic order and quantum critical points. In contrast to small changes with the T-species, electron doping in CeT(In{sub 1-x}M{sub x}){sub 5} with M = Sn and hole doping with Cd or Hg have a dramatic effect on the electronic properties at very low concentrations. The present work reports local structure measurements using the extended x-ray absorption fine-structure (EXAFS) technique that address the substituent atom distribution as a function of T, M, and x, in the vicinity of the superconducting phase. Together with previous measurements for M = Sn, the proportion of the M atom residing on the In(1) site, f{sub 1n(1)}, increases in the order M = Cd, Sn, and Hg, ranging from about 40% to 70%, showing a strong preference for each of these substituents to occupy the In(1) site (random occupation = 20%). In addition, f{sub In(1)} ranges from 70% to 100% for M = Hg in the order T = Co, Rh, and Ir. These fractions track the changes in the atomic radii of the various species, and help explain the sharp dependence of T{sub c} on substituting into the In site. However, it is difficult to reconcile the small concentrations of M with the dramatic changes in the ground state in the hole-doped materials with only an impurity scattering model. These results therefore indicate that while such substitutions have interesting local atomic structures with important electronic and magnetic consequences, other local changes in the electronic and magnetic structure are equally important in determining the bulk properties of these materials.

  15. Local structure and site occupancy of Cd and Hg substitutions in CeTIn_5 (T=Co, Rh, Ir)

    SciTech Connect

    Booth, Corwin H.; Bauer, Eric. D.; Bianchi, Andrea D.; Ronning, Fillip; Thompson, Joe D.; Sarrao, John L.; Cho, Jung Young; Chan, Julia Y.; Capan, Cigdem; Fisk, Zachary

    2009-04-22

    The CeTIn_5 superconductors (T=Co, Rh, or Ir) have generated great interest due to their relatively high transition temperatures, non-Fermi liquid behavior, and their proximity to antiferromagnetic order and quantum critical points. In contrast to small changes with the T-species, electron doping in CeT(In_1-x M_x)_5 with $M$=Sn and hole doping with Cd or Hg have a dramatic effect on the electronic properties at very low concentrations. The present work reports local structure measurements usingthe extended x-ray absorption fine-structure (EXAFS) technique that address the substituent atom distribution as a function of T, M, and x, in the vicinity of the superconducting phase. Together with previous measurements for M=Sn, the proportion of the $M$ atom residing on the In(1) site, f_\\textrm In(1), increases in the order M=Cd, Sn, and Hg, ranging from about 40\\percent to 70percent, showing a strong preference for each of these substituents to occupy the In(1) site (random occupation = 20percent). In addition, f_In(1) ranges from 70percent to 100percent for M=Hg in the order T=Co,Rh, and Ir. These fractions track the changes in the atomic radii of the various species, and help explain the sharp dependence of $T_c$ on substituting into the In site. However, it is difficult to reconcile the small concentrations of M with the dramatic changes in the ground state in the hole-doped materials with only an impurity scattering model. These results therefore indicate that while such substitutions have interesting local atomic structures with important electronic and magnetic consequences, other local changes in the electronic and magnetic structure are equally important in determining the bulk properties of these materials.

  16. Synthesis and Biological Activity of 3-N-Substituted Estrogen Derivatives as Breast Cancer Agents

    PubMed Central

    Wan, Zhongliang; Musa, Musiliyu A; Joseph, Patrick; Cooperwood, John S.

    2013-01-01

    3-N-substituted-estrogen derivatives were synthesized and characterized. Their antiproliferative activities against human ER (+) MCF-7 (Breast), ER (−) MDA-MB-231 (breast) and Ishikawa (endometrial) cancer cell lines were determined after 72 hours drug exposure employing CellTiter-Glo assay at concentrations ranging from (0.01-100,000 nM). The antiproliferative activities of these compounds were compared to tamoxifen (TAM), 4-hydroxytamoxifen (4-OHT, active metabolite of tamoxifen) and raloxifene (RAL). In vitro results indicated that compound 5 (IC50 = 12μM) displayed comparable antiproliferative activity against MDA-MB 231 cell line; while compounds 6, 7 and 13 (IC50 = 12μM) displayed higher activity against MCF-7 and Ishikawa cell lines, in comparison to TAM activity (19-33μM). PMID:22876946

  17. Synthesis and biological activity of pyrimido [1, 2-a] quinoline moiety and its 2-substituted derivatives

    NASA Astrophysics Data System (ADS)

    Jadhav, A. G.; Halikar, N. K.

    2013-04-01

    2-Amino-3-cyano quinoline (1) and bis (methylthio) methylene malononitrile (2) were refluxed in N,N-dimethyl formamide (DMF) in presence of catalytic amount of anhydrous potassium carbonate to afforded 3, 11-dicyano-4-imino-2-methylthio -4H-pyrimido [1, 2-a] quinoline (3). The latter were further reacted with different substituted aniline, phenol, hetryl amine and compound containing active methyl group. Afforded to 3, 11-dicyano-4-imino -4H-pyrimido [1, 2-a] quinoline and their 2-substuited derivatives (4a-7c). All these newly synthesized compounds were characterized by elemental analysis and spectral data, and screened for their antimicrobial activities.

  18. Synthesis, cytostatic activity and ADME properties of C-5 substituted and N-acyclic pyrimidine derivatives.

    PubMed

    Kraljević, Tatjana Gazivoda; Klika, Mateja; Kralj, Marijeta; Martin-Kleiner, Irena; Jurmanović, Stella; Milić, Astrid; Padovan, Jasna; Raić-Malić, Silvana

    2012-01-01

    The synthesis of the novel 5-alkyl pyrimidine derivatives, 5,6-dihydrofuro[2,3-d]pyrimidines and 5-alkyl N-methoxymethyl pyrimidine derivatives and evaluation of their cytostatic activities are described. The mechanism of antiproliferative effect of 5-(2-chloroethyl)-substituted pyrimidine 3 that exerted the pronounced cytostatic activity was studied in further details on colon carcinoma (HCT116) cells. The cell cycle perturbation analysis demonstrated severe DNA damage (G2/M arrest) pointing to a potential DNA alkylating ability of 3. Preliminary ADME data of 3 and its 6-methylated structural congener (6-Me-3) showed their high permeability and good metabolic stability. PMID:22130132

  19. Corrosion Research And Web Site Activities

    NASA Technical Reports Server (NTRS)

    Heidersbach, Robert H.

    2001-01-01

    This report covers corrosion-related activities at the NASA Kennedy Space Center during the summer of 2000. The NASA Kennedy Space Center's corrosion web site, corrosion.ksc.nasa.gov, was updated with new information based on feedback over the past two years. The methodology for a two-year atmospheric exposure testing program to study the effectiveness of commercial chemicals sold for rinsing aircraft and other equipment was developed and some preliminary laboratory chemical analyses are presented.

  20. Corrosion Research and Web Site Activities

    NASA Technical Reports Server (NTRS)

    Heidersbach, Robert H.

    2002-01-01

    This report covers corrosion-related activities at the NASA Kennedy Space Center during the summer of 2000. The NASA Kennedy Space Center's corrosion web site, corrosion.ksc.nasa.gov, was updated with new information based on feedback over the past two years. The methodology for a two-year atmospheric exposure testing program to study the effectiveness of commercial chemicals sold for rinsing aircraft and other equipment was developed and some preliminary laboratory chemical analyses are presented.

  1. A cautionary note on substituting spatial subunits for repeated temporal sampling in studies of site occupancy

    USGS Publications Warehouse

    Kendall, William L.; White, Gary C.

    2009-01-01

    1. Assessing the probability that a given site is occupied by a species of interest is important to resource managers, as well as metapopulation or landscape ecologists. Managers require accurate estimates of the state of the system, in order to make informed decisions. Models that yield estimates of occupancy, while accounting for imperfect detection, have proven useful by removing a potentially important source of bias. To account for detection probability, multiple independent searches per site for the species are required, under the assumption that the species is available for detection during each search of an occupied site. 2. We demonstrate that when multiple samples per site are defined by searching different locations within a site, absence of the species from a subset of these spatial subunits induces estimation bias when locations are exhaustively assessed or sampled without replacement. 3. We further demonstrate that this bias can be removed by choosing sampling locations with replacement, or if the species is highly mobile over a short period of time. 4. Resampling an existing data set does not mitigate bias due to exhaustive assessment of locations or sampling without replacement. 5. Synthesis and applications. Selecting sampling locations for presence/absence surveys with replacement is practical in most cases. Such an adjustment to field methods will prevent one source of bias, and therefore produce more robust statistical inferences about species occupancy. This will in turn permit managers to make resource decisions based on better knowledge of the state of the system.

  2. Site selective substitution Pt for Ti in KTiOPO{sub 4}:Ga crystals revealed by electron paramagnetic resonance

    SciTech Connect

    Grachev, V.; Meyer, M.; Jorgensen, J.; Malovichko, G.; Hunt, A. W.

    2014-07-28

    Electron Paramagnetic Resonance at low temperatures has been used to characterize potassium titanyl phosphate (KTiOPO{sub 4}) single crystals grown by different techniques. Irradiation with 20 MeV electrons performed at room temperature and liquid nitrogen temperature caused an appearance of electrons and holes. Platinum impurities act as electron traps in KTiOPO{sub 4} creating Pt{sup 3+} centers. Two different Pt{sup 3+} centers were observed, Pt(A) and Pt(D). The Pt(A) centers are dominant in undoped samples, whereas Pt(D)—in Ga-doped KTP crystals. Superhyperfine structure registered for Pt(D) centers was attributed to interactions of platinum electrons with {sup 39}K and two {sup 31}P nuclei in their surroundings. In both Pt(A) and Pt(D) centers, Pt{sup 3+} ions substitute for Ti{sup 4+} ions, but with a preference to one of two electrically distinct crystallographic positions. The site selective substitution can be controlled by the Ga-doping.

  3. Study on the synthesis and biological activities of α-substituted arylacetates derivatives.

    PubMed

    Liu, Jinbing; Chen, Changhong; Wu, Fengyan; Tang, Junyuan

    2016-04-01

    Anisodamine was isolated from the medicinal herb, it was used in the treatment of gastrointestinal smooth muscle spasm, infective toxic shock and organophosphorus intoxication. But there is no report about anisodamine with α-glucosidase inhibitory activity. In order to find novel α-glucosidase inhibitors, a series of α-substituted arylacetates derivatives have been synthesized based on the active unit of anisodamine. In α-glucosidase assay, compound 9 in Schiff base form and compound 22 in ester form show strong inhibition against α-glucosidase with IC50 value of 46.81μM and 83.76μM, respectively. Compounds 9 and 22 exhibit comparable good antidiabetic activities as commercial drug Glimepiride. In addition, Schiff bases of α-substituted arylacetates show antitumor activities against human cancer cell lines, where compound 9 with thiourea moiety performs the best antitumor activity. We anticipate that our research will provide potential candidate scaffolds for antidiabetic drug design. PMID:26920798

  4. Investigating the role of a backbone to substrate hydrogen bond in OMP decarboxylase using a site-specific amide to ester substitution

    PubMed Central

    Desai, Bijoy J.; Goto, Yuki; Cembran, Alessandro; Fedorov, Alexander A.; Almo, Steven C.; Gao, Jiali; Suga, Hiroaki; Gerlt, John A.

    2014-01-01

    Hydrogen bonds between backbone amide groups of enzymes and their substrates are often observed, but their importance in substrate binding and/or catalysis is not easy to investigate experimentally. We describe the generation and kinetic characterization of a backbone amide to ester substitution in the orotidine 5′-monophosphate (OMP) decarboxylase from Methanobacter thermoautotrophicum (MtOMPDC) to determine the importance of a backbone amide–substrate hydrogen bond. The MtOMPDC-catalyzed reaction is characterized by a rate enhancement (∼1017) that is among the largest for enzyme-catalyzed reactions. The reaction proceeds through a vinyl anion intermediate that may be stabilized by hydrogen bonding interaction between the backbone amide of a conserved active site serine residue (Ser-127) and oxygen (O4) of the pyrimidine moiety and/or electrostatic interactions with the conserved general acidic lysine (Lys-72). In vitro translation in conjunction with amber suppression using an orthogonal amber tRNA charged with l-glycerate (HOS) was used to generate the ester backbone substitution (S127HOS). With 5-fluoro OMP (FOMP) as substrate, the amide to ester substitution increased the value of Km by ∼1.5-fold and decreased the value of kcat by ∼50-fold. We conclude that (i) the hydrogen bond between the backbone amide of Ser-127 and O4 of the pyrimidine moiety contributes a modest factor (∼102) to the 1017 rate enhancement and (ii) the stabilization of the anionic intermediate is accomplished by electrostatic interactions, including its proximity of Lys-72. These conclusions are in good agreement with predictions obtained from hybrid quantum mechanical/molecular mechanical calculations. PMID:25275007

  5. Synthesis, antispasmodic and antidiarrheal activities of some 1-substituted imidazole derivatives.

    PubMed

    Lakshmanan, Baskar; Mazumder, Papiya Mitra; Sasmal, Dinakar; Ganguly, Swastika

    2011-06-01

    A series of 1-substituted imidazoles 1a-d and 2a-d were synthesized and screened for antispasmodic and antidiarrheal activities. Antispasmodic activity was tested at various concentrations on isolated tissue preparations; concentration-response curves were plotted and compared with atropine. All compounds were found to inhibit contraction of the guinea pig ileum. Castor oil-induced diarrhea model in rats was used for evaluation of antidiarrheal activity. Parameters such as intestinal transit and volume of intestinal fluid were measured for antidiarrheal activity at 40 mg kg-1 dose and compared with the standard drug loperamide at 6 mg kg-1 dose. Defecation frequency in the test group was found to be significantly lower (p < 0.01) compared to the control group and comparable with that of the standard. The present study reveals that the compounds exert antidiarrheal activity through possible inhibition of intestinal movement and reduction of capillary permeability in the abdominal cavity. PMID:21684849

  6. EPR investigation of Cu2+-substituted photosynthetic bacterial reaction centers: evidence for histidine ligation at the surface metal site.

    PubMed

    Utschig, L M; Poluektov, O; Tiede, D M; Thurnauer, M C

    2000-03-21

    The coordination environments of two distinct metal sites on the bacterial photosynthetic reaction center (RC) protein were probed with pulsed electron paramagnetic resonance (EPR) spectroscopy. For these studies, Cu2+ was bound specifically to a surface site on native Fe2+-containing RCs from Rhodobacter sphaeroides R-26 and to the native non-heme Fe site in biochemically Fe-removed RCs. The cw and pulsed EPR results clearly indicate two spectroscopically different Cu2+ environments. In the dark, the RCs with Cu2+ bound to the surface site exhibit an axially symmetric EPR spectrum with g(parallel) = 2.24, A(parallel) = 160 G, g(perpendicular) = 2.06, whereas the values g(parallel) = 2.31, A(parallel) = 143 G, and g(perpendicular) = 2.07 were observed when Cu(2+) was substituted in the Fe site. Examination of the light-induced spectral changes indicate that the surface Cu2+ is at least 23 A removed from the primary donor (P+) and reduced quinone acceptor (QA-). Electron spin-echo envelope modulation (ESEEM) spectra of these Cu-RC proteins have been obtained and provide the first direct solution structural information about the ligands in the surface metal site. From these pulsed EPR experiments, modulations were observed that are consistent with multiple weakly hyperfine coupled 14N nuclei in close proximity to Cu2+, indicating that two or more histidines ligate the Cu2+ at the surface site. Thus, metal and EPR analyses confirm that we have developed reliable methods for stoichiometrically and specifically binding Cu2+ to a surface site that is distinct from the well characterized Fe site and support the view that Cu2+ is bound at or near the Zn site that modulates electron transfer between the quinones QA and QB (QA-QB --> QAQB-) (Utschig, L. M., Ohigashi, Y., Thurnauer, M. C., and Tiede, D. M (1998) Biochemistry 37, 8278-8281) and proton uptake by QB- (Paddock, M. L., Graige, M. S., Feher, G., and Okamura, M. Y. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 6183

  7. Hydroxylation of p-substituted phenols by tyrosinase: further insight into the mechanism of tyrosinase activity.

    PubMed

    Muñoz-Muñoz, Jose Luis; Berna, Jose; García-Molina, María del Mar; Garcia-Molina, Francisco; Garcia-Ruiz, Pedro Antonio; Varon, Ramon; Rodriguez-Lopez, Jose N; Garcia-Canovas, Francisco

    2012-07-27

    A study of the monophenolase activity of tyrosinase by measuring the steady state rate with a group of p-substituted monophenols provides the following kinetic information: k(cat)(m) and the Michaelis constant, K(M)(m). Analysis of these data taking into account chemical shifts of the carbon atom supporting the hydroxyl group (δ) and σ(p)(+), enables a mechanism to be proposed for the transformation of monophenols into o-diphenols, in which the first step is a nucleophilic attack on the copper atom on the form E(ox) (attack of the oxygen of the hydroxyl group of C-1 on the copper atom) followed by an electrophilic attack (attack of the hydroperoxide group on the ortho position with respect to the hydroxyl group of the benzene ring, electrophilic aromatic substitution with a reaction constant ρ of -1.75). These steps show the same dependency on the electronic effect of the substituent groups in C-4. Furthermore, a study of a solvent deuterium isotope effect on the oxidation of monophenols by tyrosinase points to an appreciable isotopic effect. In a proton inventory study with a series of p-substituted phenols, the representation of [Formula: see text] / [Formula: see text] against n (atom fractions of deuterium), where [Formula: see text] is the catalytic constant for a molar fraction of deuterium (n) and [Formula: see text] is the corresponding kinetic parameter in a water solution, was linear for all substrates. These results indicate that only one of the proton transfer processes from the hydroxyl groups involved the catalytic cycle is responsible for the isotope effects. We suggest that this step is the proton transfer from the hydroxyl group of C-1 to the peroxide of the oxytyrosinase form (E(ox)). After the nucleophilic attack, the incorporation of the oxygen in the benzene ring occurs by means of an electrophilic aromatic substitution mechanism in which there is no isotopic effect. PMID:22732412

  8. Synthesis and SAR studies of novel 6,7,8-substituted 4-substituted benzyloxyquinolin-2(1H)-one derivatives for anticancer activity

    PubMed Central

    Chen, Yi-Fong; Lin, Yi-Chien; Morris-Natschke, Susan L; Wei, Chen-Fang; Shen, Ting-Chen; Lin, Hui-Yi; Hsu, Mei-Hua; Chou, Li-Chen; Zhao, Yu; Kuo, Sheng-Chu; Lee, Kuo-Hsiung; Huang, Li-Jiau

    2015-01-01

    Background and Purpose 4-Phenylquinolin-2(1H)-one (4-PQ) derivatives can induce cancer cell apoptosis. Additional new 4-PQ analogs were investigated as more effective, less toxic antitumour agents. Experimental Approach Forty-five 6,7,8-substituted 4-substituted benzyloxyquinolin-2(1H)-one derivatives were synthesized. Antiproliferative activities were evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliun bromide assay and structure–activity relationship correlations were established. Compounds 9b, 9c, 9e and 11e were also evaluated against the National Cancer Institute-60 human cancer cell line panel. Hoechst 33258 and Annexin V-FITC/PI staining assays were used to detect apoptosis, while inhibition of microtubule polymerization was assayed by fluorescence microscopy. Effects on the cell cycle were assessed by flow cytometry and on apoptosis-related proteins (active caspase-3, -8 and -9, procaspase-3, -8, -9, PARP, Bid, Bcl-xL and Bcl-2) by Western blotting. Key Results Nine 6,7,8-substituted 4-substituted benzyloxyquinolin-2(1H)-one derivatives (7e, 8e, 9b, 9c, 9e, 10c, 10e, 11c and 11e) displayed high potency against HL-60, Hep3B, H460, and COLO 205 cancer cells (IC50 < 1 μM) without affecting Detroit 551 normal human cells (IC50 > 50 μM). Particularly, compound 11e exhibited nanomolar potency against COLO 205 cancer cells. Mechanistic studies indicated that compound 11e disrupted microtubule assembly and induced G2/M arrest, polyploidy and apoptosis via the intrinsic and extrinsic signalling pathways. Activation of JNK could play a role in TRAIL-induced COLO 205 apoptosis. Conclusion and Implications New quinolone derivatives were identified as potential pro-apoptotic agents. Compound 11e could be a promising lead compound for future antitumour agent development. PMID:25363404

  9. Microwave and infrared dielectric response of monoclinic bismuth zinc niobate based pyrochlore ceramics with ion substitution in A site

    SciTech Connect

    Wang Hong; Kamba, Stanislav; Zhang Meiling; Yao Xi; Denisov, Sergey; Kadlec, Filip; Petzelt, Jan

    2006-08-01

    It is well known that the cubic pyrochlore Bi{sub 1.5}ZnNb{sub 1.5}O{sub 7} exhibits higher permittivity and dielectric loss than monoclinic Bi{sub 2}Zn{sub 2/3}Nb{sub 4/3}O{sub 7} due to structural disorder in the A sites of Bi{sub 1.5}ZnNb{sub 1.5}O{sub 7}. We have studied systematically the impact of the ion substitution in the A site of monoclinic Bi{sub 2}Zn{sub 2/3}Nb{sub 4/3}O{sub 7} on the structure and microwave dielectric properties. It is shown that the structure and permittivity of (Bi{sub 1.92}M{sub 0.08})(Zn{sub 0.64}Nb{sub 1.36})O{sub 7} (M=Zn,Ca,Cd,Sr,Ba) ceramics remain almost the same as in Bi{sub 2}Zn{sub 2/3}Nb{sub 4/3}O{sub 7}; only the Ba substituted ceramics have higher permittivity due to multiphase structure. Microwave dielectric properties were compared with complex dielectric response in terahertz and infrared frequency range of 0.1-100 THz, which allows us to estimate intrinsic and extrinsic contributions to microwave dielectric losses. The best microwave properties were obtained in (Bi{sub 1.92}Ca{sub 0.08})(Zn{sub 0.64}Nb{sub 1.36})O{sub 7} with {epsilon}=76, Qf{>=}5000 (sintered below 950 deg.C), which is promising for microwave low temperature cofiring ceramic application.

  10. Tuning of magnetic ordering by Y substitution onto Tb site in the nanocrystalline TbMnO{sub 3}

    SciTech Connect

    Chakraborty, Keka R. E-mail: smyusuf@barc.gov.in; Mukadam, M. D.; Yusuf, S. M. E-mail: smyusuf@barc.gov.in; Shukla, Rakesh; Tyagi, A. K.; Kaushik, S. D.; Siruguri, V.

    2015-10-28

    We report the magnetic properties, of nano-crystalline powders Tb{sub 1−x}Y{sub x}MnO{sub 3} (x = 0, 0.1, 0.2, 0.3 and 0.4), as perceived by neutron diffraction, and elucidate the effect of Tb site substitution on the magnetic structure of TbMnO{sub 3}. All samples crystallized in the orthorhombic structure conforming to space group Pnma, and exhibited an incommensurate collinear antiferromagnetic ordering of the Mn ions below ∼40 K. Furthermore, at T ≤ 20 K, all these samples showed a change in magnetic structure (of Mn moments) to a spiral ordering down to 2 K, the lowest measured temperature. For the samples with x = 0, 0.1, and 0.2, a short-ranged two dimensional (2D) ordering of Tb moments was also observed at 2 K. However, for the other samples (x = 0.3 and 0.4), no magnetic ordering of Tb moments was found down to 2 K. So with Y substitution, a crossover from 2D ordering to a disordering of Tb moments was observed. The moments at the Mn site were found to be lower than the full Mn{sup 3+} (4μ{sub B}) moment for all the samples below 40 K. The magnetic properties of all the samples studied by us in nano form are more pronounced than those of the reported single crystals of same compositions [V. Yu. Ivanov et al., JETP Lett. 91, 392–397 (2010)].

  11. One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative Approach to Generate Amino Acid Substitution Collections

    PubMed Central

    Mingo, Janire; Erramuzpe, Asier; Luna, Sandra; Aurtenetxe, Olaia; Amo, Laura; Diez, Ibai; Schepens, Jan T. G.; Hendriks, Wiljan J. A. J.; Cortés, Jesús M.; Pulido, Rafael

    2016-01-01

    Site-directed mutagenesis (SDM) is a powerful tool to create defined collections of protein variants for experimental and clinical purposes, but effectiveness is compromised when a large number of mutations is required. We present here a one-tube-only standardized SDM approach that generates comprehensive collections of amino acid substitution variants, including scanning- and single site-multiple mutations. The approach combines unified mutagenic primer design with the mixing of multiple distinct primer pairs and/or plasmid templates to increase the yield of a single inverse-PCR mutagenesis reaction. Also, a user-friendly program for automatic design of standardized primers for Ala-scanning mutagenesis is made available. Experimental results were compared with a modeling approach together with stochastic simulation data. For single site-multiple mutagenesis purposes and for simultaneous mutagenesis in different plasmid backgrounds, combination of primer sets and/or plasmid templates in a single reaction tube yielded the distinct mutations in a stochastic fashion. For scanning mutagenesis, we found that a combination of overlapping primer sets in a single PCR reaction allowed the yield of different individual mutations, although this yield did not necessarily follow a stochastic trend. Double mutants were generated when the overlap of primer pairs was below 60%. Our results illustrate that one-tube-only SDM effectively reduces the number of reactions required in large-scale mutagenesis strategies, facilitating the generation of comprehensive collections of protein variants suitable for functional analysis. PMID:27548698

  12. One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative Approach to Generate Amino Acid Substitution Collections.

    PubMed

    Mingo, Janire; Erramuzpe, Asier; Luna, Sandra; Aurtenetxe, Olaia; Amo, Laura; Diez, Ibai; Schepens, Jan T G; Hendriks, Wiljan J A J; Cortés, Jesús M; Pulido, Rafael

    2016-01-01

    Site-directed mutagenesis (SDM) is a powerful tool to create defined collections of protein variants for experimental and clinical purposes, but effectiveness is compromised when a large number of mutations is required. We present here a one-tube-only standardized SDM approach that generates comprehensive collections of amino acid substitution variants, including scanning- and single site-multiple mutations. The approach combines unified mutagenic primer design with the mixing of multiple distinct primer pairs and/or plasmid templates to increase the yield of a single inverse-PCR mutagenesis reaction. Also, a user-friendly program for automatic design of standardized primers for Ala-scanning mutagenesis is made available. Experimental results were compared with a modeling approach together with stochastic simulation data. For single site-multiple mutagenesis purposes and for simultaneous mutagenesis in different plasmid backgrounds, combination of primer sets and/or plasmid templates in a single reaction tube yielded the distinct mutations in a stochastic fashion. For scanning mutagenesis, we found that a combination of overlapping primer sets in a single PCR reaction allowed the yield of different individual mutations, although this yield did not necessarily follow a stochastic trend. Double mutants were generated when the overlap of primer pairs was below 60%. Our results illustrate that one-tube-only SDM effectively reduces the number of reactions required in large-scale mutagenesis strategies, facilitating the generation of comprehensive collections of protein variants suitable for functional analysis. PMID:27548698

  13. Dual Mode Antibacterial Activity of Ion Substituted Calcium Phosphate Nanocarriers for Bone Infections

    PubMed Central

    Sampath Kumar, T. S.; Madhumathi, K.; Rubaiya, Y.; Doble, Mukesh

    2015-01-01

    Nanotechnology has tremendous potential for the management of infectious diseases caused by multi-drug resistant bacteria, through the development of newer antibacterial materials and efficient modes of antibiotic delivery. Calcium phosphate (CaP) bioceramics are commonly used as bone substitutes due to their similarity to bone mineral and are widely researched upon for the treatment of bone infections associated with bone loss. CaPs can be used as local antibiotic delivery agents for bone infections and can be substituted with antibacterial ions in their crystal structure to have a wide spectrum, sustained antibacterial activity even against drug resistant bacteria. In the present work, a dual mode antibiotic delivery system with antibacterial ion substituted calcium deficient hydroxyapatite (CDHA) nanoparticles has been developed. Antibacterial ions such as zinc, silver, and strontium have been incorporated into CDHA at concentrations of 6, 0.25–0.75, and 2.5–7.5 at. %, respectively. The samples were found to be phase pure, acicular nanoparticles of length 40–50 nm and width 5–6 nm approximately. The loading and release profile of doxycycline, a commonly used antibiotic, was studied from the nanocarriers. The drug release was studied for 5 days and the release profile was influenced by the ion concentrations. The release of antibacterial ions was studied over a period of 21 days. The ion substituted CDHA samples were tested for antibacterial efficacy on Staphylococcus aureus and Escherichia coli by MIC/MBC studies and time-kill assay. AgCDHA and ZnCDHA showed high antibacterial activity against both bacteria, while SrCDHA was weakly active against S. aureus. Present study shows that the antibiotic release can provide the initial high antibacterial activity, and the sustained ion release can provide a long-term antibacterial activity. Such dual mode antibiotic and antibacterial ion release offers an efficient and potent way to treat an incumbent

  14. Structure-olfactory activity relationship in a group of substituted phenols.

    PubMed

    Kaliszan, R; Pankowski, M; Szymula, L; Lamparczyk, H; Nasal, A; Tomaszewska, B; Grzybowski, J

    1982-07-01

    Using phenol as the standard relative olfactory thresholds have been determined for a series of substituted phenols in experiments with 8--10 human subjects. Significant relations have been obtained describing the activity as a square function of the hydrophobicity parameter corrected for ionization. Chromatographic measurement of phenol polarity has been proposed based on retention indices determined on phases of different polarity. The human sense of smell system has been discussed as a model for studies on drug-receptor interactions involving the living organism as a whole. PMID:7134257

  15. Synthesis and p38 Inhibitory Activity of Some Novel Substituted N,N'-Diarylurea Derivatives.

    PubMed

    Zhu, Dianxi; Xing, Qifeng; Cao, Ruiyuan; Zhao, Dongmei; Zhong, Wu

    2016-01-01

    We have identified a novel series of substituted N,N'-diarylurea p38α inhibitors. The inhibitory activity of the target compounds against the enzyme p38α, MAPKAPK2 in BHK cells, TNF-α release in LPS-stimulated THP-1 cells and p38α binding experiments were tested. Among these compounds, 25a inhibited the p38α enzyme with an IC50 value of 0.47 nM and a KD value of 1.54 × 10(-8) and appears to be the most promising one in the series. PMID:27223276

  16. Antibacterial activity of substituted dibenzo[a,g]quinolizin-7-ium derivatives

    PubMed Central

    Parhi, Ajit; Lu, Songfeng; Kelley, Cody; Kaul, Malvika; Pilch, Daniel S.; LaVoie, Edmond J.

    2014-01-01

    Berberine is a substituted dibenzo[a,g]quinolizin-7-ium derivative whose modest antibiotic activity is derived from its disruptive impact on the function of the essential bacterial cell division protein FtsZ. The present study reveals that the presence of a biphenyl substituent at either the 2- or 12-position of structurally-related dibenzo[a,g]quinolizin-7-ium derivatives significantly enhances antibacterial potency versus Staphylococcus aureus and Enterococcus faecalis. Studies with purified S. aureus FtsZ demonstrate that both 2- and 12-biphenyl dibenzo[a,g]quinolizin-7-ium derivatives act as enhancers of FtsZ self-polymerization. PMID:23058886

  17. Active site diversification of P450cam with indole generates catalysts for benzylic oxidation reactions

    PubMed Central

    Herter, Susanne; Kranz, David C; Turner, Nicholas J

    2015-01-01

    Summary Cytochrome P450 monooxygenases are useful biocatalysts for C–H activation, and there is a need to expand the range of these enzymes beyond what is naturally available. A panel of 93 variants of active self-sufficient P450cam[Tyr96Phe]-RhFRed fusion enzymes with a broad diversity in active site amino acids was developed by screening a large mutant library of 16,500 clones using a simple, highly sensitive colony-based colorimetric screen against indole. These mutants showed distinct fingerprints of activity not only when screened in oxidations of substituted indoles but also for unrelated oxidations such as benzylic hydroxylations. PMID:26664590

  18. Preparation and antioxidant activity of xanthan oligosaccharides derivatives with similar substituting degrees.

    PubMed

    Xiong, Xiaoying; Li, Ming; Xie, Jing; Xue, Bin; Sun, Tao

    2014-12-01

    Maleoyl xanthan oligosaccharides (XGOSMAs) and phthaloyl xanthan oligosaccharides (XGOSPAs) were prepared by reacting xanthan oligosaccharides with maleic anhydride and phthalic anhydride, respectively. The substituting degrees (DSs) of XGOSMAs and XGOSPAs were determined by a neutralization reaction. XGOAMA-1 (DS=0.30), XGOSPA (DS=0.31), XGOSMA-2 (DS=0.62) and XGOSPA-2 (DS=0.60) were selected for structural characterization and antioxidant activity evaluation. Their structural changes were confirmed by Fourier transform infrared spectra (FT-IR), and their molecular weights were determined with a gel permeation chromatography method (GPC). The pyruvate acid and reducing sugar contents were determined by ultraviolet spectrophotometry and the dinitrosalicylic acid method. The antioxidant activity was evaluated by the scavenging of the superoxide anion radical (O2(-)), hydroxyl radical (OH), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and determination of reducing power. The results indicated that XGOSPA exhibited higher antioxidant activity than XGOSMA with similar substituting degrees in all the above mentioned antioxidant evaluation systems, which may be related to the fact that phthaloyl group has a stronger electron-withdrawing effect than the maleoyl group. PMID:24996297

  19. Synthesis and antitumor activity of novel N-substituted carbazole imidazolium salt derivatives

    PubMed Central

    Liu, Lan-Xiang; Wang, Xue-Quan; Zhou, Bei; Yang, Li-Juan; Li, Yan; Zhang, Hong-Bin; Yang, Xiao-Dong

    2015-01-01

    A series of novel N-substituted carbazole imidazolium salt derivatives has been prepared and investigated for their cytotoxic activity against five human tumor cell lines by MTS assay. The results indicated that the existence of 5,6-dimethyl-benzimidazole ring, substitution of the imidazolyl-3-position with a 2-bromobenzyl or naphthylacyl group, as well as alkyl chain length between carbazole and imidazole ring were important for the antitumor activity. Compound 61, bearing a 2-bromobenzyl substituent at position-3 of the 5,6-dimethyl-benzimidazole, showed powerful inhibitory activities and was more selective to HL-60, SMMC-7721, MCF-7 and SW480 cell lines with IC50 values 0.51–2.48 μM. Mechanism of action studies revealed that this new compound could remarkably induce cell cycle arrest and apoptosis in SMMC-7721 cells. This work provides alternative novel way for future drug development based on carbazole and imidazolium salt scaffolds. PMID:26287982

  20. Synthesis, Characterization, Antimicrobial Screening and Free-Radical Scavenging Activity of Some Novel Substituted Pyrazoles.

    PubMed

    Hamada, Nagwa Mohamed Mahrous; Abdo, Nadia Yousef Megally

    2015-01-01

    The present work deals with the synthesis of acetoxysulfonamide pyrazole derivatives, substituted 4,5-dihydropyrazole-1-carbothioamide and 4,5-dihydropyrazole-1-isonicotinoyl derivatives starting from substituted vanillin chalcones. Acetoxysulfonamide pyrazole derivatives were prepared from the reaction of chalcones with p-sulfamylphenylhydrazine followed by treatment with acetic anhydride. At the same time 4,5-dihydropyrazole-1-carbothioamide and 4,5-dihydropyrazole-1-isonicotinoyl derivatives were prepared from the reaction of chalcones with either thiosemicarbazide or isonicotinic acid hydrazide, respectively. The synthesized compounds were structurally characterized on the basis of IR, 1H-NMR, 13C-NMR spectral data and microanalyses. All of the newly isolated compounds were tested for their antimicrobial activities. The antimicrobial screening using the agar well-diffusion method revealed that the chloro derivatives are the most active ones. Moreover, the antioxidant and anti-inflammatory activity of these chloro derivatives are also studied using the DPPH radical scavenging and NO radical scavenging methods, respectively. PMID:26060913

  1. Synthesis of N-substituted phthalimides and their antifungal activity against Alternaria solani and Botrytis cinerea.

    PubMed

    Pan, Le; Li, Xiuzhuang; Gong, Chengwen; Jin, Hui; Qin, Bo

    2016-06-01

    As organosulfur and organophosphorus agents, phaltane and phosmet are facing great challenges for the environmental contamination, mammalian toxicity and increasing resistance with long term use. It is efficient and meaningful to develop phthalimide-based alternatives with non-sulfur and non-phosphorus groups. A series of N-substituted phthalimides were synthesized and their antifungal activity against two disastrous phytopathogenic fungi, Alternaria solani and Botrytis cinerea was evaluated in vitro. Most of them showed significant antifungal activity against both of fungi, or either of them selectively. N-vinylphthalimide (4) and 8-[4-(phthalimide-2-yl) butyloxy] quinoline (13) were identified as the most promising candidates against B. cinerea and A. solani with the IC50 values of 7.92 μg/mL and 10.85 μg/mL respectively. The brief structure-activity relationships have revealed that vinyl, quinolyl, bromide alkyl and benzyl substitutions were appropriate substituents and coupling functional moieties indirectly with optimum alkyl chain was efficient to prepare phthalimides related fungicides. PMID:27079471

  2. Functional analysis of the BRI1 receptor kinase by Thr-for-Ser substitution in a regulatory autophosphorylation site

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BRI1 becomes highly phosphorylated in vivo upon perception of the ligand, brassinolide, as a result of autophosphorylation and transphosphorylation by its co-receptor kinase, BAK1. Important autophosphorylation sites include those involved in activation of kinase activity and those that are inhibito...

  3. Hydroxylation of p-substituted phenols by tyrosinase: Further insight into the mechanism of tyrosinase activity

    SciTech Connect

    Munoz-Munoz, Jose Luis; Berna, Jose; Garcia-Molina, Maria del Mar; Garcia-Molina, Francisco; Garcia-Ruiz, Pedro Antonio; Varon, Ramon [Departamento de Quimica-Fisica, Escuela de Ingenieros Industriales de Albacete, Universidad de Castilla la Mancha, Avda. Espana s and others

    2012-07-27

    Highlights: Black-Right-Pointing-Pointer The action the copper complexes and tyrosinase on phenols is equivalent. Black-Right-Pointing-Pointer Isotope effect showed that nucleophilic attack to copper atom may be the slower step. Black-Right-Pointing-Pointer The value of {rho} (Hammett constant) supports an electrophilic aromatic substitution. Black-Right-Pointing-Pointer Data obtained in steady state pH 7 conditions support the mechanism of Scheme 1SM. -- Abstract: A study of the monophenolase activity of tyrosinase by measuring the steady state rate with a group of p-substituted monophenols provides the following kinetic information: k{sub cat}{sup m} and the Michaelis constant, K{sub M}{sup m}. Analysis of these data taking into account chemical shifts of the carbon atom supporting the hydroxyl group ({delta}) and {sigma}{sub p}{sup +}, enables a mechanism to be proposed for the transformation of monophenols into o-diphenols, in which the first step is a nucleophilic attack on the copper atom on the form E{sub ox} (attack of the oxygen of the hydroxyl group of C-1 on the copper atom) followed by an electrophilic attack (attack of the hydroperoxide group on the ortho position with respect to the hydroxyl group of the benzene ring, electrophilic aromatic substitution with a reaction constant {rho} of -1.75). These steps show the same dependency on the electronic effect of the substituent groups in C-4. Furthermore, a study of a solvent deuterium isotope effect on the oxidation of monophenols by tyrosinase points to an appreciable isotopic effect. In a proton inventory study with a series of p-substituted phenols, the representation of k{sub cat}{sup f{sub n}}/k{sub cat}{sup f{sub 0}} against n (atom fractions of deuterium), where k{sub cat}{sup f{sub n}} is the catalytic constant for a molar fraction of deuterium (n) and k{sub cat}{sup f{sub 0}} is the corresponding kinetic parameter in a water solution, was linear for all substrates. These results indicate that

  4. Bacteriorhodopsin mutants containing single substitutions of serine or threonine residues are all active in proton translocation

    SciTech Connect

    Marti, T.; Otto, H.; Mogi, T.; Roesselet, S.J.H.; Heyn, M.P.; Khorana, H.G. )

    1991-04-15

    To study their role in proton translocation by bacteriorhodopsin, 22 serine and threonine residues presumed to be located within and near the border of the transmembrane segments have been individually replaced by alanine or valine, respectively. Thr-89 was substituted by alanine, valine, and aspartic acid, and Ser-141 by alanine and cysteine. Most of the mutants showed essentially wild-type phenotype with regard to chromophore regeneration and absorption spectrum. However, replacement of Thr-89 by Val and of Ser-141 by Cys caused striking blue shifts of the chromophore by 100 and 80 nm, respectively. All substitutions of Thr-89 regenerated the chromophore at least 10-fold faster with 13-cis retinal than with all-trans retinal. The substitutions at positions 89, 90, and 141 also showed abnormal dark-light adaptation, suggesting interactions between these residues and the retinylidene chromophore. Proton pumping measurements revealed 60-75% activity for mutants of Thr-46, -89, -90, -205, and Ser-226, and about 20% for Ser-141----Cys, whereas the remaining mutants showed normal pumping. Kinetic studies of the photocycle and of proton release and uptake for mutants in which proton pumping was reduced revealed generally little alterations. The reduced activity in several of these mutants is most likely due to a lower percentage of all-trans retinal in the light-adapted state. In the mutants Thr-46----Val and Ser-226----Ala the decay of the photointer-mediate M was significantly accelerated, indicating an interaction between these residues and Asp-96 which reprotonates the Schiff base. Our results show that no single serine or threonine residue is obligatory for proton pumping.

  5. Synthesis and fungicidal activities of novel benzothiophene-substituted oxime ether strobilurins.

    PubMed

    Tu, Song; Xie, Ya-Qiang; Gui, Si-Zhe; Ye, Li-Yi; Huang, Zi-Long; Huang, Yi-Bing; Che, Li-Ming

    2014-05-01

    Twenty-one novel benzothiophene-substituted oxime ether strobilurins, which employed a benzothiophene group to stabilise the E-styryl group in Enoxastrobin (an unsaturated oxime strobilurin fungicide developed by Shenyang Research Institute of Chemical Industry, China) were designed and synthesised. The biological assay indicated that most compounds exhibited good or excellent fungicidal activities, especially against Colletotrichum lagenarium and Puccinia sorghi Schw. In addition, methyl 3-methoxypropenoate oxime ethers and N-methoxy-carbamic acid methyl esters exhibited good in vivo fungicidal activities against Erysiphe graminis, Colletotrichum lagenarium and Puccinia sorghi Schw. under the tested concentrations. Notably, (E,E)-methyl 3-methoxy-2-(2-((((6-chloro-1-(1H-benzo[b]thien-2-yl)ethylidene)amino)oxy)methyl)phenyl)propenoate (5E) exhibited more potent in vivo fungicidal activities against nearly all of the tested fungi at a concentration of 0.39 mg/L compared to Enoxastrobin. PMID:24717155

  6. Synthesis and anti-proliferative activity of fluoro-substituted chalcones.

    PubMed

    Burmaoglu, Serdar; Algul, Oztekin; Anıl, Derya Aktas; Gobek, Arzu; Duran, Gulay Gulbol; Ersan, Ronak Haj; Duran, Nizami

    2016-07-01

    A series of novel fluoro-substituted chalcone derivatives have been synthesized. All synthesized compounds were characterized by (1)H nuclear magnetic resonance (NMR), (13)C NMR, and elemental analysis. Their anti-proliferative activities were evaluated against five cancer cells lines, namely, A549, A498, HeLa, A375, and HepG2 using the MTT method. Most of the compounds showed moderate to high activity with IC50 values in the range of 0.029-0.729μM. Of all the synthesized compounds, 10 and 19 exhibited the most potent anti-proliferative activities against cancer cells, and 10 was identified as the most promising compound. PMID:27217001

  7. Site-directed mutagenesis and high-resolution NMR spectroscopy of the active site of porphobilinogen deaminase

    SciTech Connect

    Scott, A.I.; Roessner, C.A.; Stolowich, N.J.; Karuso, P.; Williams, H.J.; Grant, S.K.; Gonzalez, M.D.; Hoshino, T. )

    1988-10-18

    The active site of porphobilinogen (PBG){sup 1} deaminase from Escherichia coli has been found to contain an unusual dipyrromethane derived from four molecules of 5-aminolevulinic acid (ALA) covalently linked to Cys-242, one of the two cysteine residues conserved in E. coli and human deaminase. By use of a hemA{sup {minus}} strain of E. coli the enzyme was enriched from (5-{sup 13}C)ALA and examined by {sup 1}H-detected multiple quantum coherence spectroscopy, which revealed all of the salient features of a dipyrromethane composed of two PBG units linked heat to tail and terminating in a CH{sub 2}-S bond to a cysteine residue. Site-specific mutagenesis of Cys-99 and Cys-242, respectively, has shown that substitution of Ser for Cys-99 does not affect the enzymatic activity, whereas substitution of Ser for Cys-242 removes essentially all of the catalytic activity as measured by the conversion of the substrate PBG to uro'gen I. The NMR spectrum of the covalent complex of deaminase with the suicide inhibitor 2-bromo-(2,11-{sup 13}C{sub 2})PBG reveals that the aminomethyl terminus of the inhibitor reacts with the enzyme's cofactor at the {alpha}-free pyrrole. NMR spectroscopy of the ES{sub 2} complex confirmed a PBG-derived head-to-tail dipyrromethane attached to the {alpha}-free pyrrole position of the enzyme. A mechanistic rationale for deaminase is presented.

  8. Purification and Properties of a Protein Which Binds Cytokinin-active 6-Substituted Purines 1

    PubMed Central

    Erion, Jack L.; Fox, J. Eugene

    1981-01-01

    A protein which binds 6-substituted purines of the cytokinin type with relatively high affinity has been extensively purified from wheat germ. Conventional chromatographic techniques, as well as an affinity matrix to which a cytokinin was covalently coupled, were used in the purification. The wheat germ cytokinin-binding protein (CBF-1) has four unlike subunits and an apparent molecular weight of 183,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. CBF-1 is saturated at one cytokinin molecule per tetramer with a Kd for 6-benzylaminopurine of 5 × 10−7 molar. The protein exists both on the native wheat germ ribosome (1 molecule CBF-1 per 80S ribosome) and free in the cytosol with approximately three copies of the latter for each of the former. Data from affinity chromatography studies and cross-linking experiments strongly suggest that a specific binding site for CBF-1 occurs on the wheat germ ribosome. Images PMID:16661618

  9. Tuning the Curie temperature of L1{sub 0} ordered FePt thin films through site-specific substitution of Rh

    SciTech Connect

    Xu, Dongbin; Sun, Cheng-Jun E-mail: msecgm@nus.edu.sg; Heald, Steve M.; Chen, Jing-Sheng; Chow, Gan Moog E-mail: msecgm@nus.edu.sg; Zhou, Tie-Jun; Bergman, Anders; Sanyal, Biplab

    2014-10-14

    In structurally ordered magnetic thin films, the Curie temperature (T{sub C}) of ferromagnetic films depends on the exchange integral of the short range ordered neighboring atoms. The exchange integral may be adjusted by controlling the elemental substitutional concentration at the lattice site of interest. We show how to control the T{sub C} in high anisotropy L1{sub 0} Fe{sub 50}Pt{sub 50} magnetic thin films by substituting Rh into the Pt site. Rh substitution in L1{sub 0} FePt modified the local atomic environment and the corresponding electronic properties, while retaining the ordered L1{sub 0} phase. The analysis of extended x-ray Absorption Fine Structure spectra shows that Rh uniformly substitutes for Pt in L1{sub 0} FePt. A model of antiferromagnetic defects caused by controlled Rh substitution of the Pt site, reducing the T{sub C,} is proposed to interpret this phenomenon and its validity is further examined by ab initio density functional calculations.

  10. Active site of ribulosebisphosphate carboxylase/oxygenase

    SciTech Connect

    Hartman, F.C.; Stringer, C.D.; Milanez, S.; Lee, E.H.

    1985-01-01

    Previous affinity labeling studies and comparative sequence analyses have identified two different lysines at the active site of ribulosebisphosphate carboxylase/oxygenase and have suggested their essentiality to function. The essential lysines occupy positions 166 and 329 in the Rhodospirillum rubrum enzyme and positions 175 and 334 in the spinach enzyme. Based on the pH-dependencies of inactivations of the two enzymes by trinitrobenzene sulfonate, Lys-166 (R. rubrum enzyme) exhibits a pK/sub a/ of 7.9 and Lys-334 (spinach enzyme) exhibits a pK/sub a/ of 9.0. These low pK/sub a/ values as well as the enhanced nucleophilicities of the lysyl residues argue that both are important to catalysis rather than to substrate binding. Lys-166 may correspond to the essential base that initiates catalysis and that displays a pK/sub a/ of 7.5 in the pH-curve for V/sub max//K/sub m/. Cross-linking experiments with 4,4'-diisothiocyano-2,2'-disulfonate stilbene demonstrate that the two active-site lysines are within 12 A. 50 refs., 7 figs., 1 tab.

  11. The polyoma virus enhancer cannot substitute for DNase I core hypersensitive sites 2-4 in the human beta-globin LCR.

    PubMed Central

    Tanimoto, K; Liu, Q; Bungert, J; Engel, J D

    1999-01-01

    The polyoma virus enhancer (PyE) is capable of conferring integration position-independent expression to linked genes in stably transfected erythroid cells after joining to DNase I hypersensitive site (HS) 5 of the human beta-globin locus control region (LCR). In attempting to separate the chromatin opening activity of the LCR from its enhancer activity and to investigate contributions of the individual HS core elements to LCR function, the human beta-globin LCR HS2, HS3 and HS4 core elements were replaced with the PyE within the context of a yeast artificial chromosome (YAC) bearing the whole locus. We show here that, in contrast to its function in cultured cells, the PyE is unable to replace HS core element function in vivo. We found that the PyE substitution mutant LCR is unable to provide either chromatin opening or transcriptional potentiating activity at any erythroid developmental stage in transgenic mice. These data provide direct evidence that the human beta-globin LCR core elements specify unique functions that cannot be replaced by a ubiquitous enhancer activity. PMID:10454609

  12. A Review on the Synthesis and Anti-cancer Activity of 2-substituted Quinolines.

    PubMed

    Gopaul, Kaalin; Shintre, Suhas A; Koorbanally, Neil A

    2015-01-01

    Quinolines substituted at C-2 on the quinoline scaffold have shown interesting anticancer activity in a number of anticancer assays such as breast (MCF-7, MDA-MB 231), human cervical epithelioid (HeLa), oral squamous cell carcinoma (SAS), human stomach adenocarcinoma (AGS, MKN45), hepatocellular (SKHep, HepG-2, Hep-3B), prostate (PC-3, DU145), lung (A549, H-460), gastric (HGC, MNK-74), leukemia (K562, U937, REH, NALM6, CEM/ADR 5000), colon (Colo-205, HCT 116, SW620, Caco-2, HT29), neuroblastoma (IMR32), CNS (SF-268), oesophageal (EAC) and melanoma (A-375). They have been synthesised by a number of strategies starting with isatin, anilines, nitrobenzenes and benzamides and some even with cyclohexanone and cyclohexa-1,3-diones with ammonium acetate. Many of the synthetic strategies employ the derivatisation of quinoline precursors itself. We review here the synthesis of 145 bioactive anticancer quinolines substituted at the 2-position and their anticancer activity. PMID:25511516

  13. Water-soluble Co(III) complexes of substituted phenanthrolines with cell selective anticancer activity.

    PubMed

    Jagadeesan, Sivaraman; Balasubramanian, Vimalkumar; Baumann, Patric; Neuburger, Markus; Häussinger, Daniel; Palivan, Cornelia G

    2013-11-01

    Transition metal complexes with substituted phenanthrolines as ligands represent potential anticancer products without the drawbacks of platinum complexes that are currently marketed. Here, we report the synthesis and cell selective anticancer activity of five new water-soluble Co(III) complexes with methyl substituted phenanthroline ligands. The complexes were characterized by elemental analysis, NMR, FAB-mass spectrometry, FTIR, electronic spectroscopy, and single crystal X-ray diffraction. Possible interaction of these complexes with DNA was assessed by a combination of circular dichroism, UV-vis spectroscopy titration, and ethidium bromide displacement assay, and the results indicated that DNA interaction is weak for these complexes. Cellular uptake and cytotoxicity of complexes at low concentrations were assessed by flow cytometry on PC-3 cells, while their effect on intracellular mitochondrial function was measured by MTS assay on HeLa and PC-3 cell lines. These complexes showed selective cytotoxicity with a significantly higher effect on intracellular mitochondrial function in PC-3 cells than in HeLa cells. At low concentrations, complex 2 had the highest cytotoxic effect on PC-3 cells, inducing around 38% cell death, and the correlation of cytotoxicity of these complexes to their hydrophobicity indicates that an appropriate value of the hydrophobicity is essential for high antitumor activity. PMID:24127683

  14. Active-Site Monovalent Cations Revealed in a 1.55 Å Resolution Hammerhead Ribozyme Structure

    PubMed Central

    Anderson, Michael; Schultz, Eric P.; Martick, Monika; Scott, William G.

    2013-01-01

    We have obtained a 1.55 Å crystal structure of a hammerhead ribozyme derived from Schistosoma mansoni in conditions that permit detailed observations of Na+ ion binding in the ribozyme's active site. At least two such Na+ ions are observed. The first Na+ ion binds to the N7 of G10.1 and the adjacent A9 phosphate in a manner identical to that previously observed for divalent cations. A second Na+ ion binds to the Hoogsteen face of G12, the general base in the hammerhead cleavage reaction, thereby potentially dissipating the negative charge of the catalytically active enolate form of the nucleotide base. A potential but more ambiguous third site bridges the A9 and scissile phosphates in a manner consistent with previous predictions. Hammerhead ribozymes have been observed to be active in the presence of high concentrations of monovalent cations, including Na+, but the mechanism by which monovalent cations substitute for divalent cations in hammerhead catalysis remains unclear. Our results enable us to suggest that Na+ directly and specifically substitutes for divalent cations in the hammerhead active site. The detailed geometry of the pre-catalytic active site complex is also revealed with a new level of precision, thanks to the quality of the electron density maps obtained from what is currently the highest resolution ribozyme structure in the protein data bank. PMID:23711504

  15. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1992-02-01

    The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of transuranic (TRU) waste and active low-level waste (LLW) facilities at Oak Ridge National Laboratory (ORNL) in accordance with US Department of Energy (DOE) Order 5820.2A. Active LLW facilities in Solid Waste Storage Area (SWSA) 6 include Tumulus I and Tumulus II, the Interim Waste Management Facility (IWMF), LLW silos, high-range wells, asbestos silos, and fissile wells. The tumulus pads and IWMF are aboveground, high-strength concrete pads on which concrete vaults containing metal boxes of LLW are placed; the void space between the boxes and vaults is filled with grout. Eventually, these pads and vaults will be covered by an engineered multilayered cap. All other LLW facilities in SWSA 6 are below ground. In addition, this plan includes monitoring of the Hillcut Disposal Test Facility (HDTF) in SWSA 6, even though this facility was completed prior to the data of the DOE order. In SWSA 5 North, the TRU facilities include below-grade engineered caves, high-range wells, and unlined trenches. All samples from SWSA 6 are screened for alpha and beta activity, counted for gamma-emitting isotopes, and analyzed for tritium. In addition to these analytes, samples from SWSA 5 North are analyzed for specific transuranic elements.

  16. Yeast allosteric chorismate mutase is locked in the activated state by a single amino acid substitution

    SciTech Connect

    Schmidheini, T.; Moesch, H.U.; Braus, G. ); Evans, J.N.S. )

    1990-04-17

    Chorismate mutase, a branch-point enzyme in the aromatic amino acid pathway of Saccharomyces cerevisiae, and also a mutant chorismate mutase with a single amino acid substitution in the C-terminal part of the protein have been purified approximately 20-fold and 64-fold from overproducing strains, respectively. The wild-type enzyme is activated by tryptophan and subject to feedback inhibition by tyrosine, whereas the mutant enzyme does not respond to activation by tryptophan nor inhibition by tyrosine. Both enzymes are dimers consisting of two identical subunits of M{sub r} 30,000, each one capable of binding one substrate and one activator molecule. Each subunit of the wild-type enzyme also binds one inhibitor molecule, whereas the mutant enzyme lost this ability. The enzyme reaction was observed by {sup 1}H NMR and shows a direct and irreversible conversion of chorismate to prephenate without the accumulation of any enzyme-free intermediates. The kinetic data of the wild-type chorismate mutase show positive cooperativity toward the substrate with a Hill coefficient of 1.71 and a (S){sub 0.5} value of 4.0 mM. In the presence of the activator tryptophan, the cooperativity is lost. The enzyme has an (S){sub 0.5} value of 1.2 mM in the presence of 10 {mu}M tryptophan and an increased (S){sub 0.5} value of 8.6 mM in the presence of 300 {mu}M tyrosine. In the mutant enzyme, a loss of the cooperativity was observed, and (S){sub 0.5} was reduced to 1.0 mM. This enzyme is therefore locked in the activated state by a single amino acid substitution.

  17. Structure, bonding, and catalytic activity of monodisperse, transition-metal-substituted CeO2 nanoparticles.

    PubMed

    Elias, Joseph S; Risch, Marcel; Giordano, Livia; Mansour, Azzam N; Shao-Horn, Yang

    2014-12-10

    We present a simple and generalizable synthetic route toward phase-pure, monodisperse transition-metal-substituted ceria nanoparticles (M0.1Ce0.9O2-x, M = Mn, Fe, Co, Ni, Cu). The solution-based pyrolysis of a series of heterobimetallic Schiff base complexes ensures a rigorous control of the size, morphology and composition of 3 nm M0.1Ce0.9O2-x crystallites for CO oxidation catalysis and other applications. X-ray absorption spectroscopy confirms the dispersion of aliovalent (M(3+) and M(2+)) transition metal ions into the ceria matrix without the formation of any bulk transition metal oxide phases, while steady-state CO oxidation catalysis reveals an order of magnitude increase in catalytic activity with copper substitution. Density functional calculations of model slabs of these compounds confirm the stabilization of M(3+) and M(2+) in the lattice of CeO2. These results highlight the role of the host CeO2 lattice in stabilizing high oxidation states of aliovalent transition metal dopants that ordinarily would be intractable, such as Cu(3+), as well as demonstrating a rational approach to catalyst design. The current work demonstrates, for the first time, a generalizable approach for the preparation of transition-metal-substituted CeO2 for a broad range of transition metals with unparalleled synthetic control and illustrates that Cu(3+) is implicated in the mechanism for CO oxidation on CuO-CeO2 catalysts. PMID:25406101

  18. Identifiability of the unrooted species tree topology under the coalescent model with time-reversible substitution processes, site-specific rate variation, and invariable sites.

    PubMed

    Chifman, Julia; Kubatko, Laura

    2015-06-01

    The inference of the evolutionary history of a collection of organisms is a problem of fundamental importance in evolutionary biology. The abundance of DNA sequence data arising from genome sequencing projects has led to significant challenges in the inference of these phylogenetic relationships. Among these challenges is the inference of the evolutionary history of a collection of species based on sequence information from several distinct genes sampled throughout the genome. It is widely accepted that each individual gene has its own phylogeny, which may not agree with the species tree. Many possible causes of this gene tree incongruence are known. The best studied is the incomplete lineage sorting, which is commonly modeled by the coalescent process. Numerous methods based on the coalescent process have been proposed for the estimation of the phylogenetic species tree given DNA sequence data. However, use of these methods assumes that the phylogenetic species tree can be identified from DNA sequence data at the leaves of the tree, although this has not been formally established. We prove that the unrooted topology of the n-leaf phylogenetic species tree is generically identifiable given observed data at the leaves of the tree that are assumed to have arisen from the coalescent process under a time-reversible substitution process with the possibility of site-specific rate variation modeled by the discrete gamma distribution and a proportion of invariable sites. PMID:25791286

  19. Antimicrobial activity of meta-alkoxyphenylcarbamates containing substituted N-phenylpiperazine fragment

    PubMed Central

    Malík, Ivan; Bukovský, Marián; Andriamainty, Fils; Gališinová, Jana

    2012-01-01

    In the present investigation, the basic esters of meta-alkoxyphenylcarbamic acid bearing variously substituted N-phenylpiperazine fragment were screened for their in vitro antimicrobial activity against Staphylococcus aureus, Escherichia coli and Candida albicans, respectively. The most effective against Escherichia coli was found the compound 6d (MIC=195,3 μg/mL) bearing simultaneously para-fluoro substituent at the 4-phenylpiperazin-1-yl core and meta-methoxy side chain in the lipophilic part of the molecule. From whole analyzed set of the molecules the substance 8e with propoxy side chain forming meta-alkoxyphenylcarbamoyl fragment and lipophilic, sterically bulky meta-trifluoromethyl group attached at N-phenylpiperazine moiety was evaluated as the most active against Candida albicans (MIC=97,7 μg/mL). On the contrary, all investigated structures were practically inactive against Staphylococcus aureus (MIC>1000 μg/mL) PMID:24031913

  20. Activation of methane by transition metal-substituted aluminophosphate molecular sieves

    DOEpatents

    Iton, Lennox E.; Maroni, Victor A.

    1991-01-01

    Aluminophosphate molecular sieves substituted with cobalt, manganese or iron and having the AlPO.sub.4 -34 or AlPO.sub.4 -5, or related AlPO.sub.4 structure activate methane starting at approximately 350.degree. C. Between 400.degree. and 500.degree. C. and at methane pressures .ltoreq.1 atmosphere the rate of methane conversion increases steadily with typical conversion efficiencies at 500.degree. C. approaching 50% and selectivity to the production of C.sub.2+ hydrocarbons approaching 100%. The activation mechanism is based on reduction of the transition metal(III) form of the molecular sieve to the transition metal(II) form with accompanying oxidative dehydrogenation of the methane. Reoxidation of the - transition metal(II) form to the transition metal(III) form can be done either chemically (e.g., using O.sub.2) or electrochemically.

  1. Antitrypanosomal Activities and Cytotoxicity of Some Novel Imido-substituted 1,4-Naphthoquinone Derivatives

    PubMed Central

    Khraiwesh, Mozna H.; Lee, Clarence M.; Brandy, Yakini; Akinboye, Emmanuel S.; Berhe, Solomon; Gittens, Genelle; Abbas, Muneer M.; Ampy, Franklin R.; Ashraf, Mohammad

    2013-01-01

    The antitrypanosomal activities, cytotoxicity, and selectivity indices of eleven imido-substituted 1,4-naphthoquinone derivatives and nifurtimox have been studied. Compared to nifurtimox (IC50 = 10.67 µM), all the imido-naphthoquinone analogs (IMDNQ1-IMDNQ11) are more potent on Trypanosoma cruzi with IC50 values ranging from 0.7 µM to 6.1 µM (p < 0.05). Studies of the cytotoxic activities of these compounds on a Balb/C 3T3 mouse fibroblast cell line revealed that four of these compounds, IMDNQ1, IMDNQ2, IMDNQ3, and IMDNQ10 displayed selectivity indices of 60.25, 53.97, 31.83, and 275.3, respectively, rendering them significantly (p < 0.05) more selective in inhibiting the parasite growth than nifurtimox (selectivity index = 10.86). PMID:22297740

  2. Antitrypanosomal activities and cytotoxicity of some novel imido-substituted 1,4-naphthoquinone derivatives.

    PubMed

    Khraiwesh, Mozna H; Lee, Clarence M; Brandy, Yakini; Akinboye, Emmanuel S; Berhe, Solomon; Gittens, Genelle; Abbas, Muneer M; Ampy, Franklin R; Ashraf, Mohammad; Bakare, Oladapo

    2012-01-01

    The antitrypanosomal activities, cytotoxicity, and selectivity indices of eleven imido-substituted 1,4-naphthoquinone derivatives and nifurtimox have been studied. Compared to nifurtimox (IC(50) = 10.67 μM), all the imido-naphthoquinone analogs (IMDNQ1-IMDNQ11) are more potent on Trypanosoma cruzi with IC50 values ranging from 0.7 μM to 6.1 μM (p < 0.05). Studies of the cytotoxic activities of these compounds on a Balb/C 3T3 mouse fibroblast cell line revealed that four of these compounds, IMDNQ1, IMDNQ2, IMDNQ3, and IMDNQ10 displayed selectivity indices of 60.25, 53.97, 31.83, and 275.3, respectively, rendering them significantly (p < 0.05) more selective in inhibiting the parasite growth than nifurtimox (selectivity index = 10.86). PMID:22297740

  3. Optically active substituted polyacetylene@carbon nanotube hybrids: Preparation, characterization and infrared emissivity property study

    SciTech Connect

    Bu, Xiaohai; Zhou, Yuming Zhang, Tao; Wang, Yongjuan; Zhang, Zewu; He, Man

    2014-08-15

    Optically active substituted polyacetylene@multiwalled carbon nanotubes (SPA@MWCNTs) nanohybrids were fabricated by wrapping helical SPA copolymers onto the surface of modified nanotubes through ester bonding linkage. SPA copolymer based on chiral phenylalanine and serine was pre-polymerized by a rhodium zwitterion catalyst in THF, and evidently proved to possess strong optical activity and adopt a predominately one-handed helical conformation. Various characterizations including Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), and transmission electron microscopy (TEM) demonstrated that the SPA had been covalently grafted onto the nanotubes without destroying their original graphite structure. The wrapped SPA was found to exhibit an enhancement in thermal stability and still maintained considerable optical activity after grafting. The infrared emissivity property of the nanohybrids at 8–14 μm was investigated in addition. The results indicated that the SPA@MWCNTs hybrid matrix could possess a much lower infrared emissivity value (ε=0.707) than raw MWCNTs, which might be due to synergistic effect of the unique helical conformation of optically active SPA and strengthened interfacial interaction between the organic polymers and inorganic nanoparticles. - Graphical abstract: Optically active SPA@MWCNTs nanohybrids with low infrared emissivity. - Highlights: • Synthesis of optically active SPA copolymer derived from serine and phenylalanine. • Preparation and characterization of optically active SPA@MWCNTs nanohybrids. • Application study of the SPA@MWCNTs nanohybrids (ε=0.707) in lowering the infrared emissivity.

  4. Design, synthesis, and herbicidal activity of novel substituted 3-(pyridin-2-yl)benzenesulfonamide derivatives.

    PubMed

    Xie, Yong; Chi, Hui-Wei; Guan, Ai-Ying; Liu, Chang-Ling; Ma, Hong-Juan; Cui, Dong-Liang

    2014-12-31

    A series of novel substituted 3-(pyridin-2-yl)benzenesulfonamide derivatives were designed and synthesized using 2-phenylpridines as the lead compound by intermediate derivatization methods in an attempt to obtain novel compound candidates for weed control. The herbicidal activity assay in glasshouse tests showed several compounds (II6, II7, II8, II9, II10, II11, III2, III3, III4, and III5) could efficiently control velvet leaf, youth-and-old age, barnyard grass, and foxtail at the 37.5 g/ha active substance. Especially, the activities of II6, II7, III2, and III4 were proved roughly equivalent to the saflufenacil and better than 95% sulcotrione at the same concentration. The result of the herbicidal activity assay in field tests demonstrated that II7 at 60 g/ha active substance could give the same effect as bentazon at 1440 g/ha active substance to control dayflower and nightshade, meanwhile II7 showed better activity than oxyfluorfen to control arrowhead and security to rice. The present work indicates that II7 may be a novel compound candidate for potential herbicide. PMID:25437124

  5. Molecular Basis for Enzymatic Sulfite Oxidation -- HOW THREE CONSERVED ACTIVE SITE RESIDUES SHAPE ENZYME ACTIVITY

    SciTech Connect

    Bailey, Susan; Rapson, Trevor; Johnson-Winters, Kayunta; Astashkin, Andrei; Enemark, John; Kappler, Ulrike

    2008-11-10

    Sulfite dehydrogenases (SDHs) catalyze the oxidation and detoxification of sulfite to sulfate, a reaction critical to all forms of life. Sulfite-oxidizing enzymes contain three conserved active site amino acids (Arg-55, His-57, and Tyr-236) that are crucial for catalytic competency. Here we have studied the kinetic and structural effects of two novel and one previously reported substitution (R55M, H57A, Y236F) in these residues on SDH catalysis. Both Arg-55 and His-57 were found to have key roles in substrate binding. An R55M substitution increased Km(sulfite)(app) by 2-3 orders of magnitude, whereas His-57 was required for maintaining a high substrate affinity at low pH when the imidazole ring is fully protonated. This effect may be mediated by interactions of His-57 with Arg-55 that stabilize the position of the Arg-55 side chain or, alternatively, may reflect changes in the protonation state of sulfite. Unlike what is seen for SDHWT and SDHY236F, the catalytic turnover rates of SDHR55M and SDHH57A are relatively insensitive to pH (~;;60 and 200 s-1, respectively). On the structural level, striking kinetic effects appeared to correlate with disorder (in SDHH57A and SDHY236F) or absence of Arg-55 (SDHR55M), suggesting that Arg-55 and the hydrogen bonding interactions it engages in are crucial for substrate binding and catalysis. The structure of SDHR55M has sulfate bound at the active site, a fact that coincides with a significant increase in the inhibitory effect of sulfate in SDHR55M. Thus, Arg-55 also appears to be involved in enabling discrimination between the substrate and product in SDH.

  6. Synthesis and Antimicrobial Activity of 9-O-Substituted Palmatine Derivatives

    PubMed Central

    Li, Z. C.; Kong, X. B.; Mai, W. P.; Sun, G. C.; Zhao, S. Z.

    2015-01-01

    A series of new palmatine derivatives with alkyl or alkyl with N-heterocyclic structures were designed and synthesized at C-9-O according to the principle of association. These compounds were characterised by 1H NMR, 13C NMR, ESI-MS and elemental analysis, and tested for their antimicrobial activity in vitro to evaluate structure-activity relationships. The results indicated that 9-O-substituted palmatine derivatives exhibit varying degrees of antimicrobial activity. Antibacterial activities of compounds (3a-f) against Gram +ve bacteria increased 2- to 64-fold than that of palmatine. The compounds (3a-f) possessed relatively weaker inhibitory effects against Gram −ve bacteria and fungi than that against Gram +ve bacteria. Antimicrobial activities of compounds (5a-e) are lower than that of compounds (3a-f). Compound 3d showed the highest antimicrobial activity of all the compounds. The LD50 values of compounds (3a-f) decreased as the alkyl side chain was elongated. Compound 3f showed least toxicity. PMID:26009653

  7. Ionic liquid activated Bacillus subtilis lipase A variants through cooperative surface substitutions.

    PubMed

    Zhao, Jing; Jia, Ning; Jaeger, Karl-Erich; Bocola, Marco; Schwaneberg, Ulrich

    2015-10-01

    The interest in performing enzyme-catalyzed reactions in amphiphilic systems, e.g., imidazolium-based ionic liquids (ILs) or surfactants, has been increased over the past decades. Directed protein evolution has been successful in tailoring enzymes for desired properties. Herein, nine IL-resistant Bacillus subtilis lipase A variants, particularly an IL-activated variant M1 (M134N/N138S/L140S), were identified by directed evolution. For instance, variant M2 (M134R/L140S) showed almost doubled specific activity (16.9 vs. 9.4 U/mg) and resistance (233% vs. 111%) at 9 vol% 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([C4 mim][TfO]) compared with wild-type. The specific activities and resistance of purified individual single and double variants have been studied in five different IL-aqueous mixtures. The re-activation of lipase variant M1 (not wild-type) at high IL concentration was attributed to the cooperative effect of three surface substitutions (M134N, N138S, L140S) near the substrate-binding cleft. The presence of IL/substrate clusters under assay conditions was likely related to the re-activation effect. This study provides first example of IL-activated lipase variant generated by protein engineering, and helps to better understand the protein-IL interaction. PMID:25899108

  8. Influence of A-site substitution on ESR spectra of lanthanum manganite perovskites

    NASA Astrophysics Data System (ADS)

    Phan, T. L.; Tho, N. D.; Bau, L. V.; Phuc, N. X.; Yu, S. C.

    2006-08-01

    We studied electron spin resonance (ESR) spectra of La 0.7A' 0.3MnO 3 (A'=Sr, Ba, Pb, and Cd) perovskites in a large range of temperature, from their Curie temperature to ˜500 K. Asymmetrical EPR signals at temperatures TTmin well fits to the model of adiabatic hopping motion of small polarons ΔH(T)=ΔH0+(a/T) exp(-E/kT), where Ea and kB are the activation energy and the Boltzmann factor, respectively; Δ H0 and a are constants. It is worth noticing that, in this temperature range, the Lande factor g for the samples is about 2.00, indicating spin-spin interaction among electrons of Mn 3+ and Mn 4+ ions playing an important role.

  9. Synthesis of biologically active O-substituted derivatives of 1-[(3, 5-dichloro-2-hydroxyphenyl)sulfonyl]piperidine.

    PubMed

    Khalid, Hira; Rehman, Azizur; Abbasi, Muhammad Athar; Khan, Khalid Mohammad; Ashraf, Muhammad; Ahmad, Irshad; Ejaz, Syeda Abida

    2013-05-01

    In the present work, a series of 2-O-substituted derivatives of 1-[(3,5-dichloro-2-hydroxy phenyl) sulfonyl]piperidine (5a-j) were synthesized. These derivatives were geared up by the coupling of 3,5-dichloro-2-hydroxy benzenesulfonyl chloride (1) with piperidine (2) under dynamic pH control in aqueous media to form parent compound 1-[(3,5-dichloro-2-hydroxyphenyl)sulfonyl]piperidine (3), followed by the substitution at oxygen atom with different electrophiles (4a-j) in the presence of sodium hydride (NaH) and dimethyl formamide (DMF) to give a series of O-substituted derivatives of sulfonamides bearing piperidine nucleus 5a-j. The synthesized O-substituted sulfonamides were spectrally characterized. The bioactivity of all the synthesized compounds were evaluated against lipoxygenase (LOX), acetylcholinesterae (AChE) and butyrylcholinesterase (BChE) enzymes and found to be having talented activity against butyrylcholinesterase enzyme. PMID:23625420

  10. Synthesis, antimicrobial, and anti-inflammatory activity, of novel S-substituted and N-substituted 5-(1-adamantyl)-1,2,4-triazole-3-thiols

    PubMed Central

    Al-Abdullah, Ebtehal S; Asiri, Hanadi H; Lahsasni, Siham; Habib, Elsayed E; Ibrahim, Tarek M; El-Emam, Ali A

    2014-01-01

    The reaction of 5-(1-adamantyl)-4-phenyl-1,2,4-triazoline-3-thione (compound 5) with formaldehyde and 1-substituted piperazines yielded the corresponding N-Mannich bases 6a–f. The reaction of 5-(1-adamantyl)-4-methyl-1,2,4-triazoline-3-thione 8 with various 2-aminoethyl chloride yielded separable mixtures of the S-(2-aminoethyl) 9a–d and the N-(2-aminoethyl) 10a–d derivatives. The reaction of compound 5 with 1-bromo-2-methoxyethane, various aryl methyl halides, and ethyl bromoacetate solely yielded the S-substituted products 11, 12a–d, and 13. The new compounds were tested for activity against a panel of Gram-positive and Gram-negative bacteria and the pathogenic fungus Candida albicans. Compounds 6b, 6c, 6d, 6e, 6f, 10b, 10c, 10d, 12c, 12d, 12e, 13, and 14 displayed potent antibacterial activity. Meanwhile, compounds 13 and 14 produced good dose-dependent anti-inflammatory activity against carrageenan-induced paw edema in rats. PMID:24872681

  11. Explorations of Substituted Urea Functionality for Discovery of New Activators of the Heme Regulated Inhibitor Kinase

    PubMed Central

    Chen, Ting; Takrouri, Khuloud; Hee-Hwang, Sung; Rana, Sandeep; Yefidoff-Freedman, Revital; Halperin, Jose; Natarajan, Amarnath; Morisseau, Christophe; Hammock, Bruce; Chorev, Michael; Aktas, Bertal H.

    2014-01-01

    Heme-regulated inhibitor kinase (HRI), an eukaryotic translation initiation factor 2 alpha (eIF2α) kinase, plays critical roles in cell proliferation, differentiation, and adaptation to cytoplasmic stress. HRI is also a critical modifier of hemoglobin disorders such as β-thalassemia. We previously identified N,N′-diarylureas as potent activators of HRI suitable for studying biology of this important kinase. To expand the repertoire of chemotypes that activate HRI we screened a ~1,900 member N,N′-disubstituted urea library in the surrogate eIF2α phosphorylation assay identifying N-aryl,N′-cyclohexylphenoxyurea as a promising scaffold. We validated hit compounds as a bona-fide HRI activators in secondary assays and explored contributions of substitutions on the N-aryl and N′-cyclohexylphenoxy groups to their activity by studying focused libraries of complementing analogs. We tested these N-aryl,N′-cyclohexylphenoxyureas in the surrogate eIF2α phosphorylation and cell proliferation assays, demonstrating significantly improved bioactivities and specificities. We consider these compounds to represent lead candidates for the development of potent and specific HRI activators. PMID:24261904

  12. Tuning the Biological Activity Profile of Antibacterial Polymers via Subunit Substitution Pattern

    PubMed Central

    2015-01-01

    Binary nylon-3 copolymers containing cationic and hydrophobic subunits can mimic the biological properties of host-defense peptides, but relationships between composition and activity are not yet well understood for these materials. Hydrophobic subunits in previously studied examples have been limited mostly to cycloalkane-derived structures, with cyclohexyl proving to be particularly promising. The present study evaluates alternative hydrophobic subunits that are isomeric or nearly isomeric with the cyclohexyl example; each has four sp3 carbons in the side chains. The results show that varying the substitution pattern of the hydrophobic subunit leads to relatively small changes in antibacterial activity but causes significant changes in hemolytic activity. We hypothesize that these differences in biological activity profile arise, at least in part, from variations among the conformational propensities of the hydrophobic subunits. The α,α,β,β-tetramethyl unit is optimal among the subunits we have examined, providing copolymers with potent antibacterial activity and excellent prokaryote vs eukaryote selectivity. Bacteria do not readily develop resistance to the new antibacterial nylon-3 copolymers. These findings suggest that variation in subunit conformational properties could be generally valuable in the development of synthetic polymers for biological applications. PMID:24601599

  13. Site-directed mutagenesis substituting cysteine for serine in 2-Cys peroxiredoxin (2-Cys Prx A) of Arabidopsis thaliana effectively improves its peroxidase and chaperone functions

    PubMed Central

    Lee, Eun Mi; Lee, Seung Sik; Tripathi, Bhumi Nath; Jung, Hyun Suk; Cao, Guang Ping; Lee, Yuno; Singh, Sudhir; Hong, Sung Hyun; Lee, Keun Woo; Lee, Sang Yeol; Cho, Jae-Young; Chung, Byung Yeoup

    2015-01-01

    Background and Aims The 2-Cys peroxiredoxin (Prx) A protein of Arabidopsis thaliana performs the dual functions of a peroxidase and a molecular chaperone depending on its conformation and the metabolic conditions. However, the precise mechanism responsible for the functional switching of 2-Cys Prx A is poorly known. This study examines various serine-to-cysteine substitutions on α-helix regions of 2-Cys Prx A in Arabidopsis mutants and the effects they have on the dual function of the protein. Methods Various mutants of 2-Cys Prx A were generated by replacing serine (Ser) with cysteine (Cys) at different locations by site-directed mutagenesis. The mutants were then over-expressed in Escherichia coli. The purified protein was further analysed by size exclusion chromatography, polyacrylamide gel electrophoresis, circular dichroism spectroscopy and transmission electron microscopy (TEM) and image analysis. Peroxidase activity, molecular chaperone activity and hydrophobicity of the proteins were also determined. Molecular modelling analysis was performed in order to demonstrate the relationship between mutation positions and switching of 2-Cys Prx A activity. Key Results Replacement of Ser150 with Cys150 led to a marked increase in holdase chaperone and peroxidase activities of 2-Cys Prx A, which was associated with a change in the structure of an important domain of the protein. Molecular modelling demonstrated the relationship between mutation positions and the switching of 2-Cys Prx A activity. Examination of the α2 helix, dimer–dimer interface and C-term loop indicated that the peroxidase function is associated with a fully folded α2 helix and easy formation of a stable reduced decamer, while a more flexible C-term loop makes the chaperone function less likely. Conclusions Substitution of Cys for Ser at amino acid location 150 of the α-helix of 2-Cys Prx A regulates/enhances the dual enzymatic functions of the 2-Cys Prx A protein. If confirmed in planta, this

  14. Ion-Pair SN 2 Substitution: Activation Strain Analyses of Counter-Ion and Solvent Effects.

    PubMed

    Laloo, Jalal Z A; Rhyman, Lydia; Ramasami, Ponnadurai; Bickelhaupt, F Matthias; de Cózar, Abel

    2016-03-18

    The ion-pair SN 2 reactions of model systems Mn F(n-1) +CH3 Cl (M(+) =Li(+) , Na(+) , K(+) , and MgCl(+) ; n=0, 1) have been quantum chemically explored by using DFT at the OLYP/6-31++G(d,p) level. The purpose of this study is threefold: 1) to elucidate how the counterion M(+) modifies ion-pair SN 2 reactivity relative to the parent reaction F(-) +CH3 Cl; 2) to determine how this influences stereochemical competition between the backside and frontside attacks; and 3) to examine the effect of solvation on these ion-pair SN 2 pathways. Trends in reactivity are analyzed and explained by using the activation strain model (ASM) of chemical reactivity. The ASM has been extended to treat reactivity in solution. These findings contribute to a more rational design of tailor-made substitution reactions. PMID:26879231

  15. Synthesis, Characterization, and Antifungal Activity of Phenylpyrrole-Substituted Tetramic Acids Bearing Carbonates.

    PubMed

    Xu, Wen-Qin; Chen, Min; Wang, Kun-Yao; Ren, Zheng-Jiao; Lu, Ai-Min; Yang, Chun-Long

    2016-01-01

    For the aim of discovering new fungicide, a series of phenylpyrrole-substituted tetramic acid derivatives bearing carbonates 6a-q were designed and synthesized via 4-(2,4-dioxopyrrolidin-3-ylidene)-4-(phenylamino)butanoic acids 4a-k and the cyclized products 1',3,4,5'-tetrahydro-[2,3'-bipyrrolylidene]-2',4',5(1H)-triones 5a-k. The compounds were characterized using IR, ¹H- and (13)C-NMR spectroscopy, mass spectrometry (EI-MS), and elemental analysis. The structure of 6b was confirmed by X-ray diffraction crystallography. The title compounds 6a-q were bioassayed in vitro against the phytopathogenic fungi Fusarium graminearum, Botrytis cinerea and Rhizoctonia solani at a concentration of 100 μg/mL, respectively. Most compounds displayed good inhibitory activity. PMID:27007370

  16. Nursing Home Staffing Requirements and Input Substitution: Effects on Housekeeping, Food Service, and Activities Staff

    PubMed Central

    Bowblis, John R; Hyer, Kathryn

    2013-01-01

    Objective To study the effect of minimum nurse staffing requirements on the subsequent employment of nursing home support staff. Data Sources Nursing home data from the Online Survey Certification and Reporting (OSCAR) System merged with state nurse staffing requirements. Study Design Facility-level housekeeping, food service, and activities staff levels are regressed on nurse staffing requirements and other controls using fixed effect panel regression. Data Extraction Method OSCAR surveys from 1999 to 2004. Principal Findings Increases in state direct care and licensed nurse staffing requirements are associated with decreases in the staffing levels of all types of support staff. Conclusions Increased nursing home nurse staffing requirements lead to input substitution in the form of reduced support staffing levels. PMID:23445455

  17. New structure-activity relationships of N-acetamide substituted pyrazolopyrimidines as pharmacological ligands of TSPO.

    PubMed

    Li, Jun; Schulte, Michael L; Nickels, Michael L; Manning, H Charles

    2016-08-01

    Translocator protein (TSPO) represents an attractive target for molecular imaging and therapy due to its prevalence and critical roles played in oncology and other pathologies. Based upon our previously optimized pyrazolopyrimidine scaffold, we elucidated new structure activity relationships related to N,N-disubstitutions of the terminal acetamide on pyrazolopyrimidines and further explored the impacts of these substituents on lipophilicity and plasma protein binding. Several novel chemical probes reported here exhibited significantly increased binding affinity, suitable lipophilicity and protein binding compared with contemporary TSPO ligands. We illustrate that N,N-acetamide disubstitution affords opportunities to introduce diverse chemical moieties distal to the central pyrazolopyrimidine core, without sacrificing TSPO affinity. We anticipate that further exploration of N-acetamide substitutions may yield additional TSPO ligands capable of furthering the field of precision medicine. PMID:27353534

  18. Conformational coupling, bridge helix dynamics and active site dehydration in catalysis by RNA polymerase

    PubMed Central

    Seibold, Steve A.; Singh, Badri Nath; Zhang, Chunfen; Kireeva, Maria; Domecq, Céline; Bouchard, Annie; Nazione, Anthony M.; Feig, Michael; Cukier, Robert I.; Coulombe, Benoit; Kashlev, Mikhail; Hampsey, Michael; Burton, Zachary F.

    2010-01-01

    Molecular dynamics simulation of Thermus thermophilus (Tt) RNA polymerase (RNAP) in a catalytic conformation demonstrates that the active site dNMP-NTP base pair must be substantially dehydrated to support full active site closing and optimum conditions for phosphodiester bond synthesis. In silico mutant β R428A RNAP, which was designed based on substitutions at the homologous position (Rpb2 R512) of Saccharomyces cerevisiae (Sc) RNAP II, was used as a reference structure to compare to Tt RNAP in simulations. Long range conformational coupling linking a dynamic segment of the bridge α-helix, the extended fork loop, the active site, and the trigger loop-trigger helix is apparent and adversely affected in β R428A RNAP. Furthermore, bridge helix bending is detected in the catalytic structure, indicating that bridge helix dynamics may regulate phosphodiester bond synthesis as well as translocation. An active site “latch” assembly that includes a key trigger helix residue Tt β’ H1242 and highly conserved active site residues β E445 and R557 appears to help regulate active site hydration/dehydration. The potential relevance of these observations in understanding RNAP and DNAP induced fit and fidelity is discussed. PMID:20478425

  19. Computational study on the roles of amino acid residues in the active site formation mechanism of blue-light photoreceptors

    NASA Astrophysics Data System (ADS)

    Sato, Ryuma; Kitoh-Nishioka, Hirotaka; Ando, Koji; Yamato, Takahisa

    2015-07-01

    To examine the functional roles of the active site methionine (M-site) and glutamic acid (E-site) residues of blue-light photoreceptors, we performed in silico mutation at the M-site in a systematic manner and focused on the hydrogen bonding between the E-site and the substrate: the cyclobutane-pyrimidine dimer (CPD). Fragment molecular orbital calculations with electron correlations demonstrated that substitution of the M-site methionine with either alanine or glutamine always destabilizes the interaction energy between the E-site and the CPD by more than 12.0 kcal/mol, indicating that the methionine and glutamic acid residues cooperatively facilitate the enzymatic reaction in the active site.

  20. Nucleophilic Aromatic Substitution.

    ERIC Educational Resources Information Center

    Avila, Walter B.; And Others

    1990-01-01

    Described is a microscale organic chemistry experiment which demonstrates one feasible route in preparing ortho-substituted benzoic acids and provides an example of nucleophilic aromatic substitution chemistry. Experimental procedures and instructor notes for this activity are provided. (CW)

  1. Phase-partitioning and site-substitution patterns of molybdenum in a model Ni-Al-Mo superalloy: An atom-probe tomographic and first-principles study

    NASA Astrophysics Data System (ADS)

    Tu, Yiyou; Mao, Zugang; Seidman, David N.

    2012-09-01

    Atom-probe tomography (APT) and first-principles calculations are employed to investigate the partitioning of Mo in the γ(f.c.c.)-and γ'(L12)-phases in a model Ni-6.5Al-9.9Mo at. % superalloy. Mo is experimentally observed to partition preferentially to the γ(f.c.c.)-matrix, which is consistent with the smaller value of the γ(f.c.c.)-matrix substitutional formation-energy, with a driving force of 0.707 eV for partitioning as determined by first-principles calculations. APT measurements of the γ'(L12)-precipitate-phase composition and Al-, Mo-centered partial radial distribution functions indicate that Mo occupies the Al sublattice sites of the Ni3Al(L12) phase. The preferential site-substitution of Mo at Al sublattice sites is confirmed by first-principles calculations.

  2. Synthesis and biological activity of nitro-substituted cyclopenta-fused PAH

    SciTech Connect

    Goldring, J.M.; Ball, L.M.; Sangaiah, R.; Gold, A.

    1986-08-01

    PAH containing a peripherally fused cyclopenta ring are genotoxically active in Salmonella and mammalian cells and have been identified in combustion emissions. Since the cyclopenta ring is predicted to be susceptible to electrophilic attack, nitrosubstituted cyclopenta-fused PAH may be formed in the presence of NO(x), particularly in diesel exhaust, and hence be present in the environment. Nitro derivatives of cyclopenta (cd) pyrene and the cyclopenta-fused benzanthracene isomers were prepared by reaction of the PAH with dinitrogen tetroxide. The purifed products were characterized by mass spectroscopy, (1)H NMR, UV-vis and HPLC. In all cases, nitro substitution occurred on the cyclopenta ring in accord with expectation. Preliminary assay in S. typhimurium indicates that 4-nitro-cyclopenta(cd)pyrene is mutagenically active without exogenous metabolic activation in nitroreductase-competent strains. The results of the study have allowed us to test the hypothesis and will aid in development of structure-activity relationships of nitroPAH.

  3. Optically active substituted polyacetylene@carbon nanotube hybrids: Preparation, characterization and infrared emissivity property study

    NASA Astrophysics Data System (ADS)

    Bu, Xiaohai; Zhou, Yuming; Zhang, Tao; Wang, Yongjuan; Zhang, Zewu; He, Man

    2014-08-01

    Optically active substituted polyacetylene@multiwalled carbon nanotubes (SPA@MWCNTs) nanohybrids were fabricated by wrapping helical SPA copolymers onto the surface of modified nanotubes through ester bonding linkage. SPA copolymer based on chiral phenylalanine and serine was pre-polymerized by a rhodium zwitterion catalyst in THF, and evidently proved to possess strong optical activity and adopt a predominately one-handed helical conformation. Various characterizations including Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), and transmission electron microscopy (TEM) demonstrated that the SPA had been covalently grafted onto the nanotubes without destroying their original graphite structure. The wrapped SPA was found to exhibit an enhancement in thermal stability and still maintained considerable optical activity after grafting. The infrared emissivity property of the nanohybrids at 8-14 μm was investigated in addition. The results indicated that the SPA@MWCNTs hybrid matrix could possess a much lower infrared emissivity value (ε=0.707) than raw MWCNTs, which might be due to synergistic effect of the unique helical conformation of optically active SPA and strengthened interfacial interaction between the organic polymers and inorganic nanoparticles.

  4. The Element Effect Revisited: Factors Determining Leaving Group Ability in Activated Nucleophilic Aromatic Substitution Reactions

    PubMed Central

    Senger, Nicholas A.; Bo, Bo; Cheng, Qian; Keeffe, James R.; Gronert, Scott; Wu, Weiming

    2012-01-01

    The “element effect” in nucleophilic aromatic substitution reactions (SNAr) is characterized by the leaving group order, F > NO2 > Cl ≈ Br > I, in activated aryl halides. Multiple causes for this result have been proposed. Experimental evidence shows that the element effect order in the reaction of piperidine with 2,4-dinitrophenyl halides in methanol is governed by the differences in enthalpies of activation. Computational studies of the reaction of piperidine and dimethylamine with the same aryl halides using the polarizable continuum model (PCM) for solvation indicate that polar, polarizability, solvation, and negative hyperconjugative effects are all of some importance in producing the element effect in methanol. In addition, a reversal of polarity of the C–X bond from reactant to transition state in the case of ArCl and ArBr compared to ArF also contributes to their difference in reactivity. The polarity reversal, and hyperconjugative influences have received little or no attention in the past. Nor has differential solvation of the different transition states been strongly emphasized. An anionic nucleophile, thiolate, gives very early transition states and negative activation enthalpies with activated aryl halides. The element effect is not established for these reactions. We suggest that the leaving group order in the gas phase will be dependent on the exact combination of nucleophile, leaving group, and substrate framework. The geometry of the SNAr transition state permits useful, qualitative conceptual distinctions to be made between this reaction and other modes of nucleophilic attack. PMID:23057717

  5. Mono- and tetra-substituted zinc(II) phthalocyanines containing morpholinyl moieties: Synthesis, antifungal photodynamic activities, and structure-activity relationships.

    PubMed

    Zheng, Bi-Yuan; Ke, Mei-Rong; Lan, Wen-Liang; Hou, Lu; Guo, Jun; Wan, Dong-Hua; Cheong, Ling-Zhi; Huang, Jian-Dong

    2016-05-23

    A series of zinc(II) phthalocyanines (ZnPcs) mono-substituted and tetra-substituted with morpholinyl moieties and their quaternized derivatives have been synthesized and evaluated for their antifungal photodynamic activities toward Candida albicans. The α-substituted, quaternized, and mono-substituted ZnPcs are found to have higher antifungal photoactivity than β-substituted, neutral, and tetra-substituted counterparts. The cationic α-mono-substituted ZnPc (6a) exhibits the highest photocytotoxicity. Moreover, it is more potent than axially di-substituted analogue. The different photocytotoxicities of these compounds have also been rationalized by investigating their spectroscopic and photochemical properties, aggregation trend, partition coefficients, and cellular uptake. The IC90 value of 6a against C. albicans cells is as low as 3.3 μM with a light dose of 27 J cm(-2), meaning that 6a is a promising candidate as the antifungal photosensitizer for future investigations. PMID:27046231

  6. Modulation of Enzymatic Activity and Biological Function of Listeria monocytogenes Broad-Range Phospholipase C by Amino Acid Substitutions and by Replacement with the Bacillus cereus Ortholog

    PubMed Central

    Zückert, Wolfram R.; Marquis, Hélène; Goldfine, Howard

    1998-01-01

    The secreted broad-range phosphatidylcholine (PC)-preferring phospholipase C (PC-PLC) of Listeria monocytogenes plays a role in the bacterium’s ability to escape from phagosomes and spread from cell to cell. Based on comparisons with two orthologs, Clostridium perfringens α-toxin and Bacillus cereus PLC (PLCBc), we generated PC-PLC mutants with altered enzymatic activities and substrate specificities and analyzed them for biological function in tissue culture and mouse models of infection. Two of the conserved active-site zinc-coordinating histidines were confirmed by single amino acid substitutions H69G and H118G, which resulted in proteins inactive in broth culture and unstable intracellularly. Substitutions D4E and H56Y remodeled the PC-PLC active site to more closely resemble the PLCBc active site, while a gene replacement resulted in L. monocytogenes secreting PLCBc. All of these mutants yielded similar amounts of active enzyme as wild-type PC-PLC both in broth culture and intracellularly. D4E increased activity on and specificity for PC, while H56Y and D4E H56Y showed higher activity on both PC and sphingomyelin, with reduced specificity for PC. As expected, PLCBc expressed by L. monocytogenes was highly specific for PC. During early intracellular growth in human epithelial cells, the D4E mutant and the PLCBc-expressing strain performed significantly better than the wild type, while the H56Y and D4E H56Y mutants showed a significant defect. In assays for cell-to-cell spread, the H56Y and D4E mutants had close to wild-type characteristics, while the spreading efficiency of PLCBc was significantly lower. These studies emphasize the species-specific features of PC-PLC important for growth in mammalian cells. PMID:9746585

  7. Synthesis and Structure-Activity Relationships of Substituted Urea Derivatives on Mouse Melanocortin Receptors.

    PubMed

    Singh, Anamika; Kast, Johannes; Dirain, Marvin L S; Huang, Huisuo; Haskell-Luevano, Carrie

    2016-02-17

    The melanocortin system is involved in the regulation of several complex physiological functions. In particular, the melanocortin-3 and -4 receptors (MC3R/MC4R) have been demonstrated to regulate body weight, energy homeostasis, and feeding behavior. Synthetic and endogenous melanocortin agonists have been shown to be anorexigenic in rodent models. Herein, we report synthesis and structure-activity relationship (SAR) studies of 27 nonpeptide small molecule ligands based on an unsymmetrical substituted urea core. Three templates containing key residues from the lead compounds, showing diversity at three positions (R(1), R(2), R(3)), were designed and synthesized. The syntheses were optimized for efficient microwave-assisted chemistry that significantly reduced total syntheses time compared to a previously reported room temperature method. The pharmacological characterization of the compounds on the mouse melanocortin receptors identified compounds 1 and 12 with full agonist activity at the mMC4R, but no activity was observed at the mMC3R when tested up to 100 μM concentrations. The SAR identified compounds possessing aliphatic or saturated cyclic amines at the R(1) position, bulky aromatic groups at the R(2) position, and benzyl group at the R(3) position resulted in mMC4R selectivity over the mMC3R. The small molecule template and SAR knowledge from this series may be helpful in further design of MC3R/MC4R selective small molecule ligands. PMID:26645732

  8. Quantitative structure–activity relationship analysis of the pharmacology of para-substituted methcathinone analogues

    PubMed Central

    Bonano, J S; Banks, M L; Kolanos, R; Sakloth, F; Barnier, M L; Glennon, R A; Cozzi, N V; Partilla, J S; Baumann, M H; Negus, S S

    2015-01-01

    Background and Purpose Methcathinone (MCAT) is a potent monoamine releaser and parent compound to emerging drugs of abuse including mephedrone (4-CH3 MCAT), the para-methyl analogue of MCAT. This study examined quantitative structure–activity relationships (QSAR) for MCAT and six para-substituted MCAT analogues on (a) in vitro potency to promote monoamine release via dopamine and serotonin transporters (DAT and SERT, respectively), and (b) in vivo modulation of intracranial self-stimulation (ICSS), a behavioural procedure used to evaluate abuse potential. Neurochemical and behavioural effects were correlated with steric (Es), electronic (σp) and lipophilic (πp) parameters of the para substituents. Experimental Approach For neurochemical studies, drug effects on monoamine release through DAT and SERT were evaluated in rat brain synaptosomes. For behavioural studies, drug effects were tested in male Sprague-Dawley rats implanted with electrodes targeting the medial forebrain bundle and trained to lever-press for electrical brain stimulation. Key Results MCAT and all six para-substituted analogues increased monoamine release via DAT and SERT and dose- and time-dependently modulated ICSS. In vitro selectivity for DAT versus SERT correlated with in vivo efficacy to produce abuse-related ICSS facilitation. In addition, the Es values of the para substituents correlated with both selectivity for DAT versus SERT and magnitude of ICSS facilitation. Conclusions and Implications Selectivity for DAT versus SERT in vitro is a key determinant of abuse-related ICSS facilitation by these MCAT analogues, and steric aspects of the para substituent of the MCAT scaffold (indicated by Es) are key determinants of this selectivity. PMID:25438806

  9. A three-dimensional model of mammalian tyrosinase active site accounting for loss of function mutations.

    PubMed

    Schweikardt, Thorsten; Olivares, Concepción; Solano, Francisco; Jaenicke, Elmar; García-Borrón, José Carlos; Decker, Heinz

    2007-10-01

    Tyrosinases are the first and rate-limiting enzymes in the synthesis of melanin pigments responsible for colouring hair, skin and eyes. Mutation of tyrosinases often decreases melanin production resulting in albinism, but the effects are not always understood at the molecular level. Homology modelling of mouse tyrosinase based on recently published crystal structures of non-mammalian tyrosinases provides an active site model accounting for loss-of-function mutations. According to the model, the copper-binding histidines are located in a helix bundle comprising four densely packed helices. A loop containing residues M374, S375 and V377 connects the CuA and CuB centres, with the peptide oxygens of M374 and V377 serving as hydrogen acceptors for the NH-groups of the imidazole rings of the copper-binding His367 and His180. Therefore, this loop is essential for the stability of the active site architecture. A double substitution (374)MS(375) --> (374)GG(375) or a single M374G mutation lead to a local perturbation of the protein matrix at the active site affecting the orientation of the H367 side chain, that may be unable to bind CuB reliably, resulting in loss of activity. The model also accounts for loss of function in two naturally occurring albino mutations, S380P and V393F. The hydroxyl group in S380 contributes to the correct orientation of M374, and the substitution of V393 for a bulkier phenylalanine sterically impedes correct side chain packing at the active site. Therefore, our model explains the mechanistic necessity for conservation of not only active site histidines but also adjacent amino acids in tyrosinase. PMID:17850513

  10. Defining the Structural Parameters that Confer Anticonvulsant Activity by the Site-by-Site Modification of (R)-N′-Benzyl 2- Amino-3-methylbutanamide

    PubMed Central

    King, Amber; De Ryck, Marc; Kaminski, Rafal; Valade, Anne; Stables, James P.; Kohn, Harold

    2011-01-01

    Primary Amino Acid Derivatives (PAADs) (N′-benzyl 2-substituted 2-amino acetamides) are structurally related to Functionalized Amino Acids (FAAs) (N′-benzyl 2- substituted 2-acetamido acetamides) but differ by the absence of the terminal N-acetyl group. Both classes exhibit potent anticonvulsant activities in the maximal electroshock seizure animal model and the reported structure-activity relationships (SARs) of PAADs and FAAs differ in significant ways. Recently, we documented that PAAD efficacy was associated with a hydrocarbon moiety at the C(2)-carbon, while in the FAAs, a substituted heteroatom one atom removed from the C(2)-center was optimal. Previously in this issue, we showed that PAAD activity was dependent upon the electronic properties of the 4′-N′-benzylamide substituent, while FAA activity was insensitive to electronic changes at this site. In this study, we prepared analogs of (R)-N′-benzyl 2-amino-3-methylbutanamide to identify the structural components for maximal anticonvulsant activity. We demonstrated that the SAR of PAADs and FAAs diverged at the terminal amide site and that PAADs had considerably more structural latitude in the types of units that could be incorporated at this position, suggesting that these compounds function according to different mechanism(s). PMID:21861466

  11. Age and physical activity effects on reaction time and digit symbol substitution performance in cognitively active adults.

    PubMed

    Lupinacci, N S; Rikli, R E; Jones, C J; Ross, D

    1993-06-01

    University professors (N = 56), divided into two age groups (< 50 years and > 50 years) and two physical activity level groups (high and low), were tested on three tasks requiring increasingly complex cognitive processing--simple reaction time (SRT), choice reaction time (CRT), and on a digit symbol substitution test (DSST). A significant main effect for exercise, with high active subjects performing better than low active subjects, was found for SRT (p < .001) and CRT (p < .01) but not for DSST (p < .09). Significant main effects for age, with younger subjects performing better than older subjects, were found on DSST (p < .01) and CRT (p < .05) but not for SRT (p < .09). The observation that the effect of age was more pronounced with increasing task complexity is consistent with previous research. However, the tendency for exercise effects to decrease with increasing task complexity is not consistent with former findings, suggesting that perhaps the controlled high level of cognitive activity of subjects in this study may have offset the usual effects of exercise on information processing speed. No significant Age x Activity Level interactions were found on any of the dependent raw score data. However, compared to normative scores of the population at large, there was a slight increase in DSST percentile ranks with age for the older aerobically active professors, whereas a decrease occurred for the inactive subjects. PMID:8341837

  12. Mapping of the SecA signal peptide binding site and dimeric interface by using the substituted cysteine accessibility method.

    PubMed

    Bhanu, Meera K; Zhao, Ping; Kendall, Debra A

    2013-10-01

    SecA is an ATPase nanomotor critical for bacterial secretory protein translocation. Secretory proteins carry an amino-terminal signal peptide that is recognized and bound by SecA followed by its transfer across the SecYEG translocon. While this process is crucial for the onset of translocation, exactly where the signal peptide interacts with SecA is unclear. SecA protomers also interact among themselves to form dimers in solution, yet the oligomeric interface and the residues involved in dimerization are unknown. To address these issues, we utilized the substituted cysteine accessibility method (SCAM); we generated a library of 23 monocysteine SecA mutants and probed for the accessibility of each mutant cysteine to maleimide-(polyethylene glycol)2-biotin (MPB), a sulfhydryl-labeling reagent, both in the presence and absence of a signal peptide. Dramatic differences in MPB labeling were observed, with a select few mutants located at the preprotein cross-linking domain (PPXD), the helical wing domain (HWD), and the helical scaffold domain (HSD), indicating that the signal peptide binds at the groove formed between these three domains. The exposure of this binding site is varied under different conditions and could therefore provide an ideal mechanism for preprotein transfer into the translocon. We also identified residues G793, A795, K797, and D798 located at the two-helix finger of the HSD to be involved in dimerization. Adenosine-5'-(γ-thio)-triphosphate (ATPγS) alone and, more extensively, in conjunction with lipids and signal peptides strongly favored dimer dissociation, while ADP supports dimerization. This study provides key insight into the structure-function relationships of SecA preprotein binding and dimer dissociation. PMID:23935053

  13. Novel Anthranilic Diamide Scaffolds Containing N-Substituted Phenylpyrazole as Potential Ryanodine Receptor Activators.

    PubMed

    Liu, Jing-Bo; Li, Yu-Xin; Zhang, Xiu-Lan; Hua, Xue-Wen; Wu, Chang-Chun; Wei, Wei; Wan, Ying-Ying; Cheng, Dan-Dan; Xiong, Li-Xia; Yang, Na; Song, Hai-Bin; Li, Zheng-Ming

    2016-05-11

    To discover potent insecticides targeting ryanodine receptors (RyRs), a series of novel anthranilic diamides analogues (12a-12u) containing N-substituted phenylpyrazole were designed and synthesized. These compounds were characterized by (1)H NMR, (13)C NMR, and HRMS, and the structure of compound 12u was confirmed by X-ray diffraction. Their insecticidal activities indicated that these compounds displayed moderate to excellent activities. In particular, 12i showed 100 and 37% larvicidal activities against oriental armyworm (Mythimna separata) at 0.25 and 0.05 mg L(-1), equivalent to that of chlorantraniliprole (100%, 0.25 mg L(-1); and 33%, 0.05 mg L(-1)). The activity of 12i against diamondback moth (Plutella xylostella) was 95% at 0.05 mg L(-1), whereas the control was 100% at 0.05 mg L(-1). The calcium-imaging technique experiment results showed that the effects of 12i on the intracellular calcium ion concentration ([Ca(2+)]i) in neurons were concentration-dependent. After the central neurons of Helicoverpa armigera were dyed by loading with fluo-5N and treated with 12i, the free calcium released in endoplasmic reticulum indicated the target of compound 12i is RyRs or IP3Rs. The activation of RyRs by natural ryanodine completely blocked the calcium release induced by 12i, which indicated that RyRs in the central neurons of H. armigera third-instar larvae is the possible target of compound 12i. PMID:27109555

  14. Adamantylidene-substituted alkylphosphocholine TCAN26 is more active against Sporothrix schenckii than miltefosine

    PubMed Central

    Borba-Santos, Luana Pereira; Ishida, Kelly; Calogeropoulou, Theodora; de Souza, Wanderley; Rozental, Sonia

    2016-01-01

    Sporotrichosis is the most frequent subcutaneous mycosis in the world and its increasing incidence has led to the search for new therapeutic options for its treatment. In this study, we demonstrated that three structural analogues of miltefosine (TCAN26, TC19, and TC70) showed inhibitory activity against Sporothrix schenckii sensu stricto and that TCAN26 was more active in vitro than miltefosine against several isolates. Scanning electron microscopy showed that S. schenckii exposure to TCAN26 resulted in cells that were slightly more elongated than untreated cells. Transmission electron microscopy showed that TCAN26 treatment induced loss of the regular cytoplasmic electron-density and altered the cell envelope (disruption of the cell membrane and cell wall, and increased cell wall thickness). Additionally, TCAN26 concentrations required to kill S. schenckii cells were lower than concentrations that were cytotoxic in mammalian cells, and TCAN26 was more selective than miltefosine. Thus, the adamantylidene-substituted alkylphosphocholine TCAN26 is a promising molecule for the development of novel antifungal compounds, although further investigations are required to elucidate the mode of action of TCAN26 in S. schenckii cells. PMID:27581121

  15. Adamantylidene-substituted alkylphosphocholine TCAN26 is more active against Sporothrix schenckii than miltefosine.

    PubMed

    Borba-Santos, Luana Pereira; Ishida, Kelly; Calogeropoulou, Theodora; Souza, Wanderley de; Rozental, Sonia

    2016-07-11

    Sporotrichosis is the most frequent subcutaneous mycosis in the world and its increasing incidence has led to the search for new therapeutic options for its treatment. In this study, we demonstrated that three structural analogues of miltefosine (TCAN26, TC19, and TC70) showed inhibitory activity against Sporothrix schenckii sensu stricto and that TCAN26 was more active in vitro than miltefosine against several isolates. Scanning electron microscopy showed that S. schenckii exposure to TCAN26 resulted in cells that were slightly more elongated than untreated cells. Transmission electron microscopy showed that TCAN26 treatment induced loss of the regular cytoplasmic electron-density and altered the cell envelope (disruption of the cell membrane and cell wall, and increased cell wall thickness). Additionally, TCAN26 concentrations required to kill S. schenckii cells were lower than concentrations that were cytotoxic in mammalian cells, and TCAN26 was more selective than miltefosine. Thus, the adamantylidene-substituted alkylphosphocholine TCAN26 is a promising molecule for the development of novel antifungal compounds, although further investigations are required to elucidate the mode of action of TCAN26 in S. schenckii cells. PMID:27409840

  16. Anti-inflammatory active gold(I) complexes involving 6-substituted-purine derivatives.

    PubMed

    Trávníček, Zdeněk; Starha, Pavel; Vančo, Ján; Silha, Tomáš; Hošek, Jan; Suchý, Pavel; Pražanová, Gabriela

    2012-05-24

    The gold(I) complexes of the general formula [Au(L(n))(PPh(3))]·xH(2)O (1-8; n = 1-8 and x = 0-1.5), where L(n) stands for a deprotonated form of the benzyl-substituted derivatives of 6-benzylaminopurine, were prepared, thoroughly characterized (elemental analyses, FT-IR, Raman and multinuclear NMR spectroscopy, ESI+ mass spectrometry, conductivity, DFT calculations), and studied for their in vitro cytotoxicity and in vitro and in vivo anti-inflammatory effects on LPS-activated macrophages (derived from THP-1 cell line) and using the carrageenan-induced hind paw edema model on rats. The obtained results indicate that the representative complexes (1, 3, 6) exhibit a strong ability to reduce the production of pro-inflammatory cytokines TNF-α, IL-1β and HMGB1 without influence on the secretion of anti-inflammatory cytokine IL-1RA in the LPS-activated macrophages. The complexes also significantly influence the formation of edema, caused by the intraplantar application of polysaccharide λ-carrageenan to rats in vivo. All the tested complexes showed similar or better biological effects as compared with Auranofin, but contrary to Auranofin they were found to be less cytotoxic in vitro. The obtained results clearly indicate that the gold(I) complexes behave as very effective anti-inflammatory agents and could prove to be useful for the treatment of difficult to treat inflammatory diseases such as rheumatoid arthritis. PMID:22541000

  17. Alkyne-substituted diminazene as G-quadruplex binders with anticancer activities.

    PubMed

    Wang, Changhao; Carter-Cooper, Brandon; Du, Yixuan; Zhou, Jie; Saeed, Musabbir A; Liu, Jinbing; Guo, Min; Roembke, Benjamin; Mikek, Clinton; Lewis, Edwin A; Lapidus, Rena G; Sintim, Herman O

    2016-08-01

    G-quadruplex ligands have been touted as potential anticancer agents, however, none of the reported G-quadruplex-interactive small molecules have gone past phase II clinical trials. Recently it was revealed that diminazene (berenil, DMZ) actually binds to G-quadruplexes 1000 times better than DNA duplexes, with dissociation constants approaching 1 nM. DMZ however does not have strong anticancer activities. In this paper, using a panel of biophysical tools, including NMR, FRET melting assay and FRET competition assay, we discovered that monoamidine analogues of DMZ bearing alkyne substitutes selectively bind to G-quadruplexes. The lead DMZ analogues were shown to be able to target c-MYC G-quadruplex both in vitro and in vivo. Alkyne DMZ analogues display respectable anticancer activities (single digit micromolar GI50) against ovarian (OVCAR-3), prostate (PC-3) and triple negative breast (MDA-MB-231) cancer cell lines and represent interesting new leads to develop anticancer agents. PMID:27132164

  18. Adamantylidene-substituted alkylphosphocholine TCAN26 is more active against Sporothrix schenckii than miltefosine.

    PubMed

    Borba-Santos, Luana Pereira; Ishida, Kelly; Calogeropoulou, Theodora; Souza, Wanderley de; Rozental, Sonia

    2016-08-01

    Sporotrichosis is the most frequent subcutaneous mycosis in the world and its increasing incidence has led to the search for new therapeutic options for its treatment. In this study, we demonstrated that three structural analogues of miltefosine (TCAN26, TC19, and TC70) showed inhibitory activity against Sporothrix schenckii sensu stricto and that TCAN26 was more active in vitro than miltefosine against several isolates. Scanning electron microscopy showed that S. schenckii exposure to TCAN26 resulted in cells that were slightly more elongated than untreated cells. Transmission electron microscopy showed that TCAN26 treatment induced loss of the regular cytoplasmic electron-density and altered the cell envelope (disruption of the cell membrane and cell wall, and increased cell wall thickness). Additionally, TCAN26 concentrations required to kill S. schenckii cells were lower than concentrations that were cytotoxic in mammalian cells, and TCAN26 was more selective than miltefosine. Thus, the adamantylidene-substituted alkylphosphocholine TCAN26 is a promising molecule for the development of novel antifungal compounds, although further investigations are required to elucidate the mode of action of TCAN26 in S. schenckii cells. PMID:27581121

  19. Single substitution in bacteriophage T4 RNase H alters the ratio between its exo- and endonuclease activities.

    PubMed

    Kholod, Natalia; Sivogrivov, Dmitry; Latypov, Oleg; Mayorov, Sergey; Kuznitsyn, Rafail; Kajava, Andrey V; Shlyapnikov, Mikhail; Granovsky, Igor

    2015-11-01

    The article describes substitutions in bacteriophage T4 RNase H which provide so called das-effect. Phage T4 DNA arrest suppression (das) mutations have been described to be capable of partially suppressing the phage DNA arrest phenotype caused by a dysfunction in genes 46 and/or 47 (also known as Mre11/Rad50 complex). Genetic mapping of das13 (one of the das mutations) has shown it to be in the region of the rnh gene encoding RNase H. Here we report that Das13 mutant of RNase H has substitutions of valine 43 and leucine 242 with isoleucines. To investigate the influence of these mutations on RNase H nuclease properties we have designed a novel in vitro assay that allows us to separate and quantify exo- or endonuclease activities of flap endonuclease. The nuclease assay in vitro showed that V43I substitution increased the ratio between exonuclease/endonuclease activities of RNase H whereas L242I substitution did not affect the nuclease activity of RNase H in vitro. However, both mutations were necessary for the full das effect in vivo. Molecular modelling of the nuclease structure suggests that V43I substitution may lead to disposition of H4 helix, responsible for the interaction with the first base pairs of 5'end of branched DNA. These structural changes may affect unwinding of the first base pairs of gapped or nicked DNA generating a short flap and therefore may stabilize the DNA-enzyme complex. L242I substitution did not affect the structure of RNase H and its role in providing das-effect remains unclear. PMID:26432500

  20. Site-dependent cobalt electronic state in La-Co co-substituted magnetoplumbite-type ferrite: (59)Co nuclear magnetic resonance study.

    PubMed

    Nakamura, Hiroyuki; Shimoda, Aiko; Waki, Takeshi; Tabata, Yoshikazu; Mény, Christian

    2016-09-01

    The nuclear magnetic resonance of (59)Co was measured over a wide frequency range in a powder sample crushed from a well-characterized single crystal of La-Co co-substituted magnetoplumbite-type strontium ferrite (SrFe12O19), a familiar base material for the ferrite permanent magnet. The simultaneous observation of both high- and low-frequency resonances suggests the coexistence of both high- and low-spin states of the substituted Co or the presence of Co orbital moment at a particular site. The possible presence of trivalent Co was also investigated. The results suggest that the Co atoms are distributed across different crystallographic sites with different local environments, and that the electronic state of Co is much more subtle than the conventional understanding. PMID:27355901

  1. Site-dependent cobalt electronic state in La–Co co-substituted magnetoplumbite-type ferrite: 59Co nuclear magnetic resonance study

    NASA Astrophysics Data System (ADS)

    Nakamura, Hiroyuki; Shimoda, Aiko; Waki, Takeshi; Tabata, Yoshikazu; Mény, Christian

    2016-09-01

    The nuclear magnetic resonance of 59Co was measured over a wide frequency range in a powder sample crushed from a well-characterized single crystal of La–Co co-substituted magnetoplumbite-type strontium ferrite (SrFe12O19), a familiar base material for the ferrite permanent magnet. The simultaneous observation of both high- and low-frequency resonances suggests the coexistence of both high- and low-spin states of the substituted Co or the presence of Co orbital moment at a particular site. The possible presence of trivalent Co was also investigated. The results suggest that the Co atoms are distributed across different crystallographic sites with different local environments, and that the electronic state of Co is much more subtle than the conventional understanding.

  2. Synthesis and cytotoxic activity of 2-methylimidazo[1,2-a]pyridine- and quinoline-substituted 2-aminopyrimidine derivatives.

    PubMed

    Vilchis-Reyes, Miguel Angel; Zentella, Alejandro; Martínez-Urbina, Miguel Angel; Guzmán, Angel; Vargas, Omar; Ramírez Apan, María Teresa; Ventura Gallegos, José Luis; Díaz, Eduardo

    2010-01-01

    A series of 2-methylimidazo[1,2-a]pyridine- and quinoline-substituted 2-aminopyrimidines derivatives were synthesized using a convenient synthetic route. We evaluate the isosteric replacement of methyl groups in 4-(2-methylimidazo[1,2-a]pyridin-3-yl)-N-p-tolylpyrimidin-2-amine (compound 1) by trifluoromethyl groups and the isosteric substitution of the 2-methylimidazo[1,2-a]pyridin-3-yl scaffold by quinolin-4-yl or quinolin-3-yl moieties. The replacement of hydrogen by fluorine does not affect notably the cytotoxic activity and CDK inhibitor activity in this series. Quinolin-4-yl-substituted compound, 8, presents cytotoxic activity and is most effective and selective against CDK1/CycA than against CDK2/CycB. Compound 11, which has a quinolin-3-yl moiety is CDK inhibitor but presents null cytotoxic activity. Quinolin-4-yl-substituted compounds constitute a new lead of cytotoxic and CDK inhibitor compounds from which more compelling and selective inhibitors can be designed. PMID:19879023

  3. Electrophilic Activation of P-Alkynes in the Synthesis of P-Substituted and P-Centered Heterocycles.

    PubMed

    Gupta, Akhil; Flynn, Bernard L

    2016-05-20

    Electrophilic activation of alkynylphosphine oxides and phosphonates provides a novel approach to the synthesis of P-substituted and P-centered heterocycles. Iodocyclization affords a heteroaryl iodide that can, among other things, be used in reiterative alkyne coupling and iodocyclization to give cyclic phosphonates and other cyclization reactions to give π-rich P-heterocycles. PMID:27088459

  4. Assembly of Fe-substituted Dawson-type nanoscale selenotungstate clusters with photocatalytic H2 evolution activity.

    PubMed

    Chen, Wei-Chao; Qin, Chao; Wang, Xin-Long; Li, Yang-Guang; Zang, Hong-Ying; Jiao, Yan-Qing; Huang, Peng; Shao, Kui-Zhan; Su, Zhong-Min; Wang, En-Bo

    2014-11-11

    Two Fe-substituted Dawson-type nanoscale selenotungstate clusters, {Fe6Se6W34} and {Fe10Se8W62} involving {α-Se2W14} and {γ-Se2W14} building blocks, have been isolated, which exhibit photocatalytic H2 evolution activity. Their electrochemical behaviours and magnetic properties were also investigated. PMID:25232933

  5. Synthesis and structure activity relationship study of N-substituted 3,5-diarylidenepiperidin- 4-ones as potential antitumor agents.

    PubMed

    El-Nassan, Hala Bakr

    2014-02-01

    A new series of N-substituted diarylidenepiperidin-4-ones was synthesized and screened for their possible anticancer activity at the NCI Developmental Therapeutic Program. Almost all the synthesized compounds showed more potent antiproliferative activity than curcumin. The most active compound in this study was 3,5-bis(4-bromobenzylidene)-1-propanoylpiperidin-4-one (8a) with MG-MID GI50, TGI, and LC50 values of 0.35, 1.62 and 9.12 µM, respectively. Compound 8a displayed broad spectrum antiproliferative activity with GI50 values below 1 µM in 81% of the tested cell lines and was found to be two folds more potent than EF-24. A detailed study of the structure activity relationship of the N-substitution was also described. PMID:24102314

  6. Sites of Regulated Phosphorylation that Control K-Cl Cotransporter Activity

    PubMed Central

    Rinehart, Jesse; Maksimova, Yelena D.; Tanis, Jessica E.; Stone, Kathryn L.; Hodson, Caleb A.; Zhang, Junhui; Risinger, Mary; Pan, Weijun; Wu, Dianqing; Colangelo, Christopher M.; Forbush, Biff; Joiner, Clinton H.; Gulcicek, Erol E.; Gallagher, Patrick G.; Lifton, Richard P.

    2010-01-01

    Summary Modulation of intracellular chloride concentration ([Cl−]i) plays a fundamental role in cell volume regulation and neuronal response to GABA. Cl− exit via K-Cl cotransporters (KCCs) is a major determinant of [Cl−]I; however, mechanisms governing KCC activities are poorly understood. We identified two sites in KCC3 that are rapidly dephosphorylated in hypotonic conditions in cultured cells and human red blood cells in parallel with increased transport activity. Alanine substitutions at these sites result in constitutively active cotransport. These sites are highly phosphorylated in plasma membrane KCC3 in isotonic conditions, suggesting that dephosphorylation increases KCC3's intrinsic transport activity. Reduction of WNK1 expression via RNA interference reduces phosphorylation at these sites. Homologous sites are phosphorylated in all human KCCs. KCC2 is partially phosphorylated in neonatal mouse brain and dephosphorylated in parallel with KCC2 activation. These findings provide insight into regulation of [Cl−]i and have implications for control of cell volume and neuronal function. PMID:19665974

  7. Acid-catalyzed synthesis of 10-substituted triazolyl artemisinins and their growth inhibitory activity against various cancer cells.

    PubMed

    Oh, Sangtae; Shin, Woon-Seob; Ham, Jungyeob; Lee, Seokjoon

    2010-07-15

    A diastereomeric and regioisomeric library of 10-substituted triazolyl artemisinin compounds (6a-6h, 7a-7h, and 8a-8h) with a potent growth inhibitory activities against various cancer cell lines was established. These compounds were synthesized by a reaction with dihydroartemisinin (2) and various substituted triazoles (5a-5h) in methylene chloride using a BF(3)Et(2)O catalyst. Most of the compounds exhibited a strong potency in the submicromolar range, and, in particular, 6f, 7f, and 8f, which have a pentylphenyltriazole moiety, proved to be promising candidates for preclinical trials. PMID:20538462

  8. Synthesis and cytotoxic activity of N-substituted thiosemicarbazones of 3-(3,4-methylenedioxy)phenylpropanal.

    PubMed

    Joselice e Silva, M; Alves, A J; Do Nascimento, S C

    1998-03-01

    Five new N-substituted thiosemicarbazones of 3-(3,4-methylenedioxy)phenylpropanal were synthesized. Safrole, a natural product obtained from sassafras oil (Ocotea pretiosa), was oxidized to alcohol using BH3-THF and H2O2, followed by oxidation to aldehyde using pyridinium dichromate (PDC) and condensation with five N-substituted derivatives of thiosemicarbazide. Tests were performed to evaluate the cytotoxic activity with continuous chain KB cells (epidermoide carcinoma of the floor of the mouth). Compounds 5 and 6 showed IC50 values of 1.5 and 4.6 micrograms/ml, respectively. PMID:9639871

  9. Dissecting the active site of a photoreceptor protein

    NASA Astrophysics Data System (ADS)

    Hoff, Wouter; Hara, Miwa; Ren, Jie; Moghadam, Farzaneh; Xie, Aihua; Kumauchi, Masato

    While enzymes are quite large molecules, functionally important chemical events are often limited to a small region of the protein: the active site. The physical and chemical properties of residues at such active sites are often strongly altered compared to the same groups dissolved in water. Understanding such effects is important for unraveling the mechanisms underlying protein function and for protein engineering, but has proven challenging. Here we report on our ongoing efforts on using photoactive yellow protein (PYP), a bacterial photoreceptor, as a model system for such effects. We will report on the following questions: How many residues affect active site properties? Are these residues in direct physical contact with the active site? Can functionally important residues be recognized in the crystal structure of a protein? What structural resolution is needed to understand active sites? What spectroscopic techniques are most informative? Which weak interactions dominate active site properties?

  10. Solvent substitution

    SciTech Connect

    Not Available

    1990-01-01

    The DOE Environmental Restoration and Waste Management Office of Technology Development and the Air Force Engineering and Services Center convened the First Annual International Workshop on Solvent Substitution on December 4--7, 1990. The primary objectives of this joint effort were to share information and ideas among attendees in order to enhance the development and implementation of required new technologies for the elimination of pollutants associated with industrial use of hazardous and toxic solvents; and to aid in accelerating collaborative efforts and technology transfer between government and industry for solvent substitution. There were workshop sessions focusing on Alternative Technologies, Alternative Solvents, Recovery/Recycling, Low VOC Materials and Treatment for Environmentally Safe Disposal. The 35 invited papers presented covered a wide range of solvent substitution activities including: hardware and weapons production and maintenance, paint stripping, coating applications, printed circuit boards, metal cleaning, metal finishing, manufacturing, compliance monitoring and process control monitoring. This publication includes the majority of these presentations. In addition, in order to further facilitate information exchange and technology transfer, the US Air Force and DOE solicited additional papers under a general Call for Papers.'' These papers, which underwent review and final selection by a peer review committee, are also included in this combined Proceedings/Compendium. For those involved in handling, using or managing hazardous and toxic solvents, this document should prove to be a valuable resource, providing the most up-to-date information on current technologies and practices in solvent substitution. Individual papers are abstracted separated.

  11. Mouse skin tumor-initiating activity of 5-, 7-, and 12-methyl- and fluorine-substituted benz(a)anthracenes

    SciTech Connect

    Wood, A.W.; Levin, W.; Chang, R.L.; Conney, A.H.; Slaga, T.J.; O'Malley, R.F.; Newman, M.S.; Buhler, D.R.; Jerina, D.M.

    1982-09-01

    Eleven methyl- and/or fluorine-substitued benz(a)anthracenes were evaluated for tumor-initating activity on mouse skin. Outbred CD-1 and outbred Sencar mice received a single topical application of the hydrocarbons followed by twice weekly application of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate for 16-26 weeks. 7, 12-DMBA was almost two orders of magnitude more active as a tumor-initator than 7- and 12-methylbenz(a)anthracene. Methyl substitution at the 7- and 7,12-positions of benz(a)anthracence was significantly more effective in the enhancement of tumorigenic activity than fluorine substitution at these positions. Although 7-fluorobenz(a)anthracene, 12-fluorobenz(a)anthracene, and 7,12-difluorobenz(a)anthracene had only 0.15, 0.26, and less than 0.005 times the tumor-initiating activity of their respective methyl-substituted derivatives, they were severalfold more active than benz(a)anthracene. 7-Fluorobenz(a)anthracene was slightly less active than 12-fluorobenz(a)anthracene, whereas 7-methylbenz(a)anthracene was about twofold more active than 12-methylbenz(a)anthracene. For 7,12-disubstituted benz(a)anthracenes, 7-methyl-12-fluorobenz(a)anthracene was more than twice as tumorigenic as 7-fluoro-12-methylbenz(a)anthracene, but each was individually more active than 7-methylbenz(a)anthracene and 12-methylbenz(a)anthracene, respectively. Both fluorinated compounds were much less active than 7,12-DMBA. Substitution of fluorine or methyl at the 5-position of 7-methylbenz(a)anthracene and substition of fluorine at the 5-position of 12-methylbenz(a)anthracene dramatically reduced their tumorigenic activity.

  12. Substitution of Wheat for Corn in Beef Cattle Diets: Digestibility, Digestive Enzyme Activities, Serum Metabolite Contents and Ruminal Fermentation.

    PubMed

    Liu, Y F; Zhao, H B; Liu, X M; You, W; Cheng, H J; Wan, F C; Liu, G F; Tan, X W; Song, E L; Zhang, X L

    2016-10-01

    The objective of this study was to evaluate the effect of diets containing different amounts of wheat, as a partial or whole substitute for corn, on digestibility, digestive enzyme activities, serum metabolite contents and ruminal fermentation in beef cattle. Four Limousin×LuXi crossbred cattle with a body weight (400±10 kg), fitted with permanent ruminal, proximal duodenal and terminal ileal cannulas, were used in a 4×4 Latin square design with four treatments: Control (100% corn), 33% wheat (33% substitution for corn), 67% wheat (67% substitution for corn), and 100% wheat (100% substitution for corn) on a dry matter basis. The results showed that replacing corn with increasing amounts of wheat increased the apparent digestibility values of dry matter, organic matter, and crude protein (p<0.05). While the apparent digestibility of acid detergent fiber and neutral detergent fiber were lower with increasing amounts of wheat. Digestive enzyme activities of lipase, protease and amylase in the duodenum were higher with increasing wheat amounts (p<0.05), and showed similar results to those for the enzymes in the ileum except for amylase. Increased substitution of wheat for corn increased the serum alanine aminotransferase concentration (p<0.05). Ruminal pH was not different between those given only corn and those given 33% wheat. Increasing the substitution of wheat for corn increased the molar proportion of acetate and tended to increase the acetate-to-propionate ratio. Cattle fed 100% wheat tended to have the lowest ruminal NH3-N concentration compared with control (p<0.05), whereas no differences were observed among the cattle fed 33% and 67% wheat. These findings indicate that wheat can be effectively used to replace corn in moderate amounts to meet the energy and fiber requirements of beef cattle. PMID:26954111

  13. A substitution of cysteine for glycine 748 of the alpha 1 chain produces a kink at this site in the procollagen I molecule and an altered N-proteinase cleavage site over 225 nm away.

    PubMed

    Vogel, B E; Doelz, R; Kadler, K E; Hojima, Y; Engel, J; Prockop, D J

    1988-12-15

    In previous work (Vogel, B. E., Minor, R. R., Freund, M., and Prockop, D. J. (1987) J. Biol. Chem. 262, 14737-14744), we identified a single-base mutation that converted the glycine at position 748 of the alpha 1 chain of type I procollagen to a cysteine in a proband with a lethal variant of osteogenesis imperfecta. In addition to posttranslational overmodification, the abnormal molecules displayed decreased thermal stability and a decreased rate of secretion. An unexplained finding was that procollagen was poorly processed to pCcollagen in postconfluent cultures of skin fibroblasts. Here, we show that the procollagen synthesized by the proband's cells is resistant to cleavage by procollagen N-proteinase, a conformation-sensitive enzyme. Since the only detectable defect in the molecule was the cysteine for glycine substitution, we assembled several space-filling models to try to explain how the structure of the N-proteinase cleavage site can be affected by an amino acid substitution over 700 amino acid residues or 225 nm away. The models incorporated a phase shift of a tripeptide unit in one or both of the alpha 1 chains. The most satisfactory models produced a flexible kink of 30 degrees or 60 degrees at the site of the cysteine substitution. Therefore, we examined the procollagen by electron microscopy. About 25% of the molecules had a kink not seen in control samples, and the kink was at the site of the cysteine substitution. PMID:3198624

  14. Alanine substitution of conserved residues in the cytoplasmic tail of herpes simplex virus gB can enhance or abolish cell fusion activity and viral entry

    SciTech Connect

    Ruel, Nancy . E-mail: n-ruel@northwestern.edu; Zago, Anna . E-mail: anna_zago@acgtinc.com; Spear, Patricia G. . E-mail: p-spear@northwestern.edu

    2006-03-01

    Herpes simplex virus (HSV) glycoprotein B (gB) is one of the four viral glycoproteins required for viral entry and cell fusion and is highly conserved among herpesviruses. Mutants of HSV type 2 gB were generated by substituting conserved residues in the cytoplasmic tail with alanine or by deleting 41 amino acids from the C-terminus. Some of the mutations abolished cell fusion activity and also prevented transport of gB to the cell surface, identifying residues in the gB cytoplasmic tail that are critical for intracellular transport of this glycoprotein. These mutations also prevented production of infectious virus, possibly because the mutant forms of gB were not transported to the site of envelopment. Other mutations, particularly the deletion, significantly enhanced cell fusion activity. These mutations, as well as others described previously, identify regions of the gB cytoplasmic domain that modulate cell fusion activity.

  15. Mars Surveyor Project Landing Site Activities

    NASA Technical Reports Server (NTRS)

    Gulick, Virginia C.; Briggs, Geoffrey; Saunders, R. Stephen; Gilmore, Martha; Soderblom, Larry

    1999-01-01

    The Mars Surveyor Program --now a cooperative program led by NASA and CNES along with other international partners -- is underway. It has the primary science objective of furthering our understanding of the biological potential and possible biological history of Mars and has the complementary objective of improving our understanding of martian climate evolution and planetary history The missions will develop technology and acquire data necessary for eventual human Exploration. Launches of orbiters, landers and rovers will take place in 2001 and in 2003; in 2005 a complete system will be launched capable of returning samples to Earth by 2008. A key aspect of the program is the selection of landing sites. This abstract 1) reports on the status of the landing site selection process that begins with the 2001 lander mission and 2) outlines be opportunities for the Mars community to provide input into the landing site selection process.

  16. Mars Surveyor Project Landing Site Activities

    NASA Technical Reports Server (NTRS)

    Gulick, V. C.; Briggs, Geoffrey; Saunders, R. Stephen; Gilmore, Martha; Soderblom, Larry

    1999-01-01

    The Mars Surveyor Program -- now a cooperative program led by NASA and CNES along with other international partners -- is underway. It has the primary science objective of furthering our understanding of the biological potential and possible biological history of Mars and has the complementary objective of improving our understanding of martian climate evolution and planetary history. The missions will develop technology and acquire data necessary for eventual human exploration. Launches of orbiters, landers and rovers will take place in 2001 and in 2003; in 2005 a complete system will be launched capable of returning samples to Earth by 2008. A key aspect of the program is the selection of landing sites. This abstract 1) reports on the status of the landing site selection process that begins with the 2001 lander mission and 2) outlines the opportunities for the Mars community to provide input into the landing site selection process.

  17. Probing the active site tryptophan of Staphylococcus aureus thioredoxin with an analog

    PubMed Central

    Englert, Markus; Nakamura, Akiyoshi; Wang, Yane-Shih; Eiler, Daniel; Söll, Dieter; Guo, Li-Tao

    2015-01-01

    Genetically encoded non-canonical amino acids are powerful tools of protein research and engineering; in particular they allow substitution of individual chemical groups or atoms in a protein of interest. One such amino acid is the tryptophan (Trp) analog 3-benzothienyl-l-alanine (Bta) with an imino-to-sulfur substitution in the five-membered ring. Unlike Trp, Bta is not capable of forming a hydrogen bond, but preserves other properties of a Trp residue. Here we present a pyrrolysyl-tRNA synthetase-derived, engineered enzyme BtaRS that enables efficient and site-specific Bta incorporation into proteins of interest in vivo. Furthermore, we report a 2.1 Å-resolution crystal structure of a BtaRS•Bta complex to show how BtaRS discriminates Bta from canonical amino acids, including Trp. To show utility in protein mutagenesis, we used BtaRS to introduce Bta to replace the Trp28 residue in the active site of Staphylococcus aureus thioredoxin. This experiment showed that not the hydrogen bond between residues Trp28 and Asp58, but the bulky aromatic side chain of Trp28 is important for active site maintenance. Collectively, our study provides a new and robust tool for checking the function of Trp in proteins. PMID:26582921

  18. The bifunctional active site of s-adenosylmethionine synthetase. Roles of the active site aspartates.

    PubMed

    Taylor, J C; Markham, G D

    1999-11-12

    S-Adenosylmethionine (AdoMet) synthetase catalyzes the biosynthesis of AdoMet in a unique enzymatic reaction. Initially the sulfur of methionine displaces the intact tripolyphosphate chain (PPP(i)) from ATP, and subsequently PPP(i) is hydrolyzed to PP(i) and P(i) before product release. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site contains four aspartate residues. Aspartate residues Asp-16* and Asp-271 individually provide the sole protein ligand to one of the two required Mg(2+) ions (* denotes a residue from a second subunit); aspartates Asp-118 and Asp-238* are proposed to interact with methionine. Each aspartate has been changed to an uncharged asparagine, and the metal binding residues were also changed to alanine, to assess the roles of charge and ligation ability on catalytic efficiency. The resultant enzyme variants all structurally resemble the wild type enzyme as indicated by circular dichroism spectra and are tetramers. However, all have k(cat) reductions of approximately 10(3)-fold in AdoMet synthesis, whereas the MgATP and methionine K(m) values change by less than 3- and 8-fold, respectively. In the partial reaction of PPP(i) hydrolysis, mutants of the Mg(2+) binding residues have >700-fold reduced catalytic efficiency (k(cat)/K(m)), whereas the D118N and D238*N mutants are impaired less than 35-fold. The catalytic efficiency for PPP(i) hydrolysis by Mg(2+) site mutants is improved by AdoMet, like the wild type enzyme. In contrast AdoMet reduces the catalytic efficiency for PPP(i) hydrolysis by the D118N and D238*N mutants, indicating that the events involved in AdoMet activation are hindered in these methionyl binding site mutants. Ca(2+) uniquely activates the D271A mutant enzyme to 15% of the level of Mg(2+), in contrast to the approximately 1% Ca(2+) activation of the wild type enzyme. This indicates that the Asp-271 side chain size is a discriminator between the activating ability of Ca(2+) and the

  19. Exploring pharmacological activities and signaling of morphinans substituted in position 6 as potent agonists interacting with the μ opioid receptor

    PubMed Central

    2014-01-01

    Background Opioid analgesics are the most effective drugs for the treatment of moderate to severe pain. However, they also produce several adverse effects that can complicate pain management. The μ opioid (MOP) receptor, a G protein-coupled receptor, is recognized as the opioid receptor type which primarily mediates the pharmacological actions of clinically used opioid agonists. The morphinan class of analgesics including morphine and oxycodone are of main importance as therapeutically valuable drugs. Though the natural alkaloid morphine contains a C-6-hydroxyl group and the semisynthetic derivative oxycodone has a 6-carbonyl function, chemical approaches have uncovered that functionalizing position 6 gives rise to a range of diverse activities. Hence, position 6 of N-methylmorphinans is one of the most manipulated sites, and is established to play a key role in ligand binding at the MOP receptor, efficacy, signaling, and analgesic potency. We have earlier reported on a chemically innovative modification in oxycodone resulting in novel morphinans with 6-acrylonitrile incorporated substructures. Results This study describes in vitro and in vivo pharmacological activities and signaling of new morphinans substituted in position 6 with acrylonitrile and amido functions as potent agonists and antinociceptive agents interacting with MOP receptors. We show that the presence of a 6-cyano group in N-methylmorphinans has a strong influence on the binding to the opioid receptors and post-receptor signaling. One 6-cyano-N-methylmorphinan of the series was identified as the highest affinity and most selective MOP agonist, and very potent in stimulating G protein coupling and intracellular calcium release through the MOP receptor. In vivo, this MOP agonist showed to be greatly effective against thermal and chemical nociception in mice with marked increased antinociceptive potency than the lead molecule oxycodone. Conclusion Development of such novel chemotypes by targeting

  20. Synthesis and Antiplatelet Aggregation Activity Evaluation of some 2-Aminopyrimidine and 2-Substituted-4,6-diaminopyrimidine Derivatives

    PubMed Central

    Esfahanizadeh, Marjan; Mohebbi, Shohreh; Dasht Bozorg, Behnam; Amidi, Salimeh; Gudarzi, Ali; Ayatollahi, Seyed Abdolmajid; Kobarfard, Farzad

    2015-01-01

    A series of novel 2-aminopyrimidine and 2-Substituted-4,6-diaminopyrimidine derivatives have been synthesized and their antiplatelet aggregation activities were assessed against ADP and arachidonic acid-induced platelet aggregation in human plasma using light transmission aggregometry. Among the tested derivatives, compounds Ia, Ib, IB and II16 exhibited the highest antiplatelet aggregation activity (36.75, 72.4, 62.5 and 80 µM). None of the compounds showed satisfactory activity against the aggregation induced by ADP but acceptable activities were observed against the aggregation induced by arachidonic acid. 2- aminopyrimidines were more active than 4,6- diaminopyrimidines in this respect. PMID:25901148

  1. Synthesis and Antiplatelet Aggregation Activity Evaluation of some 2-Aminopyrimidine and 2-Substituted-4,6-diaminopyrimidine Derivatives.

    PubMed

    Esfahanizadeh, Marjan; Mohebbi, Shohreh; Dasht Bozorg, Behnam; Amidi, Salimeh; Gudarzi, Ali; Ayatollahi, Seyed Abdolmajid; Kobarfard, Farzad

    2015-01-01

    A series of novel 2-aminopyrimidine and 2-Substituted-4,6-diaminopyrimidine derivatives have been synthesized and their antiplatelet aggregation activities were assessed against ADP and arachidonic acid-induced platelet aggregation in human plasma using light transmission aggregometry. Among the tested derivatives, compounds Ia, Ib, IB and II16 exhibited the highest antiplatelet aggregation activity (36.75, 72.4, 62.5 and 80 µM). None of the compounds showed satisfactory activity against the aggregation induced by ADP but acceptable activities were observed against the aggregation induced by arachidonic acid. 2- aminopyrimidines were more active than 4,6- diaminopyrimidines in this respect. PMID:25901148

  2. Synthesis and structure-activity relationship studies of 2-(N-substituted)-aminobenzimidazoles as potent negative gating modulators ofsmall conductance Ca2+-activated K+ channels.

    PubMed

    Sørensen, Ulrik S; Strøbaek, Dorte; Christophersen, Palle; Hougaard, Charlotte; Jensen, Marianne L; Nielsen, Elsebet Ø; Peters, Dan; Teuber, Lene

    2008-12-11

    Small conductance Ca2+-activated K+ channels (SK channels) participate in the control of neuronal excitability, in the shaping of action potential firing patterns, and in the regulation of synaptic transmission.SK channel inhibitors have the potential of becoming new drugs for treatment of various psychiatric and neurological diseases such as depression, cognition impairment, and Parkinson's disease. In the present study we describe the structure-activity relationship (SAR) of a class of 2-(N-substituted)-2-aminobenzimidazoles that constitute a novel class of selective SK channel inhibitors that, in contrast to classical SK inhibitors, do not block the pore of the channel. The pore blocker apamin is not displaced by these compounds in binding studies, and they still inhibit SK channels in which the apamin binding site has been abolished by point mutations. These novel SK inhibitors shift the concentration-response curve for Ca2+ toward higher values and represent the first example of negative gating modulation as a mode-of-action for inhibition of SK channels. The first described compound in this class is NS8593 (14), and the most potent analogue identified in this study is the racemic compound 39 (NS11757), which reversibly inhibits SK3-mediated currents with a K(d) value of 9 nM. PMID:18998663

  3. Effect of active synthetic 2-substituted quinazolinones on anti-platelet aggregation and the inhibition of superoxide anion generation by neutrophils.

    PubMed

    Chang, Fang-Rong; Wu, Chin-Chung; Hwang, Tsong-Long; Patnam, Ramesh; Kuo, Reen-Yen; Wang, Wei-Ya; Lan, Yu-Hsuan; Wu, Yang-Chang

    2003-07-01

    Quinazolinones, 2-substituted and 3-substituted, mainly synthesized by microwave irradiation, were subjected to anti-platelet aggregation and inhibition of superoxide anion generation assays. Interestingly, 2-phenyl-4-quinazolinone (4) exhibited significant inhibitory activities toward platelet aggregation and neutrophil activation, and it might therefore serve as a prototype lead compound. PMID:12934640

  4. Reduction of urease activity by interaction with the flap covering the active site.

    PubMed

    Macomber, Lee; Minkara, Mona S; Hausinger, Robert P; Merz, Kenneth M

    2015-02-23

    With the increasing appreciation for the human microbiome coupled with the global rise of antibiotic resistant organisms, it is imperative that new methods be developed to specifically target pathogens. To that end, a novel computational approach was devised to identify compounds that reduce the activity of urease, a medically important enzyme of Helicobacter pylori, Proteus mirabilis, and many other microorganisms. Urease contains a flexible loop that covers its active site; Glide was used to identify small molecules predicted to lock this loop in an open conformation. These compounds were screened against the model urease from Klebsiella aerogenes, and the natural products epigallocatechin and quercetin were shown to inhibit at low and high micromolar concentrations, respectively. These molecules exhibit a strong time-dependent inactivation of urease that was not due to their oxygen sensitivity. Rather, these compounds appear to inactivate urease by reacting with a specific Cys residue located on the flexible loop. Substitution of this cysteine by alanine in the C319A variant increased the urease resistance to both epigallocatechin and quercetin, as predicted by the computational studies. Protein dynamics are integral to the function of many enzymes; thus, identification of compounds that lock an enzyme into a single conformation presents a useful approach to define potential inhibitors. PMID:25594724

  5. Reduction of Urease Activity by Interaction with the Flap Covering the Active Site

    PubMed Central

    Macomber, Lee; Minkara, Mona S.; Hausinger, Robert P.; Merz, Kenneth M.

    2015-01-01

    With the increasing appreciation for the human microbiome coupled with the global rise of antibiotic resistant organisms, it is imperative that new methods be developed to specifically target pathogens. To that end, a novel computational approach was devised to identify compounds that reduce the activity of urease, a medically important enzyme of Helicobacter pylori, Proteus mirabilis, and many other microorganisms. Urease contains a flexible loop that covers its active site; Glide was used to identify small molecules predicted to lock this loop in an open conformation. These compounds were screened against the model urease from Klebsiella aerogenes and the natural products epigallocatechin and quercetin were shown to inhibit at low and high micromolar concentrations, respectively. These molecules exhibit a strong time-dependent inactivation of urease that was not due to their oxygen sensitivity. Rather, these compounds appear to inactivate urease by reacting with a specific Cys residue located on the flexible loop. Substitution of this cysteine by alanine in the C319A variant increased the urease resistance to both epigallocatechin and quercetin, as predicted by the computational studies. Protein dynamics are integral to the function of many enzymes; thus, identification of compounds that lock an enzyme into a single conformation presents a useful approach to define potential inhibitors. PMID:25594724

  6. Deep Sequencing of Random Mutant Libraries Reveals the Active Site of the Narrow Specificity CphA Metallo-β-Lactamase is Fragile to Mutations.

    PubMed

    Sun, Zhizeng; Mehta, Shrenik C; Adamski, Carolyn J; Gibbs, Richard A; Palzkill, Timothy

    2016-01-01

    CphA is a Zn(2+)-dependent metallo-β-lactamase that efficiently hydrolyzes only carbapenem antibiotics. To understand the sequence requirements for CphA function, single codon random mutant libraries were constructed for residues in and near the active site and mutants were selected for E. coli growth on increasing concentrations of imipenem, a carbapenem antibiotic. At high concentrations of imipenem that select for phenotypically wild-type mutants, the active-site residues exhibit stringent sequence requirements in that nearly all residues in positions that contact zinc, the substrate, or the catalytic water do not tolerate amino acid substitutions. In addition, at high imipenem concentrations a number of residues that do not directly contact zinc or substrate are also essential and do not tolerate substitutions. Biochemical analysis confirmed that amino acid substitutions at essential positions decreased the stability or catalytic activity of the CphA enzyme. Therefore, the CphA active - site is fragile to substitutions, suggesting active-site residues are optimized for imipenem hydrolysis. These results also suggest that resistance to inhibitors targeted to the CphA active site would be slow to develop because of the strong sequence constraints on function. PMID:27616327

  7. Electronic structure origin of conductivity and oxygen reduction activity changes in low-level Cr-substituted (La,Sr)MnO{sub 3}

    SciTech Connect

    Tsekouras, George Boudoire, Florent; Braun, Artur; Pal, Banabir; Sarma, D. D.; Vondráček, Martin; Prince, Kevin C.

    2015-09-21

    The electronic structure of the (La{sub 0.8}Sr{sub 0.2}){sub 0.98}Mn{sub 1−x}Cr{sub x}O{sub 3} model series (x = 0, 0.05, or 0.1) was measured using soft X-ray synchrotron radiation at room and elevated temperature. O K-edge near-edge X-ray absorption fine structure (NEXAFS) spectra showed that low-level chromium substitution of (La,Sr)MnO{sub 3} resulted in lowered hybridisation between O 2p orbitals and M 3d and M 4sp valance orbitals. Mn L{sub 3}-edge resonant photoemission spectroscopy measurements indicated lowered Mn 3d–O 2p hybridisation with chromium substitution. Deconvolution of O K-edge NEXAFS spectra took into account the effects of exchange and crystal field splitting and included a novel approach whereby the pre-peak region was described using the nominally filled t{sub 2g}↑ state. 10% chromium substitution resulted in a 0.17 eV lowering in the energy of the t{sub 2g}↑ state, which appears to provide an explanation for the 0.15 eV rise in activation energy for the oxygen reduction reaction, while decreased overlap between hybrid O 2p–Mn 3d states was in qualitative agreement with lowered electronic conductivity. An orbital-level understanding of the thermodynamically predicted solid oxide fuel cell cathode poisoning mechanism involving low-level chromium substitution on the B-site of (La,Sr)MnO{sub 3} is presented.

  8. Electronic structure origin of conductivity and oxygen reduction activity changes in low-level Cr-substituted (La,Sr)MnO3

    NASA Astrophysics Data System (ADS)

    Tsekouras, George; Boudoire, Florent; Pal, Banabir; Vondráček, Martin; Prince, Kevin C.; Sarma, D. D.; Braun, Artur

    2015-09-01

    The electronic structure of the (La0.8Sr0.2)0.98Mn1-xCrxO3 model series (x = 0, 0.05, or 0.1) was measured using soft X-ray synchrotron radiation at room and elevated temperature. O K-edge near-edge X-ray absorption fine structure (NEXAFS) spectra showed that low-level chromium substitution of (La,Sr)MnO3 resulted in lowered hybridisation between O 2p orbitals and M 3d and M 4sp valance orbitals. Mn L3-edge resonant photoemission spectroscopy measurements indicated lowered Mn 3d-O 2p hybridisation with chromium substitution. Deconvolution of O K-edge NEXAFS spectra took into account the effects of exchange and crystal field splitting and included a novel approach whereby the pre-peak region was described using the nominally filled t2g↑ state. 10% chromium substitution resulted in a 0.17 eV lowering in the energy of the t2g↑ state, which appears to provide an explanation for the 0.15 eV rise in activation energy for the oxygen reduction reaction, while decreased overlap between hybrid O 2p-Mn 3d states was in qualitative agreement with lowered electronic conductivity. An orbital-level understanding of the thermodynamically predicted solid oxide fuel cell cathode poisoning mechanism involving low-level chromium substitution on the B-site of (La,Sr)MnO3 is presented.

  9. Antimicrobial/cytolytic peptides from the venom of the North African scorpion, Androctonus amoreuxi: biochemical and functional characterization of natural peptides and a single site-substituted analog.

    PubMed

    Almaaytah, Ammar; Zhou, Mei; Wang, Lei; Chen, Tianbao; Walker, Brian; Shaw, Chris

    2012-06-01

    The venoms of scorpions are complex cocktails of polypeptide toxins that fall into two structural categories: those that contain cysteinyl residues with associated disulfide bridges and those that do not. As the majority of lethal toxins acting upon ion channels fall into the first category, most research has been focused there. Here we report the identification and structural characterization of two novel 18-mer antimicrobial peptides from the venom of the North African scorpion, Androctonus amoreuxi. Named AamAP1 and AamAP2, both peptides are C-terminally amidated and differ in primary structure at just two sites: Leu-->Pro at position 2 and Phe-->Ile at position 17. Synthetic replicates of both peptides exhibited a broad-spectrum of antimicrobial activity against a Gram-positive bacterium (Staphylococcus aureus), a Gram-negative bacterium (Escherichia coli) and a yeast (Candida albicans), at concentrations ranging between 20 μM and 150 μM. In this concentration range, both peptides produced significant degrees of hemolysis. A synthetic replicate of AamAP1 containing a single substitution (His-->Lys) at position 8, generated a peptide (AamAP-S1) with enhanced antimicrobial potency (3-5 μM) against the three test organisms and within this concentration range, hemolytic effects were negligible. In addition, this His-->Lys variant exhibited potent growth inhibitory activity (ID(50) 25-40 μm) against several human cancer cell lines and endothelial cells that was absent in both natural peptides. Natural bioactive peptide libraries, such as those that occur in scorpion venoms, thus constitute a unique source of novel lead compounds with drug development potential whose biological properties can be readily manipulated by simple synthetic chemical means. PMID:22484288

  10. Synthesis and antibacterial activity of novel levofloxacin derivatives containing a substituted thienylethyl moiety

    PubMed Central

    2012-01-01

    Background and the purpose of the study Piperazinyl quinolones such as ciprofloxacin, ofloxacin and levofloxacin are an important group of quinolone antimicrobials which are widely used in the treatment of various infectious diseases. In the present study, we synthesized a new series of levofloxacin derivatives and evaluated their antibacterial activities. Methods The N-substituted analogs of levofloxacin 6a–j were prepared by nucleophilic reaction of N-desmethyl levofloxacin 11 with thienylethyl bromide derivatives 8 or 9. All target compounds were tested using conventional agar dilution method in comparison to levofloxacin and N-desmethyl levofloxacin and their MIC values were determined against a panel of Gram-positive and Gram-negative bacteria. Results All compounds showed significant antibacterial activities against Gram-positive bacteria (MIC = 0.04-6.25 μg/mL); however, the activity against Gram-negative bacteria was lower (MIC = 1.56–100 μg/mL). As is evident from the data, oxime derivatives 6e, 6h and 6i are superior in inhibiting the growth of Gram-positive bacteria (MIC = 0.04–0.19 μg/mL), and their activities were found to be 5–25 times better than N-desmethyl levofloxacin 11 and equal or better than levofloxacin 4. Conclusion We have designed and synthesized novel quinolone derivatives bearing functionalized thienylethyl moiety on the piperazine ring of levofloxacin. The results of antibacterial screening against Gram-positive and Gram-negative bacteria revealed that the introduction of functionalized thienylethyl moiety on the piperazine ring of levofloxacin can improve the activity against Gram-positive bacteria. Gram-positive bacteria are responsible for a wide range of infectious diseases, and rising resistance in this group is causing increasing concern. Thus, this study introduces structural features of levofloxacin scaffold for development of new candidates in the field of anti-Gram positive chemotherapy PMID:23351676

  11. The active site of ribulose-bisphosphate carboxylase/oxygenase

    SciTech Connect

    Hartman, F.C.

    1991-01-01

    The active site of ribulose-bisphosphate carboxylase/oxygenase requires interacting domains of adjacent, identical subunits. Most active-site residues are located within the loop regions of an eight-stranded {beta}/{alpha}-barrel which constitutes the larger C-terminal domain; additional key residues are located within a segment of the smaller N-terminal domain which partially covers the mouth of the barrel. Site-directed mutagenesis of the gene encoding the enzyme from Rhodospirillum rubrum has been used to delineate functions of active-site residues. 6 refs., 2 figs.

  12. A study on the flexibility of enzyme active sites

    PubMed Central

    2011-01-01

    Background A common assumption about enzyme active sites is that their structures are highly conserved to specifically distinguish between closely similar compounds. However, with the discovery of distinct enzymes with similar reaction chemistries, more and more studies discussing the structural flexibility of the active site have been conducted. Results Most of the existing works on the flexibility of active sites focuses on a set of pre-selected active sites that were already known to be flexible. This study, on the other hand, proposes an analysis framework composed of a new data collecting strategy, a local structure alignment tool and several physicochemical measures derived from the alignments. The method proposed to identify flexible active sites is highly automated and robust so that more extensive studies will be feasible in the future. The experimental results show the proposed method is (a) consistent with previous works based on manually identified flexible active sites and (b) capable of identifying potentially new flexible active sites. Conclusions This proposed analysis framework and the former analyses on flexibility have their own advantages and disadvantage, depending on the cause of the flexibility. In this regard, this study proposes an alternative that complements previous studies and helps to construct a more comprehensive view of the flexibility of enzyme active sites. PMID:21342563

  13. Safety Oversight of Decommissioning Activities at DOE Nuclear Sites

    SciTech Connect

    Zull, Lawrence M.; Yeniscavich, William

    2008-01-15

    The Defense Nuclear Facilities Safety Board (Board) is an independent federal agency established by Congress in 1988 to provide nuclear safety oversight of activities at U.S. Department of Energy (DOE) defense nuclear facilities. The activities under the Board's jurisdiction include the design, construction, startup, operation, and decommissioning of defense nuclear facilities at DOE sites. This paper reviews the Board's safety oversight of decommissioning activities at DOE sites, identifies the safety problems observed, and discusses Board initiatives to improve the safety of decommissioning activities at DOE sites. The decommissioning of former defense nuclear facilities has reduced the risk of radioactive material contamination and exposure to the public and site workers. In general, efforts to perform decommissioning work at DOE defense nuclear sites have been successful, and contractors performing decommissioning work have a good safety record. Decommissioning activities have recently been completed at sites identified for closure, including the Rocky Flats Environmental Technology Site, the Fernald Closure Project, and the Miamisburg Closure Project (the Mound site). The Rocky Flats and Fernald sites, which produced plutonium parts and uranium materials for defense needs (respectively), have been turned into wildlife refuges. The Mound site, which performed R and D activities on nuclear materials, has been converted into an industrial and technology park called the Mound Advanced Technology Center. The DOE Office of Legacy Management is responsible for the long term stewardship of these former EM sites. The Board has reviewed many decommissioning activities, and noted that there are valuable lessons learned that can benefit both DOE and the contractor. As part of its ongoing safety oversight responsibilities, the Board and its staff will continue to review the safety of DOE and contractor decommissioning activities at DOE defense nuclear sites.

  14. DOE site performance assessment activities. Radioactive Waste Technical Support Program

    SciTech Connect

    Not Available

    1990-07-01

    Information on performance assessment capabilities and activities was collected from eight DOE sites. All eight sites either currently dispose of low-level radioactive waste (LLW) or plan to dispose of LLW in the near future. A survey questionnaire was developed and sent to key individuals involved in DOE Order 5820.2A performance assessment activities at each site. The sites surveyed included: Hanford Site (Hanford), Idaho National Engineering Laboratory (INEL), Los Alamos National Laboratory (LANL), Nevada Test Site (NTS), Oak Ridge National Laboratory (ORNL), Paducah Gaseous Diffusion Plant (Paducah), Portsmouth Gaseous Diffusion Plant (Portsmouth), and Savannah River Site (SRS). The questionnaire addressed all aspects of the performance assessment process; from waste source term to dose conversion factors. This report presents the information developed from the site questionnaire and provides a comparison of site-specific performance assessment approaches, data needs, and ongoing and planned activities. All sites are engaged in completing the radioactive waste disposal facility performance assessment required by DOE Order 5820.2A. Each site has achieved various degrees of progress and have identified a set of critical needs. Within several areas, however, the sites identified common needs and questions.

  15. Savannah River Site prioritization of transition activities

    SciTech Connect

    Finley, R.H.

    1993-11-01

    Effective management of SRS conversion from primarily a production facility to other missions (or Decontamination and Decommissioning (D&D)) requires a systematic and consistent method of prioritizing the transition activities. This report discusses the design of a prioritizing method developed to achieve systematic and consistent methods of prioritizing these activities.

  16. Effect of substitutions at the nickel site on the electronic structure of LaNi{sub 5} and its hydrides

    SciTech Connect

    Gupta, M.

    1998-12-31

    The effect of Ni substitution in LaNi{sub 5} by 3d and s-p elements on the electronic structure of the intermetallic and its hydrides has been investigated using the self consistent linear muffin tin orbital (LMTO) method in the atomic sphere approximation (ASA). The Fermi level, E{sub F}, of LaN{sub 14}M (M = Fe, Co, Mn) is found to lie in the narrow additional M 3d subband above the Ni d states, leading to an increase in the density of states (DOS) at E{sub F}. In contrast, the substitution of Ni by an s element of the 3d series, Cu, or by an s-p element: Al or Sn results in a progressive filling of the Ni-d bands and in a decrease of the DOS at E{sub F}. In all the substituted intermetallic compounds, the author finds that the lattice expansion accounts for less than 50% of the observed decreased stability, this shows the importance of the effect of chemical substitution. They also discuss the factors which affect the electronic structure and the stability of the hydrides and compare the results with available experimental data.

  17. Synthesis and theoretical calculations of carbazole substituted chalcone urea derivatives and studies their polyphenol oxidase enzyme activity.

    PubMed

    Nixha, Arleta Rifati; Arslan, Mustafa; Atalay, Yusuf; Gençer, Nahit; Ergün, Adem; Arslan, Oktay

    2013-08-01

    Synthesis of carbazole substituted chalcone urea derivatives and their polyphenol oxidase enzyme activity effects on the diphenolase activity of banana tyrosinase were evaluated. Tyrosinase has been purified from banana on an affinity gel comprised of Sepharose 4B-L-tyrosine-p-aminobenzoic acid. The results showed that most of the compounds (3,4,5a,5d-h) inhibited and some of them (5c,5i-l) activated the tyrosinase enzyme activity. The molecular calculations were performed using Gaussian software for the synthesized compounds to explain the experimental results. PMID:22803668

  18. Codon repeats in genes associated with human diseases: fewer repeats in the genes of nonhuman primates and nucleotide substitutions concentrated at the sites of reiteration.

    PubMed Central

    Djian, P; Hancock, J M; Chana, H S

    1996-01-01

    Five human diseases are due to an excessive number of CAG repeats in the coding regions of five different genes. We have analyzed the repeat regions in four of these genes from nonhuman primates, which are not known to suffer from the diseases. These primates have CAG repeats at the same sites as in human alleles, and there is similar polymorphism of repeat number, but this number is smaller than in the human genes. In some of the genes, the segment of poly(CAG) has expanded in nonhuman primates, but the process has advanced further in the human lineage than in other primate lineages, thereby predisposing to diseases of CAG reiteration. Adjacent to stretches of homogeneous present-day codon repeats, previously existing codons of the same kind have undergone nucleotide substitutions with high frequency. Where these lead to amino acid substitutions, the effect will be to reduce the length of the original homopolymeric stretch in the protein. PMID:8552651

  19. Influence of Ni substitution at B-site for Fe3+ ions on morphological, optical, and magnetic properties of HoFeO3 ceramics

    NASA Astrophysics Data System (ADS)

    Habib, Zubida; Majid, Kowsar; Ikram, Mohd.; Sultan, Khalid; Mir, Sajad Ahmad; Asokan, K.

    2016-05-01

    Present study reports the effect of Ni substitution at B-site in HoFeO3 on the morphological, optical and magnetic properties. These compounds were prepared by solid-state reaction method. Scanning electron microscope reveals an increase in average grain sizes with Ni concentration. Absorption and emission spectra show redshift in band gap with increase in Ni ion concentrations. The Tauc plots show direct allowed transitions. Temperature-dependent magnetization studies on these compounds revealed the transition from ferromagnetism to paramagnetism. There is separation between temperature at which zero-field-cooled and field-cooled occurs at varied temperature with Ni substitution. The separation effect is related to the impact of the paramagnetic Ho3+ ions, whose magnitude becomes more prominent at higher temperature. The value of squareness ratio in these materials is below 0.5 indicating presence of multidomain structures.

  20. Ionizable Side Chains at Catalytic Active Sites of Enzymes

    PubMed Central

    Jimenez-Morales, David; Liang, Jie

    2012-01-01

    Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1072 Å3. The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes. PMID:22484856

  1. Structural and antibacterial activity of hydroxyapatite and fluorohydroxyapatite co-substituted with zirconium-cerium ions

    NASA Astrophysics Data System (ADS)

    Sanyal, Vijayalakshmi; Raja, C. Ramachandra

    2016-02-01

    The effect of zirconium (Zr) and cerium (Ce) ions co-substituted in hydroxyapatite (HA) and fluorohydroxyapatite (FHA), by sol-gel method, is reported in this technical article. Addition of Zr4+ and Ce3+ ions into HA and FHA reportedly transforms t-ZrO2 (tetragonal) to m-ZrO2 (monoclinic) phases resulting in HA/CSZ (Ce-stabilized zirconia) composites which improves the toughness of the materials widely used in biomedical applications. Samples with compositions 5Zr/5Ce-HA, 5Zr/5Ce-FHA, 40Zr/XCe-FHA (where X = 5, 10 and 20 mol% of Ce ions) were prepared and calcined at 800 °C. Incorporation of Ce ion stabilizes the zirconia, and fluorine ion improves thermal stability. Crystallite size, crystallinity and presence of m-ZrO2 and t-ZrO2 phases were analysed by XRD. The FTIR spectrum confirmed the presence of Zr-O stretching band. SEM reveals agglomerated spherical particles of 50-300 nm size, and ionic doping on HA was analysed through EDAX. The TG/DSC curves confirmed the formation of m-ZrO2 and t-ZrO2 phases and thermal stability of the samples. Higher concentrations of Zr and Ce ions in FHA resulted in reduction in crystallite size. The antibacterial activities of the samples were evaluated against the micro-organisms, Staphylococcus aureus and Escherichia coli exhibiting a inhibition zone diameter as 42 and 37 mm, respectively, for 40Zr/20Ce-FHA.

  2. Species and gender differences in the formation of an active metabolite of a substituted 2,4-thiazolidinedione insulin sensitizer.

    PubMed

    Beconi, M; Mao, A; Creighton, M; Hop, C E C A; Chiu, S H L; Eydelloth, R; Franklin, R; Tang, F; Yu, N; Vincent, S

    2003-07-01

    1. The metabolism of a substituted 2,4-thiazolidinedione (P1) with dual PPARalpha/gamma activity was evaluated in male and female rats, dogs and monkeys. A para-hydroxylated metabolite (M1) with potent PPARgamma-selective agonist, was a major circulating drug-related component in female rats, dogs and monkeys, but not in male rats (M1-to-P1 exposure ratio of <1, 3-5, 5 and 5-11 in male rat, monkey, female rat, and dog, respectively). 2. M1 (%) formed in vitro (5, 53, 57-65, 67 and 67% in male rat, monkey, female rat, dog, and human liver microsomes, respectively), rank ordered with M1 (%) formed in vivo (24-45, 53-57, 78, 75-85%, for male rat, monkey, female rat and dog, respectively, after oral administration of P1). 3. The plasma clearance of M1 was higher in male rats (32 ml min(-1) kg(-1) compared with 6, 7 and 2 ml min(-1) kg(-1) in female rat, male monkey and male dogs, respectively). 4. The low amounts of M1 observed in male rats, with the appearance of products of the cleavage of the propyl group between the phenyl groups was probably due to the presence of the sex-specific CYP2C11, which cleaves P1 at the propyl bridge. None of the CYPs present in female rats cleaved P1 at this site and M1 was only produced by CYP2C6. In humans, only CYP2C8 and the polymorphic CYP2C19 produced M1. PMID:12893525

  3. Synthesis and structure--activity relationships of substituted cinnamic acids and amide analogues: a new class of herbicides.

    PubMed

    Vishnoi, Shipra; Agrawal, Vikash; Kasana, Virendra K

    2009-04-22

    In the present investigation, substituted cinnamic acids (3-hydroxy, 4-hydroxy, 2-nitro, 3-nitro, 4-nitro, 3-chloro, and 4-methoxy) and their amide analogues with four different types of substituted anilines have been synthesized. The synthesized compounds have been screened for their germination inhibition activity on radish (Raphanus sativus L. var. Japanese White) seeds at 50, 100, and 200 ppm concentrations, and the activity was compared with standard herbicide, metribuzin formulation (sencor). Significant activity was exhibited by all of the compounds. It was observed that with the increase in concentration of the test solution, the activity also increased. All of the compounds showed more than 70% inhibition at 100 ppm concentration except 4-hydroxy cinnamanilide. The compound, 2-chloro (4'-hydroxy) cinnamanilide was the best among the tested compounds, and it was found to be at par with the standard, metribuzin at all concentrations. Thus, it can be concluded that substituted cinnamic acids and their amide analogues may be developed as potential herbicides. PMID:19368353

  4. Crystallinity-dependent substitutional nitrogen doping in ZnO and its improved visible light photocatalytic activity.

    PubMed

    Yu, Zongbao; Yin, Li-Chang; Xie, Yingpeng; Liu, Gang; Ma, Xiuliang; Cheng, Hui-Ming

    2013-06-15

    Increasing visible light absorption of wide-bandgap photocatalysts (for example, ZnO and TiO2) plays a pivotal role in improving their photocatalytic activity. In this work, we show that substitutional nitrogen doping can be realized in semi-crystalline zinc oxide (ZnO) nanoparticles but fails for highly crystalline ZnO by heating the ZnO at a temperature of 400°C in gaseous ammonia atmosphere. The results suggest that substitutional nitrogen for lattice oxygen is strongly dependent on the crystallinity of ZnO. The nitrogen doped ZnO obtained shows an improved visible light photocatalytic activity in the degradation of organic dyes due to its increased visible light absorption. The origin of the increased visible light absorption is theoretically attributed to the formed N 2p localized states in the bandgap. PMID:23561822

  5. Computational insight into the structure-activity relationship of novel N-substituted phthalimides with gibberellin-like activity.

    PubMed

    Li, Dongling; Du, Shaoqing; Tan, Weiming; Duan, Hongxia

    2015-10-01

    N-substituted phthalimides (NSPs) that show multiple gibberellin (GA)-like effects on the growth and development of higher plants have been reported. These NSPs may represent a potential alternative to commercial GAs. Therefore, in this work, molecular docking and molecular dynamics simulations were used to explore the mode of interaction between some NSPs and the GA receptor GID1A in order to clarify the relationship between structure and GA-like activity in the NSPs. The results obtained demonstrate that both a multiple-hydrogen-bond network and a "hat-shaped" hydrophobic interaction play important roles in the binding of the NSPs to GID1A. The carbonyl group of a phthalimide fragment in the NSPs acted in a similar manner to the pharmacophore group 6-COOH in GAs, forming multiple-hydrogen-bond interactions with residues Ser191 and Tyr322 in the binding domain of GID1A. Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were used to further study the 3D quantitative structure-activity relationship (3D-QSAR) of the NSPs. It was confirmed that the GA-like activity of these NSPs is strongly linked to a few H-bond donor and acceptor field contributions of the NSPs to the H-bond interactions with GID1A. Five new NSP molecules D1-D5 were designed using the binding domain of GID1A and then docked into the receptor. D1 and D4 were shown to have good docking scores due to enhanced hydrophobic contact. We hope that these results will provide useful guidance in the rational design of new NSPs. PMID:26412055

  6. In Operando Identification of Geometrical-Site-Dependent Water Oxidation Activity of Spinel Co3O4.

    PubMed

    Wang, Hsin-Yi; Hung, Sung-Fu; Chen, Han-Yi; Chan, Ting-Shan; Chen, Hao Ming; Liu, Bin

    2016-01-13

    Spinel Co3O4, comprising two types of cobalt ions: one Co(2+) in the tetrahedral site (Co(2+)(Td)) and the other two Co(3+) in the octahedral site (Co(3+)(Oh)), has been widely explored as a promising oxygen evolution reaction (OER) catalyst for water electrolysis. However, the roles of two geometrical cobalt ions toward the OER have remained elusive. Here, we investigated the geometrical-site-dependent OER activity of Co3O4 catalyst by substituting Co(2+)(Td) and Co(3+)(Oh) with inactive Zn(2+) and Al(3+), respectively. Following a thorough in operando analysis by electrochemical impedance spectroscopy and X-ray absorption spectroscopy, it was revealed that Co(2+)Td site is responsible for the formation of cobalt oxyhydroxide (CoOOH), which acted as the active site for water oxidation. PMID:26710084

  7. EXAFS, XANES, and DFT study of the mixed-valence compound YMn2O5 : Site-selective substitution of Fe for Mn

    NASA Astrophysics Data System (ADS)

    Wunderlich, F.; Leisegang, T.; Weißbach, T.; Zschornak, M.; Stöcker, H.; Dshemuchadse, J.; Lubk, A.; Führlich, T.; Welter, E.; Souptel, D.; Gemming, S.; Seifert, G.; Meyer, D. C.

    2010-07-01

    In YMn2O5 , the Mn atoms occupy two nonequivalent Wyckoff sites within the unit cell exhibiting different oxygen coordinations, i.e., the system can be characterized as a mixed-valence compound. For the formation of the orthorhombic crystal structure, Jahn-Teller distortions are assumed to play an important role. In this study, we aimed at the investigation of the crystal structure changes upon the substitution of Mn by the non-Jahn-Teller cation Fe3+ . Therefore, we synthesized a series of YMn2-xFexO5 powder samples with x=0 , 0.5, and 1 by a citrate technique. We utilized extended x-ray absorption fine structure (EXAFS) and x-ray absorption near-edge structure (XANES) analysis as well as density-functional theory (DFT) to investigate the two nonequivalent Wyckoff sites within the orthorhombic crystal structure (confirmed for all compositions) occupied by transition-metal atoms. For quantitative determination of structural short-range order, all plausible options of substitution of Fe for Mn are discussed. On the basis of these evaluations, the EXAFS and XANES behavior is analyzed and appropriate crystallographic weights are assigned to the subset of structural models in accordance with the experimental data. From EXAFS analysis, using multiple-scattering theory, we conclude only the 4h Wyckoff site to be occupied by Fe [occupancy refined is (100±3)% in case of x=1 ]. Furthermore, taking the XANES spectra into account, we are able to verify the EXAFS results and additionally explain the differences in the MnK XANES spectra in dependence on x to be caused by changes in the dipole transitions to 4p final states. From quantitative pre-edge analysis an oxidation number of +4 for the Mn atom for x=1 is determined whereas the Fe valence is shown to be unchanged. Since the substitution process only involves one Wyckoff site, the experimentally observed limit to a maximum amount of x=1 is explained. Additionally, a possible disorder, discussed in the literature, is not

  8. 4-Substituted boro-proline dipeptides: synthesis, characterization, and dipeptidyl peptidase IV, 8, and 9 activities.

    PubMed

    Wu, Wengen; Liu, Yuxin; Milo, Lawrence J; Shu, Ying; Zhao, Peng; Li, Youhua; Woznica, Iwona; Yu, Gengli; Sanford, David G; Zhou, Yuhong; Poplawski, Sarah E; Connolly, Beth A; Sudmeier, James L; Bachovchin, William W; Lai, Jack H

    2012-09-01

    The boroProline-based dipeptidyl boronic acids were among the first DPP-IV inhibitors identified, and remain the most potent known. We introduced various substitutions at the 4-position of the boroProline ring regioselectively and stereoselectively, and incorporated these aminoboronic acids into a series of 4-substituted boroPro-based dipeptides. Among these dipeptidyl boronic acids, Arg-(4S)-boroHyp (4q) was the most potent inhibitor of DPP-IV, DPP8 and DPP9, while (4S)-Hyp-(4R)-boroHyp (4o) exhibited the most selectivity for DPP-IV over DPP8 and DPP9. PMID:22853995

  9. Design and synthesis of a novel, orally active, brain penetrant, tri-substituted thiophene based JNK inhibitor

    SciTech Connect

    Bowers, Simeon; Truong, Anh P.; Neitz, R. Jeffrey; Neitzel, Martin; Probst, Gary D.; Hom, Roy K.; Peterson, Brian; Galemmo, Jr., Robert A.; Konradi, Andrei W.; Sham, Hing L.; Tóth, Gergley; Pan, Hu; Yao, Nanhua; Artis, Dean R.; Brigham, Elizabeth F.; Quinn, Kevin P.; Sauer, John-Michael; Powell, Kyle; Ruslim, Lany; Ren, Zhao; Bard, Frédérique; Yednock, Ted A.; Griswold-Prenner, Irene

    2012-02-28

    The SAR of a series of tri-substituted thiophene JNK3 inhibitors is described. By optimizing both the N-aryl acetamide region of the inhibitor and the 4-position of the thiophene we obtained single digit nanomolar compounds, such as 47, which demonstrated an in vivo effect on JNK activity when dosed orally in our kainic acid mouse model as measured by phospho-c-jun reduction.

  10. Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity

    PubMed Central

    Hartmann, Jens; Linke, Dennis; Bönniger, Christine; Tholey, Andreas; Sauter, Margret

    2015-01-01

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser696 and Ser698 in the JM (juxtamembrane) region and probably Ser886 and/or Ser893 in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser717 in the JM, and at Ser733, Thr752, Ser783, Ser864, Ser911, Ser958 and Thr998 in the kinase domain. The LC–ESI–MS/MS spectra provided support that up to three sites (Thr890, Ser893 and Thr894) in the AL were likely to be phosphorylated in vitro. These sites are evolutionarily highly conserved in PSK receptors, indicative of a conserved function. Site-directed mutagenesis of the four conserved residues in the activation segment, Thr890, Ser893, Thr894 and Thr899, differentially altered kinase activity in vitro and growth-promoting activity in planta. The T899A and the quadruple-mutated TSTT-A (T890A/S893A/T894A/T899A) mutants were both kinase-inactive, but PSKR1(T899A) retained growth-promoting activity. The T890A and S893A/T894A substitutions diminished kinase activity and growth promotion. We hypothesize that phosphorylation within the AL activates kinase activity and receptor function in a gradual and distinctive manner that may be a means to modulate the PSK response. PMID:26472115

  11. Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity.

    PubMed

    Hartmann, Jens; Linke, Dennis; Bönniger, Christine; Tholey, Andreas; Sauter, Margret

    2015-12-15

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser(696) and Ser(698) in the JM (juxtamembrane) region and probably Ser(886) and/or Ser(893) in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser(717) in the JM, and at Ser(733), Thr(752), Ser(783), Ser(864), Ser(911), Ser(958) and Thr(998) in the kinase domain. The LC-ESI-MS/MS spectra provided support that up to three sites (Thr(890), Ser(893) and Thr(894)) in the AL were likely to be phosphorylated in vitro. These sites are evolutionarily highly conserved in PSK receptors, indicative of a conserved function. Site-directed mutagenesis of the four conserved residues in the activation segment, Thr(890), Ser(893), Thr(894) and Thr(899), differentially altered kinase activity in vitro and growth-promoting activity in planta. The T899A and the quadruple-mutated TSTT-A (T890A/S893A/T894A/T899A) mutants were both kinase-inactive, but PSKR1(T899A) retained growth-promoting activity. The T890A and S893A/T894A substitutions diminished kinase activity and growth promotion. We hypothesize that phosphorylation within the AL activates kinase activity and receptor function in a gradual and distinctive manner that may be a means to modulate the PSK response. PMID:26472115

  12. In vitro biological activities of a series of 2 beta-substituted analogues of 1 alpha,25-dihydroxyvitamin D3.

    PubMed

    Tsugawa, N; Nakagawa, K; Kurobe, M; Ono, Y; Kubodera, N; Ozono, K; Okano, T

    2000-01-01

    Biological activities of a series of 2beta-substituted analogues of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] were evaluated in vitro in terms of their binding affinity with regard to calf thymus cytosolic vitamin D receptor (VDR) and rat plasma vitamin D-binding protein (DBP). Additionally, reporter gene luciferase activities using either a rat 25-hydroxyvitamin D3-24-hydroxylase gene promoter, including two vitamin D-responsive elements (VDREs), in transfected rat osteoblast-like ROS17/2.8 cells, or a human VDR-GAL4 modified two-hybrid system in transfected human epitheloid carcinoma, cervix HeLa cells were examined. Binding affinity for VDR, transactivation potency on the target gene and VDR-mediated gene regulation of the hydroxyalkyl and hydroxyalkoxy 2beta-substituted analogues were almost comparable to those of 1alpha,25(OH)2D3, while the alkyl and alkenyl analogues were much less active than 1alpha,25(OH)2D3. This study investigated the biological evaluation of a series of 2beta-substituted analogues at the molecular level, with regard to the structural differences of alkyl, alkenyl, hydroxyalkyl, hydroxyalkoxy, alkoxy, hydroxy and chloro substituents at the 2beta-position of 1alpha,25(OH)2D3. PMID:10706413

  13. Trp-262 is a key residue for the hydrolytic and transglucosidic reactivity of the Aspergillus niger family 3 beta-glucosidase: substitution results in enzymes with mainly transglucosidic activity.

    PubMed

    Seidle, Heather F; McKenzie, Kyle; Marten, Ira; Shoseyov, Oded; Huber, Reuben E

    2005-12-01

    Trp-262 of the Aspergillus niger family 3 beta-glucosidase is shown in this report to be a key residue for determining the ratio of this enzyme's hydrolytic and transglucosidic activities. TLC showed that when cellobiose was both the substrate and the acceptor, beta-glucosidases with substitutions (Phe, Ala, Leu, and Cys) for Trp-262 formed very high amounts of transglucosidic adducts. When pNPGlc was the substrate and the acceptor of the substituted beta-glucosidases, only transglucosidic adducts and pNP were produced. Little or no Glc could be detected, indicating that the reactions occurring were mainly transglucosidic. GLC studies with cellobiose quantitatively showed that one Glc was transferred for each free Glc produced. Since this is the maximum level of transglucosidation possible, this again showed that the reaction is predominantly transglucosidic. Analyses of the K(m) and K(i) values of cello-oligosaccharides of increasing length, of the K(i) values of Glc and of the transglucosidic activity at low acceptor concentration, showed that substitution for Trp-262 causes poor binding at the binding site for the non-reducing Glc of the substrate while the affinity for other Glc units is only minimally affected. The acceptor sites become saturated with substrate (acceptor) at the concentrations needed for glucosidic bond cleavage and thus only transglucosidic reactions occur. In addition, the data indicate that substitution for Trp-262 causes the rate of the hydrolysis step (k(3)) to be small. PMID:16274659

  14. Active Sites Environmental Monitoring Program FY 1996 annual report

    SciTech Connect

    Morrissey, C.M.; Marshall, D.S.; Cunningham, G.R.

    1997-11-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) from October 1995 through September 1996. The Radioactive Solid Waste Operations Group (RSWOG) of the Waste Management and Remedial Action Division (WMRAD) and the Environmental Sciences Division (ESD) at Oak Ridge National Laboratory (ORNL) established ASEMP in 1989. The purpose of the program is to provide early detection and performance monitoring at active low-level waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 North as required by Chapters 2 and 3 of US Department of Energy Order 5820.2A.

  15. Active sites environmental monitoring Program - Program Plan: Revision 2

    SciTech Connect

    Morrissey, C.M.; Hicks, D.S.; Ashwood, T.L.; Cunningham, G.R.

    1994-05-01

    The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of active low-level-waste (LLW) and transuranic (TRU) waste facilities at Oak Ridge National Laboratory (ORNL). Several changes have recently occurred in regard to the sites that are currently used for waste storage and disposal. These changes require a second set of revisions to the ASEMP program plan. This document incorporates those revisions. This program plan presents the organization and procedures for monitoring the active sites. The program plan also provides internal reporting levels to guide the evaluation of monitoring results.

  16. Structural Characterization of Human 8-Oxoguanine DNA Glycosylase Variants Bearing Active Site Mutations

    SciTech Connect

    Radom,C.; Banerjee, A.; Verdine, G.

    2007-01-01

    The human 8-oxoguanine DNA glycosylase (hOGG1) protein is responsible for initiating base excision DNA repair of the endogenous mutagen 8-oxoguanine. Like nearly all DNA glycosylases, hOGG1 extrudes its substrate from the DNA helix and inserts it into an extrahelical enzyme active site pocket lined with residues that participate in lesion recognition and catalysis. Structural analysis has been performed on mutant versions of hOGG1 having changes in catalytic residues but not on variants having altered 7,8-dihydro-8-oxoguanine (oxoG) contact residues. Here we report high resolution structural analysis of such recognition variants. We found that Ala substitution at residues that contact the phosphate 5 to the lesion (H270A mutation) and its Watson-Crick face (Q315A mutation) simply removed key functionality from the contact interface but otherwise had no effect on structure. Ala substitution at the only residue making an oxoG-specific contact (G42A mutation) introduced torsional stress into the DNA contact surface of hOGG1, but this was overcome by local interactions within the folded protein, indicating that this oxoG recognition motif is 'hardwired'. Introduction of a side chain intended to sterically obstruct the active site pocket (Q315F mutation) led to two different structures, one of which (Q315F{sup *149}) has the oxoG lesion in an exosite flanking the active site and the other of which (Q315F{sup *292}) has the oxoG inserted nearly completely into the lesion recognition pocket. The latter structure offers a view of the latest stage in the base extrusion pathway yet observed, and its lack of catalytic activity demonstrates that the transition state for displacement of the lesion base is geometrically demanding.

  17. Modulating the pH-activity profile of cellulase by substitution: replacing the general base catalyst aspartate with cysteinesulfinate in cellulase A from Cellulomonas fimi.

    PubMed

    Cockburn, Darrell W; Vandenende, Chris; Clarke, Anthony J

    2010-03-01

    Cellulase A (CenA) from Cellulomonas fimi is an inverting glycoside hydrolase and a member of family 6 of the CAZy database classification system. We replaced its putative catalytic base aspartyl residues, Aps392 and Asp216, with cysteinesulfinate using a combination of site-directed mutagenesis and chemical modification to investigate the applicability of this approach for the modulation of enzymatic properties. The substituted cysteinyl residues were oxidized to cysteinesulfinic acid with hydrogen peroxide, and the resulting protein products were demonstrated to retain their native structure. Oxidation of the Asp392Cys mutant enzyme restored 52% of wild-type activity when assessed at pH 7.5, whereas Asp216Cys CenA remained inactive. This suggests that Asp216 is not the catalytic base and provides further support for Asp392 performing this role. Similar substitution of the catalytic acid residue Asp252 or the catalytic nucleophile of the retaining enzyme Cel5A from Thermobifida fusca failed to produce active enzymes. This indicates a potential utility of this approach for uniquely identifying catalytic base residues. The replacement of Asp392 with cysteinesulfinate induced an acidic shift in the pH profile of the enzyme such that this enzyme derivative was more active than wild-type CenA below pH 5.5. These data demonstrate the potential of combining site-directed mutagenesis with chemical modification as a viable approach for the modulation of cellulases, and potentially other glycoside hydrolases, at low pH. PMID:20136145

  18. The active site behaviour of electrochemically synthesised gold nanomaterials.

    PubMed

    Plowman, Blake J; O'Mullane, Anthony P; Bhargava, Suresh K

    2011-01-01

    Even though gold is the noblest of metals, a weak chemisorber and is regarded as being quite inert, it demonstrates significant electrocatalytic activity in its nanostructured form. It is demonstrated here that nanostructured and even evaporated thin films of gold are covered with active sites which are responsible for such activity. The identification of these sites is demonstrated with conventional electrochemical techniques such as cyclic voltammetry as well as a large amplitude Fourier transformed alternating current (FT-ac) method under acidic and alkaline conditions. The latter technique is beneficial in determining if an electrode process is either Faradaic or capacitive in nature. The observed behaviour is analogous to that observed for activated gold electrodes whose surfaces have been severely disrupted by cathodic polarisation in the hydrogen evolution region. It is shown that significant electrochemical oxidation responses occur at discrete potential values well below that for the formation of the compact monolayer oxide of bulk gold and are attributed to the facile oxidation of surface active sites. Several electrocatalytic reactions are explored in which the onset potential is determined by the presence of such sites on the surface. Significantly, the facile oxidation of active sites is used to drive the electroless deposition of metals such as platinum, palladium and silver from their aqueous salts on the surface of gold nanostructures. The resultant surface decoration of gold with secondary metal nanoparticles not only indicates regions on the surface which are rich in active sites but also provides a method to form interesting bimetallic surfaces. PMID:22455038

  19. Nicotinamide Cofactors Suppress Active-Site Labeling of Aldehyde Dehydrogenases.

    PubMed

    Stiti, Naim; Chandrasekar, Balakumaran; Strubl, Laura; Mohammed, Shabaz; Bartels, Dorothea; van der Hoorn, Renier A L

    2016-06-17

    Active site labeling by (re)activity-based probes is a powerful chemical proteomic tool to globally map active sites in native proteomes without using substrates. Active site labeling is usually taken as a readout for the active state of the enzyme because labeling reflects the availability and reactivity of active sites, which are hallmarks for enzyme activities. Here, we show that this relationship holds tightly, but we also reveal an important exception to this rule. Labeling of Arabidopsis ALDH3H1 with a chloroacetamide probe occurs at the catalytic Cys, and labeling is suppressed upon nitrosylation and oxidation, and upon treatment with other Cys modifiers. These experiments display a consistent and strong correlation between active site labeling and enzymatic activity. Surprisingly, however, labeling is suppressed by the cofactor NAD(+), and this property is shared with other members of the ALDH superfamily and also detected for unrelated GAPDH enzymes with an unrelated hydantoin-based probe in crude extracts of plant cell cultures. Suppression requires cofactor binding to its binding pocket. Labeling is also suppressed by ALDH modulators that bind at the substrate entrance tunnel, confirming that labeling occurs through the substrate-binding cavity. Our data indicate that cofactor binding adjusts the catalytic Cys into a conformation that reduces the reactivity toward chloroacetamide probes. PMID:26990764

  20. Conformational Change in the Active Site of Streptococcal Unsaturated Glucuronyl Hydrolase Through Site-Directed Mutagenesis at Asp-115.

    PubMed

    Nakamichi, Yusuke; Oiki, Sayoko; Mikami, Bunzo; Murata, Kousaku; Hashimoto, Wataru

    2016-08-01

    Bacterial unsaturated glucuronyl hydrolase (UGL) degrades unsaturated disaccharides generated from mammalian extracellular matrices, glycosaminoglycans, by polysaccharide lyases. Two Asp residues, Asp-115 and Asp-175 of Streptococcus agalactiae UGL (SagUGL), are completely conserved in other bacterial UGLs, one of which (Asp-175 of SagUGL) acts as a general acid and base catalyst. The other Asp (Asp-115 of SagUGL) also affects the enzyme activity, although its role in the enzyme reaction has not been well understood. Here, we show substitution of Asp-115 in SagUGL with Asn caused a conformational change in the active site. Tertiary structures of SagUGL mutants D115N and D115N/K370S with negligible enzyme activity were determined at 2.00 and 1.79 Å resolution, respectively, by X-ray crystallography. The side chain of Asn-115 is drastically shifted in both mutants owing to the interaction with several residues, including Asp-175, by formation of hydrogen bonds. This interaction between Asn-115 and Asp-175 probably prevents the mutants from triggering the enzyme reaction using Asp-175 as an acid catalyst. PMID:27402448

  1. Photoluminescence of A- and B-site Eu3+-substituted (SrxBa1-x)2CaWyMo1-yO6 phosphors

    NASA Astrophysics Data System (ADS)

    Sletnes, M.; Lindgren, M.; Valmalette, J. C.; Wagner, N. P.; Grande, T.; Einarsrud, M.-A.

    2016-05-01

    The photoluminescence of two series of A- and B-site Eu3+ substituted (SrxBa1-x)2CaWyMo1-yO6 double perovskite phosphor materials, (SrxBa1-x)1.96Eu0.02K0.02CaWyMo1-yO6 and (SrxBa1-x)2Ca0.96Eu0.02Li0.02WyMo1-yO6 (x and y=0, 0.25, 0.50, 0.75, and 1), were studied systematically as a function of stoichiometry and crystal structure. The Eu3+ lattice sites controlled by co-doping with either K or Li were confirmed by Raman spectroscopy. The variation in integrated emission intensity and emission colour over the experimental matrix was examined using statistical tools, and the observed trends were rationalized based on the physical and electronic structure of the phosphors. Phosphors with Eu on B-site with maximum Sr content had remarkably higher emission intensities than all other materials, but the emission was more orange than red due to domination of the 5D0-7F1 (595 nm) transition of Eu3+. The relative intensities of the 5D0-7F2 (615 nm) and 5D0-7F1 transitions of Eu3+, and thus the red-shift of the emission, decreased linearly with increasing Sr content in the A-site Eu-substituted phosphors, and reached a maximum for Sr1.96Eu0.02K0.02CaW0.25Mo0.75O6. A maximum external quantum efficiency of 17% was obtained for the phosphor Sr2Ca0.7Eu0.15Li0.15W0.5Mo0.5O6 with Eu on B-site.

  2. Active site - a site of binding of affinity inhibitors in baker's yeast inorganic pyrophosphatase

    SciTech Connect

    Svyato, I.E.; Sklyankina, V.A.; Avaeva, S.M.

    1986-03-20

    The interaction of the enzyme-substrate complex with methyl phosphate, O-phosphoethanolamine, O-phosphopropanolamine, N-acetylphosphoserine, and phosphoglyolic acid, as well as pyrophosphatase, modified by monoesters of phosphoric acid, with pyrophosphate and tripolyphosphate, was investigated. It was shown that the enzyme containing the substrate in the active site does not react with monophosphates, but modified pyrophosphatase entirely retains the ability to bind polyanions to the regulatory site. It is concluded that the inactivation of baker's yeast inorganic pyrophosphatase by monoesters of phosphoric acid, which are affinity inhibitors of it, is the result of modification of the active site of the enzyme.

  3. Improving the activity of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation.

    PubMed

    Weng, Meizhi; Deng, Xiongwei; Bao, Wei; Zhu, Li; Wu, Jieyuan; Cai, Yongjun; Jia, Yan; Zheng, Zhongliang; Zou, Guolin

    2015-09-25

    Nattokinase (NK), a bacterial serine protease from Bacillus subtilis var. natto, is a potential cardiovascular drug exhibiting strong fibrinolytic activity. To broaden its commercial and medical applications, we constructed a single-mutant (I31L) and two double-mutants (M222A/I31L and T220S/I31L) by site-directed mutagenesis. Active enzymes were expressed in Escherichia coli with periplasmic secretion and were purified to homogeneity. The kinetic parameters of enzymes were examined by spectroscopy assay and isothermal titration calorimetry (ITC), and their fibrinolytic activities were determined by fibrin plate method. The substitution of Leu(31) for Ile(31) resulted in about 2-fold enhancement of catalytic efficiency (Kcat/KM) compared with wild-type NK. The specific activities of both double-mutants (M222A/I31L and T220S/I31L) were significantly increased when compared with the single-mutants (M222A and T220S) and the oxidative stability of M222A/I31L mutant was enhanced with respect to wild-type NK. This study demonstrates the feasibility of improving activity of NK by site-directed mutagenesis and shows successful protein engineering cases to improve the activity of NK as a potent therapeutic agent. PMID:26291268

  4. Ligand substitutions between ruthenium–cymene compounds can control protein versus DNA targeting and anticancer activity

    PubMed Central

    Adhireksan, Zenita; Davey, Gabriela E.; Campomanes, Pablo; Groessl, Michael; Clavel, Catherine M.; Yu, Haojie; Nazarov, Alexey A.; Yeo, Charmian Hui Fang; Ang, Wee Han; Dröge, Peter; Rothlisberger, Ursula; Dyson, Paul J.; Davey, Curt A.

    2014-01-01

    Ruthenium compounds have become promising alternatives to platinum drugs by displaying specific activities against different cancers and favourable toxicity and clearance properties. Nonetheless, their molecular targeting and mechanism of action are poorly understood. Here we study two prototypical ruthenium-arene agents—the cytotoxic antiprimary tumour compound [(η6-p-cymene)Ru(ethylene-diamine)Cl]PF6 and the relatively non-cytotoxic antimetastasis compound [(η6-p-cymene)Ru(1,3,5-triaza-7-phosphaadamantane)Cl2]—and discover that the former targets the DNA of chromatin, while the latter preferentially forms adducts on the histone proteins. Using a novel ‘atom-to-cell’ approach, we establish the basis for the surprisingly site-selective adduct formation behaviour and distinct cellular impact of these two chemically similar anticancer agents, which suggests that the cytotoxic effects arise largely from DNA lesions, whereas the protein adducts may be linked to the other therapeutic activities. Our study shows promise for developing new ruthenium drugs, via ligand-based modulation of DNA versus protein binding and thus cytotoxic potential, to target distinguishing epigenetic features of cancer cells. PMID:24637564

  5. Eight Nucleotide Substitutions Inhibit Splicing to HPV-16 3′-Splice Site SA3358 and Reduce the Efficiency by which HPV-16 Increases the Life Span of Primary Human Keratinocytes

    PubMed Central

    Li, Xiaoze; Johansson, Cecilia; Cardoso Palacios, Carlos; Mossberg, Anki; Dhanjal, Soniya; Bergvall, Monika; Schwartz, Stefan

    2013-01-01

    The most commonly used 3′-splice site on the human papillomavirus type 16 (HPV-16) genome named SA3358 is used to produce HPV-16 early mRNAs encoding E4, E5, E6 and E7, and late mRNAs encoding L1 and L2. We have previously shown that SA3358 is suboptimal and is totally dependent on a downstream splicing enhancer containingmultiple potential ASF/SF2 binding sites. Here weshow that only one of the predicted ASF/SF2 sites accounts for the majority of the enhancer activity. We demonstrate that single nucleotide substitutions in this predicted ASF/SF2 site impair enhancer function and that this correlates with less efficient binding to ASF/SF2 in vitro. We provide evidence that HPV-16 mRNAs that arespliced to SA3358 interact with ASF/SF2 in living cells. In addition,mutational inactivation of the ASF/SF2 site weakened the enhancer at SA3358 in episomal forms of the HPV-16 genome, indicating that the enhancer is active in the context of the full HPV-16 genome.This resulted in induction of HPV-16 late gene expression as a result of competition from late splice site SA5639. Furthermore, inactivation of the ASF/SF2 site of the SA3358 splicing enhancer reduced the ability of E6- and E7-encoding HPV-16 plasmids to increase the life span of primary keratinocytes in vitro, demonstrating arequirement for an intact splicing enhancer of SA3358 forefficient production of the E6 and E7 mRNAs. These results link the strength of the HPV-16 SA3358 splicing enhancer to expression of E6 and E7 and to the pathogenic properties of HPV-16. PMID:24039800

  6. Eight nucleotide substitutions inhibit splicing to HPV-16 3'-splice site SA3358 and reduce the efficiency by which HPV-16 increases the life span of primary human keratinocytes.

    PubMed

    Li, Xiaoze; Johansson, Cecilia; Cardoso Palacios, Carlos; Mossberg, Anki; Dhanjal, Soniya; Bergvall, Monika; Schwartz, Stefan

    2013-01-01

    The most commonly used 3'-splice site on the human papillomavirus type 16 (HPV-16) genome named SA3358 is used to produce HPV-16 early mRNAs encoding E4, E5, E6 and E7, and late mRNAs encoding L1 and L2. We have previously shown that SA3358 is suboptimal and is totally dependent on a downstream splicing enhancer containingmultiple potential ASF/SF2 binding sites. Here weshow that only one of the predicted ASF/SF2 sites accounts for the majority of the enhancer activity. We demonstrate that single nucleotide substitutions in this predicted ASF/SF2 site impair enhancer function and that this correlates with less efficient binding to ASF/SF2 in vitro. We provide evidence that HPV-16 mRNAs that arespliced to SA3358 interact with ASF/SF2 in living cells. In addition,mutational inactivation of the ASF/SF2 site weakened the enhancer at SA3358 in episomal forms of the HPV-16 genome, indicating that the enhancer is active in the context of the full HPV-16 genome.This resulted in induction of HPV-16 late gene expression as a result of competition from late splice site SA5639. Furthermore, inactivation of the ASF/SF2 site of the SA3358 splicing enhancer reduced the ability of E6- and E7-encoding HPV-16 plasmids to increase the life span of primary keratinocytes in vitro, demonstrating arequirement for an intact splicing enhancer of SA3358 forefficient production of the E6 and E7 mRNAs. These results link the strength of the HPV-16 SA3358 splicing enhancer to expression of E6 and E7 and to the pathogenic properties of HPV-16. PMID:24039800

  7. A small ribozyme with dual-site kinase activity

    PubMed Central

    Biondi, Elisa; Maxwell, Adam W.R.; Burke, Donald H.

    2012-01-01

    Phosphoryl transfer onto backbone hydroxyls is a recognized catalytic activity of nucleic acids. We find that kinase ribozyme K28 possesses an unusually complex active site that promotes (thio)phosphorylation of two residues widely separated in primary sequence. After allowing the ribozyme to radiolabel itself by phosphoryl transfer from [γ-32P]GTP, DNAzyme-mediated cleavage yielded two radiolabeled cleavage fragments, indicating phosphorylation sites within each of the two cleavage fragments. These sites were mapped by alkaline digestion and primer extension pausing. Enzymatic digestion and mutational analysis identified nucleotides important for activity and established the active structure as being a constrained pseudoknot with unusual connectivity that may juxtapose the two reactive sites. Nuclease sensitivities for nucleotides near the pseudoknot core were altered in the presence of GTPγS, indicating donor-induced folding. The 5′ target site was more strongly favored in full-length ribozyme K28 (128 nt) than in truncated RNAs (58 nt). Electrophoretic mobilities of self-thiophosphorylated products on organomercurial gels are distinct from the 5′ mono-thiophosphorylated product produced by reaction with polynucleotide kinase, potentially indicating simultaneous labeling of both sites within individual RNA strands. Our evidence supports a single, compact structure with local dynamics, rather than global rearrangement, as being responsible for dual-site phosphorylation. PMID:22618879

  8. Tuning the geometry and biomimetic catalytic activity of manganese(III)-tetrabromocatecholate based robust platforms by introducing substitution at pyridine.

    PubMed

    Jana, Narayan Ch; Brandão, Paula; Panja, Anangamohan

    2016-06-01

    The present report describes synthesis, characterization, crystal structures and catecholase activity of a series of five new manganese(III) complexes (1-5) derived from redox-noninnocent tetrabromocatecholate ligand in combination with different substituted pyridines. X-ray crystallography reveals that the geometry of manganese(III) centers in 1 and 2 is square pyramidal and they are pseudo-dimeric in the solid state resulting from the weak bonding of manganese(III) with a catecholate oxygen atom from the adjacent manganese(III) unit together with other weak interactions like hydrogen bonding and π⋯π stacking interactions. On the other hand, complexes 3-5 are discrete octahedral structures. All the complexes exhibit strong catecholase activity and their diverse catalytic activity can nicely be explained by the nature of substitution at pyridine ring - better electron donor inhibits the reduction of the metal center thereby lowering catecholase activity and vice versa (1 and 2 vs. 3-5). Besides the donor property of ancillary ligands, the structural distortion has also significant role in the biomimetic catalytic activity (1 vs. 2). PMID:26970727

  9. In vitro modification of substituted cysteines as tool to study receptor functionality and structure-activity relationships.

    PubMed

    Rathmann, Daniel; Pedragosa-Badia, Xavier; Beck-Sickinger, Annette G

    2013-08-15

    Mutagenic investigations of expressed membrane proteins are routine, but the variety of modifications is limited by the twenty canonical amino acids. We describe an easy and effective cysteine substitution mutagenesis method to modify and investigate distinct amino acids in vitro. The approach combines the substituted cysteine accessibility method (SCAM) with a functional signal transduction readout system using different thiol-specific reagents. We applied this approach to the prolactin-releasing peptide receptor (PrRPR) to facilitate biochemical structure-activity relationship studies of eight crucial positions. Especially for D(6.59)C, the treatment with the positively charged methanethiosulfonate (MTS) ethylammonium led to an induced basal activity, whereas the coupling of the negatively charged MTS ethylsulfonate nearly reconstituted full activity, obviously by mimicking the wild-type charged side chain. At E(5.26)C, W(5.28)C, Y(5.38)C, and Q(7.35)C, accessibility was observed but hindered transfer into the active receptor conformation. Accordingly, the combination of SCAM and signaling assay is feasible and can be adapted to other G-protein-coupled receptors (GPCRs). This method circumvents the laborious way of inserting non-proteinogenic amino acids to investigate activity and ligand binding, with rising numbers of MTS reagents allowing selective side chain modification. This method pinpoints to residues being accessible but also presents potential molecular positions to investigate the global conformation. PMID:23624320

  10. Key substrate recognition residues in the active site of a plant cytochrome P450, CYP73A1. Homology guided site-directed mutagenesis.

    PubMed

    Schoch, Guillaume A; Attias, Roger; Le Ret, Monique; Werck-Reichhart, Danièle

    2003-09-01

    CYP73 enzymes are highly conserved cytochromes P450 in plant species that catalyse the regiospecific 4-hydroxylation of cinnamic acid to form precursors of lignin and many other phenolic compounds. A CYP73A1 homology model based on P450 experimentally solved structures was used to identify active site residues likely to govern substrate binding and regio-specific catalysis. The functional significance of these residues was assessed using site-directed mutagenesis. Active site modelling predicted that N302 and I371 form a hydrogen bond and hydrophobic contacts with the anionic site or aromatic ring of the substrate. Modification of these residues led to a drastic decrease in substrate binding and metabolism without major perturbation of protein structure. Changes to residue K484, which is located too far in the active site model to form a direct contact with cinnamic acid in the oxidized enzyme, did not influence initial substrate binding. However, the K484M substitution led to a 50% loss in catalytic activity. K484 may affect positioning of the substrate in the reduced enzyme during the catalytic cycle, or product release. Catalytic analysis of the mutants with structural analogues of cinnamic acid, in particular indole-2-carboxylic acid that can be hydroxylated with different regioselectivities, supports the involvement of N302, I371 and K484 in substrate docking and orientation. PMID:12950252

  11. Structure/function correlations over binuclear non-heme iron active sites.

    PubMed

    Solomon, Edward I; Park, Kiyoung

    2016-09-01

    Binuclear non-heme iron enzymes activate O2 to perform diverse chemistries. Three different structural mechanisms of O2 binding to a coupled binuclear iron site have been identified utilizing variable-temperature, variable-field magnetic circular dichroism spectroscopy (VTVH MCD). For the μ-OH-bridged Fe(II)2 site in hemerythrin, O2 binds terminally to a five-coordinate Fe(II) center as hydroperoxide with the proton deriving from the μ-OH bridge and the second electron transferring through the resulting μ-oxo superexchange pathway from the second coordinatively saturated Fe(II) center in a proton-coupled electron transfer process. For carboxylate-only-bridged Fe(II)2 sites, O2 binding as a bridged peroxide requires both Fe(II) centers to be coordinatively unsaturated and has good frontier orbital overlap with the two orthogonal O2 π* orbitals to form peroxo-bridged Fe(III)2 intermediates. Alternatively, carboxylate-only-bridged Fe(II)2 sites with only a single open coordination position on an Fe(II) enable the one-electron formation of Fe(III)-O2 (-) or Fe(III)-NO(-) species. Finally, for the peroxo-bridged Fe(III)2 intermediates, further activation is necessary for their reactivities in one-electron reduction and electrophilic aromatic substitution, and a strategy consistent with existing spectral data is discussed. PMID:27369780

  12. Tissue reactions to particles of bone-substitute materials in intraosseous and heterotopic sites in rats: discrimination of osteoinduction, osteocompatibility, and inflammation.

    PubMed

    Eid, K; Zelicof, S; Perona, B P; Sledge, C B; Glowacki, J

    2001-09-01

    Two rat models were used to characterize tissue-specific reactions to particles of bone-substitute materials: one for osteocompatibility in a healing tibial wound and the other in a heterotopic, subcutaneous site. Small, unicortical tibial wounds in rats healed spontaneously, beginning with the rapid proliferation of intramedullary woven bone. That temporary bone was resorbed by osteoclasts and finally, the cortical wound was healed with lamellar bone and the medullary space was repopulated with marrow. When various particulate materials were implanted into fresh wounds, three types of reactions were observed. (1) Demineralized bone powder (DBP) and non-resorbable calcium phosphate (nrCP) were incorporated into the reactive medullary and cortical bone. (2) Polymethylmetlhacrylate (PMMA) particles were surrounded with a fibrous layer, but did not impair bone healing. (3) Polyethylene (PE) shards and resorbable calcium phosphates (rCPs) were inflammatory and inhibited osseous repair. Subcutaneous sites showed osteoinductive, fibrotic, or inflammatory responses to these materials. Only DBP induced endochondral osteogenesis subcutaneously. The nrCP evoked a fibrous reaction. In contrast, rCPs, PMMA, and PE shards generated inflammatory reactions with each particle being surrounded by fibrous tissue and large multinucleated giant cells. In conclusion, only DBP showed osteoinductive as well as osteocompatible properties. The nrCP was osteocompatible. The rCPs stimulated various degrees of inflammatory responses. PMMA was osteocompatible and did not interfere with the bone healing process. PE was not osteocompatible and generated foreign body reactions in both sites. Use of the two sites distinguishes osteoinductive, osteocompatible, and inflammatory properties of particles of bone-substitute materials. PMID:11562148

  13. Synthesis, anti-inflammatory, analgesic, COX-1/2 inhibitory activities and molecular docking studies of substituted 2-mercapto-4(3H)-quinazolinones.

    PubMed

    Abdel-Aziz, Alaa A-M; Abou-Zeid, Laila A; ElTahir, Kamal Eldin H; Ayyad, Rezk R; El-Sayed, Magda A-A; El-Azab, Adel S

    2016-10-01

    A new series of 2-substituted mercapto-4(3H)-quinazolinone 1-26 were synthesized and assessed for in vivo anti-inflammatory and analgesic activities and in vitro inhibition of cyclooxygenase COX-1/COX-2. A new series of 2-substituted mercapto-4(3H)-quinazolinone 1-26 were synthesized and assessed for in vivo anti-inflammatory and analgesic activities. The potent anti-inflammatory compounds were subjected to in vitro cyclooxygenase COX-1/COX-2 inhibition assays. Compounds 1, 3, 5, 11, 12, 13, 15, 17, and 25 exhibited potent anti-inflammatory effects, with half-maximal effective dose (ED50) values of 65.7-102.4 mg/kg, (0.16-0.36 mmol/kg), and strong analgesic activities, with ED50 values of 33.3-104.6 mg/kg, (0.07-0.34 mmol/kg). These values were compared with those of diclofenac sodium [ED50 values: 112.2 and 100.4 mg/kg, (0.35 and 0.31 mmol/kg)], and celecoxib [ED50 values: 84.3 and 71.6 mg/kg (0.22 and 0.19 mmol/kg)], respectively as reference drugs. Compounds 1, 11, 12, 13, 15, 17, and 25 exhibited effective COX-2 inhibitory activity, with half-maximal inhibitor concentration (IC50) values of 0.70-2.0 μM and selectivity index (SI) values of more than 50-142.9 compared with celecoxib as reference drugs (IC50 = 0.30 μM and COX-2 SI: >333). Potent COX-2 inhibitors, i.e., compounds 15, 11, and 17 were docked into the binding site pockets of COX-1 and COX-2. These compounds exhibited strong interactions at the COX-2 binding site and poor interactions at COX-1 active site pocket. PMID:27318118

  14. Bisphenol S and F: A Systematic Review and Comparison of the Hormonal Activity of Bisphenol A Substitutes

    PubMed Central

    Bolden, Ashley L.

    2015-01-01

    Background Increasing concern over bisphenol A (BPA) as an endocrine-disrupting chemical and its possible effects on human health have prompted the removal of BPA from consumer products, often labeled “BPA-free.” Some of the chemical replacements, however, are also bisphenols and may have similar physiological effects in organisms. Bisphenol S (BPS) and bisphenol F (BPF) are two such BPA substitutes. Objectives This review was carried out to evaluate the physiological effects and endocrine activities of the BPA substitutes BPS and BPF. Further, we compared the hormonal potency of BPS and BPF to that of BPA. Methods We conducted a systematic review based on the Office of Health Assessment and Translation (OHAT) protocol. Results We identified the body of literature to date, consisting of 32 studies (25 in vitro only, and 7 in vivo). The majority of these studies examined the hormonal activities of BPS and BPF and found their potency to be in the same order of magnitude and of similar action as BPA (estrogenic, antiestrogenic, androgenic, and antiandrogenic) in vitro and in vivo. BPS also has potencies similar to that of estradiol in membrane-mediated pathways, which are important for cellular actions such as proliferation, differentiation, and death. BPS and BPF also showed other effects in vitro and in vivo, such as altered organ weights, reproductive end points, and enzyme expression. Conclusions Based on the current literature, BPS and BPF are as hormonally active as BPA, and they have endocrine-disrupting effects. Citation Rochester JR, Bolden AL. 2015. Bisphenol S and F: a systematic review and comparison of the hormonal activity of bisphenol A substitutes. Environ Health Perspect 123:643–650; http://dx.doi.org/10.1289/ehp.1408989 PMID:25775505

  15. Synthesis of Diverse N-Substituted Muramyl Dipeptide Derivatives and Their Use in a Study of Human NOD2 Stimulation Activity.

    PubMed

    Chen, Kuo-Ting; Huang, Duen-Yi; Chiu, Cheng-Hsin; Lin, Wan-Wan; Liang, Pi-Hui; Cheng, Wei-Chieh

    2015-08-17

    A flexible synthetic strategy toward the preparation of diverse N-substituted muramyl dipeptides (N-substituted MDPs) from different protected monosaccharides is described. The synthetic MDPs include N-acetyl MDP and N-glycolyl MDP, known NOD2 ligands, and this methodology allows for structural variation at six positions, including the muramic acid, peptide, and N-substituted moieties. The capacity of these molecules to activate human NOD2 in the innate immune response was also investigated. It was found that addition of the methyl group at the C1 position of N-glycolyl MDP significantly enhanced the NOD2 stimulating activity. PMID:26226896

  16. Free energy simulations of active-site mutants of dihydrofolate reductase.

    PubMed

    Doron, Dvir; Stojković, Vanja; Gakhar, Lokesh; Vardi-Kilshtain, Alexandra; Kohen, Amnon; Major, Dan Thomas

    2015-01-22

    This study employs hybrid quantum mechanics-molecular mechanics (QM/MM) simulations to investigate the effect of mutations of the active-site residue I14 of E. coli dihydrofolate reductase (DHFR) on the hydride transfer. Recent kinetic measurements of the I14X mutants (X = V, A, and G) indicated slower hydride transfer rates and increasingly temperature-dependent kinetic isotope effects (KIEs) with systematic reduction of the I14 side chain. The QM/MM simulations show that when the original isoleucine residue is substituted in silico by valine, alanine, or glycine (I14V, I14A, and I14G DHFR, respectively), the free energy barrier height of the hydride transfer reaction increases relative to the wild-type enzyme. These trends are in line with the single-turnover rate measurements reported for these systems. In addition, extended dynamics simulations of the reactive Michaelis complex reveal enhanced flexibility in the mutants, and in particular for the I14G mutant, including considerable fluctuations of the donor-acceptor distance (DAD) and the active-site hydrogen bonding network compared with those detected in the native enzyme. These observations suggest that the perturbations induced by the mutations partly impair the active-site environment in the reactant state. On the other hand, the average DADs at the transition state of all DHFR variants are similar. Crystal structures of I14 mutants (V, A, and G) confirmed the trend of increased flexibility of the M20 and other loops. PMID:25382260

  17. VARIABLE ACTIVE SITE LOOP CONFORMATIONS ACCOMMODATE THE BINDING OF MACROCYCLIC LARGAZOLE ANALOGUES TO HDAC8

    PubMed Central

    Decroos, Christophe; Clausen, Dane J.; Haines, Brandon E.; Wiest, Olaf; Williams, Robert M.; Christianson, David W.

    2015-01-01

    The macrocyclic depsipeptide Largazole is a potent inhibitor of metal-dependent histone deacetylases (HDACs), some of which are drug targets for cancer chemotherapy. Indeed, Largazole partially resembles Romidepsin (FK228), a macrocyclic depsipeptide already approved for clinical use. Each inhibitor contains a pendant side chain thiol that coordinates to the active site Zn2+ ion, as observed in the X-ray crystal structure of the HDAC8–Largazole complex [Cole, K. E.; Dowling, D. P.; Boone, M. A.; Phillips, A. J.; Christianson, D. W. J. Am. Chem. Soc. 2011, 133, 12474]. Here, we report the X-ray crystal structures of HDAC8 complexed with three synthetic analogues of Largazole in which the depsipeptide ester is replaced with a rigid amide linkage. In two of these analogues, a 6-membered pyridine ring is also substituted (with two different orientations) for the 5-membered thiazole ring in the macrocycle skeleton. The side chain thiol group of each analogue coordinates to the active site Zn2+ ion with nearly ideal geometry, thereby preserving the hallmark structural feature of inhibition by Largazole. Surprisingly, in comparison with the binding of Largazole, these analogues trigger alternative conformational changes in the L1 and L2 loops flanking the active site. However, despite these structural differences, inhibitory potency is generally comparable to, or just moderately less than, the inhibitory potency of Largazole. Thus, this study reveals important new structure-affinity relationships for the binding of macrocyclic inhibitors to HDAC8. PMID:25793284

  18. Synthesis, biological activity evaluation and molecular docking studies of novel coumarin substituted thiazolyl-3-aryl-pyrazole-4-carbaldehydes.

    PubMed

    Vaarla, Krishnaiah; Kesharwani, Rajesh Kumar; Santosh, Karnewar; Vedula, Rajeswar Rao; Kotamraju, Srigiridhar; Toopurani, Murali Krishna

    2015-12-15

    A novel series of coumarin substituted thiazolyl-3-aryl-pyrazole-4-carbaldehydes (4a-o) were synthesized via an efficient, one-pot multicomponent approach involving 3-(2-bromoacetyl)coumarins (1a-g), thiosemicarbazide (2) and substituted acetophenones (3a-c) utilizing Vilsmeier-Haack reaction condition with good yields. The title compounds structure was elucidated by spectroscopic data (IR, NMR and Mass) and elemental analysis. All the synthesized compounds were screened for their in vitro cytotoxic activity against MCF-7, DU-145 and HeLa cell lines and studied detailed about molecular interaction of probable target protein human microsomal cytochrome CYP450 2A6 using docking simulation. These coumarin derivatives were exhibiting moderate to appreciable cytotoxic activities. The compounds 4m and 4n exhibited significant cytotoxic activity with IC50 values having 5.75 and 6.25μM against HeLa cell line. Similarly compound 4n also exhibiting good anti cancer property and antibacterial activity against DU-145 cell line and Gram negative bacterial strains. PMID:26542964

  19. Dashboard applications to monitor experiment activities at sites

    NASA Astrophysics Data System (ADS)

    Andreeva, Julia; Belforte, Stefano; Boehm, Max; Casajus, Adrian; Flix, Josep; Gaidioz, Benjamin; Grigoras, Costin; Kokoszkiewicz, Lukasz; Lanciotti, Elisa; Rocha, Ricardo; Saiz, Pablo; Santinelli, Roberto; Sidorova, Irina; Sciabà, Andrea; Tsaregorodtsev, Andrei

    2010-04-01

    In the framework of a distributed computing environment, such as WLCG, monitoring has a key role in order to keep under control activities going on in sites located in different countries and involving people based in many different sites. To be able to cope with such a large scale heterogeneous infrastructure, it is necessary to have monitoring tools providing a complete and reliable view of the overall performance of the sites. Moreover, the structure of a monitoring system critically depends on the object to monitor and on the users it is addressed to. In this article we will describe two different monitoring systems both aimed to monitor activities and services provided in the WLCG framework, but designed in order to meet the requirements of different users: Site Status Board has an overall view of the services available in all the sites supporting an experiment, whereas Siteview provides a complete view of all the activities going on at a site, for all the experiments supported by the site.

  20. Architecture and active site of particulate methane monooxygenase

    PubMed Central

    Culpepper, Megen A.; Rosenzweig, Amy C.

    2012-01-01

    Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria, organisms that live on methane gas as their sole carbon source. Understanding pMMO function has important implications for bioremediation applications and for the development of new, environmentally friendly catalysts for the direct conversion of methane to methanol. Crystal structures of pMMOs from three different methanotrophs reveal a trimeric architecture, consisting of three copies each of the pmoB, pmoA, and pmoC subunits. There are three distinct metal centers in each protomer of the trimer, mononuclear and dinuclear copper sites in the periplasmic regions of pmoB and a mononuclear site within the membrane that can be occupied by copper or zinc. Various models for the pMMO active site have been proposed within these structural constraints, including dicopper, tricopper, and diiron centers. Biochemical and spectroscopic data on pMMO and recombinant soluble fragments, denoted spmoB proteins, indicate that the active site involves copper and is located at the site of the dicopper center in the pmoB subunit. Initial spectroscopic evidence for O2 binding at this site has been obtained. Despite these findings, questions remain about the active site identity and nuclearity and will be the focus of future studies. PMID:22725967

  1. Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site.

    PubMed

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-04-20

    Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2domains reveal that the (HhH)2domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)2domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)2domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide. PMID:26908655

  2. Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site

    PubMed Central

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-01-01

    Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2 domains reveal that the (HhH)2 domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)2 domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)2 domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2 domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide. PMID:26908655

  3. N-Substituted Quinolinonyl Diketo Acid Derivatives as HIV Integrase Strand Transfer Inhibitors and Their Activity against RNase H Function of Reverse Transcriptase.

    PubMed

    Pescatori, Luca; Métifiot, Mathieu; Chung, Suhman; Masoaka, Takashi; Cuzzucoli Crucitti, Giuliana; Messore, Antonella; Pupo, Giovanni; Madia, Valentina Noemi; Saccoliti, Francesco; Scipione, Luigi; Tortorella, Silvano; Di Leva, Francesco Saverio; Cosconati, Sandro; Marinelli, Luciana; Novellino, Ettore; Le Grice, Stuart F J; Pommier, Yves; Marchand, Christophe; Costi, Roberta; Di Santo, Roberto

    2015-06-11

    Bifunctional quinolinonyl DKA derivatives were first described as nonselective inhibitors of 3'-processing (3'-P) and strand transfer (ST) functions of HIV-1 integrase (IN), while 7-aminosubstituted quinolinonyl derivatives were proven IN strand transfer inhibitors (INSTIs) that also displayed activity against ribonuclease H (RNase H). In this study, we describe the design, synthesis, and biological evaluation of new quinolinonyl diketo acid (DKA) derivatives characterized by variously substituted alkylating groups on the nitrogen atom of the quinolinone ring. Removal of the second DKA branch of bifunctional DKAs, and the amino group in position 7 of quinolinone ring combined with a fine-tuning of the substituents on the benzyl group in position 1 of the quinolinone, increased selectivity for IN ST activity. In vitro, the most potent compound was 11j (IC50 = 10 nM), while the most active compounds against HIV infected cells were ester derivatives 10j and 10l. In general, the activity against RNase H was negligible, with only a few compounds active at concentrations higher than 10 μM. The binding mode of the most potent IN inhibitor 11j within the IN catalytic core domain (CCD) is described as well as its binding mode within the RNase H catalytic site to rationalize its selectivity. PMID:25961960

  4. Antibacterial and anticancer activity of a series of novel peptides incorporating cyclic tetra-substituted C(α) amino acids.

    PubMed

    Hicks, Rickey P

    2016-09-15

    Eleven antimicrobial peptides (AMP) based on the incorporation of cyclic tetra substituted C(α) amino acids, as well as other unnatural amino acids were designed, synthesized and screened for in vitro activity against 18 strains of bacteria as well as 12 cancer cell lines. The AMPs discussed herein are derived from the following peptide sequence: Ac-GF(X)G(X)B(X)G(X)F(X)G(X)GB(X)BBBB-amide, X=any one of the following residues, A5c, A6c, Tic or Oic and B=any one of the following residues, Arg, Lys, Orn, Dpr or Dab. A diversity of in vitro inhibitory activity was observed for these AMPs. Several analogs exhibited single digit μM activity against drug resistant bacteria including; multiple drug resistant Mycobacterium tuberculosis, extremely drug resistant Mycobacterium tuberculosis and MRSA. The physicochemical properties of the basic amino acid residues incorporated into these AMPs seem to play a major role in defining antibacterial activity. Overall hydrophobicity seems to play a limited role in defining antibacterial activity. The ESKAPE pathogens were used to compare the activity of these AMPs to another family of synthetic AMPs incorporating the unnatural amino acids Tic and Oic. In most cases similarly substituted members of both families exhibited similar inhibitory activity against the ESKAPE pathogens. In specific cases differences in activity as high as 15 fold were observed between analogs. In addition four of these AMPs exhibited promising IC50 (<7.5μM) values against 12 different and diverse cancer cell lines. Five other AMPs exhibited promising IC50 (<7.5μM) values against selected cancer cell lines. PMID:27387357

  5. Structure/antileishmanial activity relationship study of naphthoquinones and dependency of the mode of action on the substitution patterns.

    PubMed

    Ali, Ahmad; Assimopoulou, Andreana Nikolaos; Papageorgiou, Vassilios Peter; Kolodziej, Herbert

    2011-12-01

    A series of naphthoquinones was tested for activity against both extracellular promastigote and intracellular amastigote Leishmania major GFP in vitro. In parallel, the compounds were evaluated for cytotoxic effects against bone marrow-derived macrophages (BMM Φ) as a mammalian host cell control. Most of the compounds noticeably inhibited the growth of extracellular parasites (IC (50) 0.5 to 6 µM) and the intracellular survival of L. major GFP amastigotes (IC (50) 1 to 7 µM) when compared with the antileishmanial drug amphotericin B (IC (50) of 2.5 and 0.2 µM, respectively). In general, antiprotozoal activity and host cell cytotoxicity seemed to increase in parallel. Conspicuously, the cytotoxic effect was less pronounced on infected host cells when compared with that on noninfected cells. Concerning structure/activity relationships for the tested naphthoquinones, some interesting structural features emerged from this study. Introduction of a methyl or methoxyl group at C-2 of the parent 1,4-naphthoquinone slightly increased the antileishmanial activity against clinically relevant amastigotes, while the presence of a hydroxyl function in this position dramatically reduced the effectiveness. In contrast, hydroxylation at C-5 and dihydroxy substitution at C-5 and C-8 significantly enhanced the antiprotozoal activity. Similarly, the presence of a side chain hydroxyl group PERI to a carbonyl function as represented in the series of shikonin/alkannin derivatives increased the activity when compared with substituted analogs. Within the series of naphthoquinones tested, the dimeric mixture of vaforhizin and isovaforhizin showed the highest activity IN VITRO against the clinically relevant intracellular amastigote with an IC (50) of 1.1 µM. With IC (50) values mostly in the range of 1-3 µM, the shikonin/alkannin derivatives proved to be similarly considerably leishmanicidal. None of the compounds tested was capable to induce NO production known to play a

  6. Construction of Hexahydrophenanthrenes By Rhodium(I)-Catalyzed Cycloisomerization of Benzylallene-Substituted Internal Alkynes through C-H Activation.

    PubMed

    Kawaguchi, Yasuaki; Yasuda, Shigeo; Mukai, Chisato

    2016-08-22

    The treatment of benzylallene-substituted internal alkynes with [RhCl(CO)2 ]2 effects a novel cycloisomerization by C(sp(2) )-H bond activation to produce hexahydrophenanthrene derivatives. The reaction likely proceeds through consecutive formation of a rhodabicyclo[4.3.0] intermediate, σ-bond metathesis between the C(sp(2) )-H bond on the benzene ring and the C(sp(2) )-Rh(III) bond, and isomerization between three σ-, π-, and σ-allylrhodium(III) species, which was proposed based on experiments with deuterated substrates. PMID:27467443

  7. Antiproliferative activity and synthesis of 8-prenylnaringenin derivatives by demethylation of 7-O- and 4'-O-substituted isoxanthohumols.

    PubMed

    Anioł, Mirosław; Swiderska, Anna; Stompor, Monika; Zołnierczyk, Anna Katarzyna

    2012-12-01

    Several analogues of 7-O- and 4'-O-substituted isoxanthohumol and 8-prenylnaringenin, the strongest known phytoestrogen and potential anticancerogenic agent, were synthesized. Acyl, alkyl, and allyl derivatives of isoxanthohumol underwent the demethylation process using MgI(2 )× 2Et(2)O in anhydrous THF with the yields of 61-89%. Some of the compounds approached the international criteria of antiproliferative activity (4 μg/ml) for synthetic agents against the human cancer cell lines. PMID:23087590

  8. Structure-Activity Relationship Studies of Amino Acid Substitutions in Radiolabeled Neurotensin Conjugates.

    PubMed

    Mascarin, Alba; Valverde, Ibai E; Mindt, Thomas L

    2016-01-01

    Radiolabeled derivatives of the peptide neurotensin (NT) and its binding sequence NT(8-13) have been studied as potential imaging probes and therapeutics for NT-1-receptor-positive cancer. However, a direct comparison of reported NT analogues, even if radiolabeled with the same radionuclide, is difficult because different techniques and models have been used for preclinical evaluations. In an effort to identify a suitable derivative of NT(8-13) for radiotracer development, we herein report a side-by-side in vitro comparison of radiometallated NT derivatives bearing some of the most commonly reported amino acid substitutions in their sequence. Performed investigations include cell internalization experiments, determinations of receptor affinity, measurements of the distribution coefficient, and blood serum stability studies. Of the [(177)Lu]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-labeled examples studied, analogues of NT(8-13) containing a short hydrophilic tetraethylene glycol (PEG4 ) spacer between the peptide and the radiometal complex, and a minimum number of substitutions of amino acid residues, exhibited the most promising properties in vitro. PMID:26593062

  9. Molecular Imprint of Enzyme Active Site by Camel Nanobodies

    PubMed Central

    Li, Jiang-Wei; Xia, Lijie; Su, Youhong; Liu, Hongchun; Xia, Xueqing; Lu, Qinxia; Yang, Chunjin; Reheman, Kalbinur

    2012-01-01

    Screening of inhibitory Ab1 antibodies is a critical step for producing catalytic antibodies in the anti-idiotypic approach. However, the incompatible surface of the active site of the enzyme and the antigen-binding site of heterotetrameric conventional antibodies become the limiting step. Because camelid-derived nanobodies possess the potential to preferentially bind to the active site of enzymes due to their small size and long CDR3, we have developed a novel approach to produce antibodies with alliinase activities by exploiting the molecular mimicry of camel nanobodies. By screening the camelid-derived variable region of the heavy chain cDNA phage display library with alliinase, we obtained an inhibitory nanobody VHHA4 that recognizes the active site. Further screening with VHHA4 from the same variable domain of the heavy chain of a heavy-chain antibody library led to a higher incidence of anti-idiotypic Ab2 abzymes with alliinase activities. One of the abzymes, VHHC10, showed the highest activity that can be inhibited by Ab1 VHHA4 and alliinase competitive inhibitor penicillamine and significantly suppressed the B16 tumor cell growth in the presence of alliin in vitro. The results highlight the feasibility of producing abzymes via anti-idiotypic nanobody approach. PMID:22374998

  10. Active Sites Environmental Monitoring Program: Mid-FY 1991 report

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1991-10-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) from October 1990 through March 1991. The ASEMP was established in 1989 by Solid Waste Operations and the Environmental Sciences Division to provide early detection and performance monitoring at active low-level radioactive waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 as required by chapters II and III of US Department of Energy Order 5820.2A. Monitoring results continue to demonstrate the no LLW is being leached from the storage vaults on the tumulus pads. Loading of vaults on Tumulus II began during this reporting period and 115 vaults had been loaded by the end of March 1991.

  11. An active-site peptide from pepsin C

    PubMed Central

    Kay, J.; Ryle, A. P.

    1971-01-01

    Porcine pepsin C is inactivated rapidly and irreversibly by diazoacetyl-dl-norleucine methyl ester in the presence of cupric ions at pH values above 4.5. The inactivation is specific in that complete inactivation accompanies the incorporation of 1mol of inhibitor residue/mol of enzyme and evidence has been obtained to suggest that the reaction occurs with an active site residue. The site of reaction is the β-carboxyl group of an aspartic acid residue in the sequence Ile-Val-Asp-Thr. This sequence is identical with the active-site sequence in pepsin and the significance of this in terms of the different activities of the two enzymes is discussed. PMID:4942834

  12. Tricyclic covalent inhibitors selectively target Jak3 through an active site thiol.

    PubMed

    Goedken, Eric R; Argiriadi, Maria A; Banach, David L; Fiamengo, Bryan A; Foley, Sage E; Frank, Kristine E; George, Jonathan S; Harris, Christopher M; Hobson, Adrian D; Ihle, David C; Marcotte, Douglas; Merta, Philip J; Michalak, Mark E; Murdock, Sara E; Tomlinson, Medha J; Voss, Jeffrey W

    2015-02-20

    The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. We have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). We found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC50 < 100 nm) inhibit Jak3 activity in cell-based assays. These results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases. PMID:25552479

  13. Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol*

    PubMed Central

    Goedken, Eric R.; Argiriadi, Maria A.; Banach, David L.; Fiamengo, Bryan A.; Foley, Sage E.; Frank, Kristine E.; George, Jonathan S.; Harris, Christopher M.; Hobson, Adrian D.; Ihle, David C.; Marcotte, Douglas; Merta, Philip J.; Michalak, Mark E.; Murdock, Sara E.; Tomlinson, Medha J.; Voss, Jeffrey W.

    2015-01-01

    The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. We have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). We found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC50 < 100 nm) inhibit Jak3 activity in cell-based assays. These results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases. PMID:25552479

  14. Are substitution rates and RNA editing correlated?

    PubMed Central

    2010-01-01

    Background RNA editing is a post-transcriptional process that, in seed plants, involves a cytosine to uracil change in messenger RNA, causing the translated protein to differ from that predicted by the DNA sequence. RNA editing occurs extensively in plant mitochondria, but large differences in editing frequencies are found in some groups. The underlying processes responsible for the distribution of edited sites are largely unknown, but gene function, substitution rate, and gene conversion have been proposed to influence editing frequencies. Results We studied five mitochondrial genes in the monocot order Alismatales, all showing marked differences in editing frequencies among taxa. A general tendency to lose edited sites was observed in all taxa, but this tendency was particularly strong in two clades, with most of the edited sites lost in parallel in two different areas of the phylogeny. This pattern is observed in at least four of the five genes analyzed. Except in the groups that show an unusually low editing frequency, the rate of C-to-T changes in edited sites was not significantly higher that in non-edited 3rd codon positions. This may indicate that selection is not actively removing edited sites in nine of the 12 families of the core Alismatales. In all genes but ccmB, a significant correlation was found between frequency of change in edited sites and synonymous substitution rate. In general, taxa with higher substitution rates tend to have fewer edited sites, as indicated by the phylogenetically independent correlation analyses. The elimination of edited sites in groups that lack or have reduced levels of editing could be a result of gene conversion involving a cDNA copy (retroprocessing). If so, this phenomenon could be relatively common in the Alismatales, and may have affected some groups recurrently. Indirect evidence of retroprocessing without a necessary correlation with substitution rate was found mostly in families Alismataceae and Hydrocharitaceae

  15. Synthesis, characterization and antimicrobial activities of novel silver(I) complexes with coumarin substituted N-heterocyclic carbene ligands.

    PubMed

    Karataş, Mert Olgun; Olgundeniz, Begüm; Günal, Selami; Özdemir, İlknur; Alıcı, Bülent; Çetinkaya, Engin

    2016-02-15

    Eight new coumarin substituted silver(I) N-heterocyclic carbene (NHC) complexes were synthesized by the interaction of the corresponding imidazolium or benzimidazolium chlorides and Ag2O in dichloromethane at room temperature. Structures of these complexes were established on the basis of elemental analysis, (1)H NMR, (13)C NMR, IR and mass spectroscopic techniques. The antimicrobial activities of carbene precursors and silver NHC complexes were tested against standard strains: Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and the fungi Candida albicans and Candida tropicalis. Results showed that all the compounds inhibited the growth of the all bacteria and fungi strains and some complexes performed good activities against different microorganisms. Among all the compounds, the most lipophilic complex bis[1-(4-methylene-6,8-dimethyl-2H-chromen-2-one)-3-(naphthalene-2-ylmethyl)benzimidazol-2-ylidene]silver(I) dichloro argentate (5e) was found out as the most active one. PMID:26740157

  16. Multi-Enhanced-Phonon Scattering Modes in Ln-Me-A Sites co-substituted LnMeA11O19 Ceramics

    PubMed Central

    Lu, Haoran; Wang, Chang-An; Huang, Yong; Xie, Huimin

    2014-01-01

    Authors reported an effective path to decrease the thermal conductivity while to increase the coefficient of thermal expansion, thus enhancing the thermo-physical properties of the LnMeA11O19-type magnetoplumbite LaMgAl11O19 by simultaneously substituting La3+, Mg2+ and Al3+ ions with large ionic radius Ba2+, Zn2+ and Ti4+, respectively. The mechanism behind the lowered thermal conductivity was mainly due to the multi-enhanced-phonon scattering modes in Ln-Me-A sites co-substituted LnMeA11O19 ceramics. These modes involve the following four aspects, namely, point defect mechanism, the intrinsic scattering in the complex crystal cell and materials with stepped surface to localize phonon vibrational modes, as well as nano-platelet-like structure to incorporate additional grain boundary scattering. This study provides novel thoughts for promising candidate materials of even lower thermal conductivity for the next generation thermal barrier coatings. PMID:25351166

  17. Active chemisorption sites in functionalized ionic liquids for carbon capture.

    PubMed

    Cui, Guokai; Wang, Jianji; Zhang, Suojiang

    2016-07-25

    Development of novel technologies for the efficient and reversible capture of CO2 is highly desired. In the last decade, CO2 capture using ionic liquids has attracted intensive attention from both academia and industry, and has been recognized as a very promising technology. Recently, a new approach has been developed for highly efficient capture of CO2 by site-containing ionic liquids through chemical interaction. This perspective review focuses on the recent advances in the chemical absorption of CO2 using site-containing ionic liquids, such as amino-based ionic liquids, azolate ionic liquids, phenolate ionic liquids, dual-functionalized ionic liquids, pyridine-containing ionic liquids and so on. Other site-containing liquid absorbents such as amine-based solutions, switchable solvents, and functionalized ionic liquid-amine blends are also investigated. Strategies have been discussed for how to activate the existent reactive sites and develop novel reactive sites by physical and chemical methods to enhance CO2 absorption capacity and reduce absorption enthalpy. The carbon capture mechanisms of these site-containing liquid absorbents are also presented. Particular attention has been paid to the latest progress in CO2 capture in multiple-site interactions by amino-free anion-functionalized ionic liquids. In the last section, future directions and prospects for carbon capture by site-containing ionic liquids are outlined. PMID:27243042

  18. Structure–function studies on the active site of the coelenterazine-dependent luciferase from Renilla

    PubMed Central

    Woo, Jongchan; Howell, Matthew H.; von Arnim, Albrecht G.

    2008-01-01

    Renilla luciferase (RLUC) is a versatile tool for gene expression assays and in vivo biosensor applications, but its catalytic mechanism remains to be elucidated. RLUC is evolutionarily related to the α/β hydrolase family. Its closest known homologs are bacterial dehalogenases, raising the question of how a protein with a hydrolase fold can function as a decarboxylating oxygenase. Molecular docking simulations with the coelenterazine substrate against an RLUC homology model as well as a recently determined RLUC crystal structure were used to build hypotheses to identify functionally important residues, which were subsequently tested by site-directed mutagenesis, heterologous expression, and bioluminescence emission spectroscopy. The data highlighted two triads of residues that are critical for catalysis. The putative catalytic triad residues D120, E144, and H285 bear only limited resemblance to those found in the active site of aequorin, a coelenterazine-utilizing photoprotein, suggesting that the reaction scheme employed by RLUC differs substantially from the one established for aequorin. The role of H285 in catalysis was further supported by inhibition using diethylpyrocarbonate. Multiple substitutions of N53, W121, and P220—three other residues implicated in product binding in the homologous dehalogenase Sphingomonas LinB—also supported their involvement in catalysis. Together with luminescence spectra, our data lead us to propose that the conserved catalytic triad of RLUC is directly involved in the decarboxylation reaction of coelenterazine to produce bioluminescence, while the other active-site residues are used for binding of the substrate. PMID:18359861

  19. Design, Synthesis, Biological Evaluation, and Antioxidant and Cytotoxic Activity of Heteroatom-Substituted 1,4-Naphtho- and Benzoquinones.

    PubMed

    Deniz, Nahide Gülşah; Ibis, Cemil; Gokmen, Zeliha; Stasevych, Maryna; Novikov, Volodymyr; Komarovska-Porokhnyavets, Olena; Ozyurek, Mustafa; Guclu, Kubilay; Karakas, Didem; Ulukaya, Engin

    2015-01-01

    In the present paper, we report the synthesis, characterization, and biological evaluation as antifungal, antibacterial, antioxidant, and cytotoxic/anticancer agents of N-, S-, O-substituted-1,4-naphtho- and 2,5-bis(amino-substituted)-1,4-benzoquinone derivatives. In the synthesized compounds, antimicrobial activity at low concentrations against Escherichia coli B-906, Staphylococcus aureus 209-P, and Mycobacterium luteum B-917 bacteria and Candida tenuis VKM Y-70 and Aspergillus niger F-1119 fungi in comparison with controls was identified. 2-(N-Diphenylmethylpiperazin-1-yl)-3-chloro-1,4-naphthoquinone 9a was the most potent, with a minimum inhibitory concentration value of 3.9 µg/mL against test culture M. luteum. The synthesized compounds were screened for their antioxidant capacity using the cupric-reducing antioxidant capacity (CUPRAC) method. 2,2'-[1-(2-Aminoethyl)piperazin-1-yl]-3,3'-dichloro-bis(1,4-naphthoquinone) 10 showed the highest antioxidant capacity, with a 0.455 CUPRAC-trolox equivalent antioxidant capacity (TEAC) coefficient. Other parameters of antioxidant activity (scavenging effects on OH(·), O2(·-), and H2O2) of these compounds were also determined. The cytotoxic activity of the compounds was investigated by employing the sulforhodamine B cell viability assay against A549 (lung), MCF-7 (breast), DU145 (prostate), and HT-29 (colon) cancer cell lines. Compound 10 exhibited the most powerful cytotoxic activity at a concentration of 20 µM against all cell lines. In addition to the strongest antioxidant activity of compound 10, it also had lowest IC50 values (<3 µM), warranting further in vivo studies due to its anticancer activity. PMID:26633024

  20. MUTATION SPECTRA OF GLU-P-1 IN SALMONELLA: INDUCTION OF HOTSPOT FRAMESHIFTS AND SITE-SPECIFIC BASE SUBSTITUTIONS

    EPA Science Inventory

    The mutations induced in approximately 2,000 mutants of Salmonella by the heterocyclic@ amine Glu-P-1 were determined by colony probe hybridization and PCR/DNA sequence analysis. ll of the mutations were at sites containing guanine, which is the base at which Glu-P-1 forms DNA ad...

  1. Synthesis and evaluation of dual antiplatelet activity of bispidine derivatives of N-substituted pyroglutamic acids.

    PubMed

    Misra, Ankita; Anil Kumar, K S; Jain, Manish; Bajaj, Kirti; Shandilya, Shyamali; Srivastava, Smriti; Shukla, Pankaj; Barthwal, Manoj K; Dikshit, Madhu; Dikshit, Dinesh K

    2016-03-01

    N-aralkylpyroglutamides of substituted bispidine were prepared and evaluated for their ability to inhibit collagen induced platelet aggregation, both in vivo and in vitro. Some compounds showed high anti-platelet efficacy (in vitro) of which six inhibited both collagen as well as U46619 induced platelet aggregation with concentration dependent anti-platelet efficacy through dual mechanism. In particular, the compound 4j offered significant protection against collagen epinephrine induced pulmonary thromboembolism as well as ferric chloride induced arterial thrombosis, without affecting bleeding tendency in mice. Therefore, the present study suggests that the compound 4j displays a remarkable antithrombotic efficacy much better than aspirin and clopidogrel. PMID:26807542

  2. Synthesis of high specific activity (+)- and (-)-6-( sup 18 F)fluoronorepinephrine via the nucleophilic aromatic substitution reaction

    SciTech Connect

    Ding, Y.S.; Fowler, J.S.; Gatley, S.J.; Dewey, S.L.; Wolf, A.P. )

    1991-02-01

    The first example of a no-carrier-added {sup 18}F-labeled catecholamine, 6-({sup 18}F)fluoronorepinephrine (6-({sup 18}F)FNE), has been synthesized via nucleophilic aromatic substitution. The racemic mixture was resolved on a chiral HPLC column to obtain pure samples of (-)-6-({sup 18}F)FNE and (+)6-({sup 18}F)FNE. Radiochemical yields of 20% at the end of bombardment (EOB) for the racemic mixture (synthesis time 93 min), 6% for each enantiomer (synthesis time 128 min) with a specific activity of 2-5 Ci/mumol at EOB were obtained. Chiral HPLC peak assignment for the resolved enantiomers was achieved by using two independent methods: polarimetric determination and reaction with dopamine beta-hydroxylase. Positron emission tomography (PET) studies with racemic 6-({sup 18}F)FNE show high uptake and retention in the baboon heart. This work demonstrates that nucleophilic aromatic substitution by ({sup 18}F)fluoride ion is applicable to systems having electron-rich aromatic rings, leading to high specific activity radiopharmaceuticals. Furthermore, the suitably protected dihydroxynitrobenzaldehyde 1 may serve as a useful synthetic precursor for the radiosynthesis of other complex {sup 18}F-labeled radiotracers.

  3. Site- and Enantioselective Formation of Allene-Bearing Tertiary or Quaternary Carbon Stereogenic Centers through NHC–Cu-Catalyzed Allylic Substitution

    PubMed Central

    Jung, Byunghyuck; Hoveyda, Amir H.

    2012-01-01

    Catalytic enantioselective allylic substitutions that result in exclusive addition of an allenyl group (<2% propargyl addition) and formation of tertiary or quaternary C–C bonds are described. Commercially available allenylboronic acid pinacol ester is used (preparation of a more reactive but less stable boronate derivative not required). Reactions are promoted by 5.0–10 mol % of sulfonate-bearing chiral bidentate N-heterocyclic carbene (NHC) complexes of copper, which exhibit the unique ability to furnish chiral products arising from SN2′ mode of addition. The desired allenyl-containing products are generated in up to 95% yield, >98% SN2′ selectivity and 99:1 enantiomeric ratio (er). Site-selective NHC–Cu-catalyzed hydroboration of enantiomerically enriched allenes and conversion to the corresponding β-vinyl ketones demonstrates utility. PMID:22214185

  4. Rat intestinal trehalase. Studies of the active site.

    PubMed

    Chen, C C; Guo, W J; Isselbacher, K J

    1987-11-01

    Rat intestinal trehalase was solubilized, purified and reconstituted into proteoliposomes. With octyl glucoside as the solubilizing detergent, the purified protein appeared as a single band on SDS/polyacrylamide-gel electrophoresis with an apparent molecular mass of 67 kDa. Kinetic studies indicated that the active site of this enzyme can be functionally divided into two adjacent regions, namely a binding site (with pKa 4.8) and a catalytic site (with pKa 7.2). Other findings suggested that the catalytic site contains a functional thiol group, which is sensitive to inhibition by N-ethylmaleimide, Hg2+ and iodoacetate. Substrate protection and iodoacetate labelling of the thiol group demonstrated that only a protein of 67 kDa was labelled. Furthermore, sucrose and phlorizin protected the thiol group, but Tris-like inhibitors did not. Structure-inhibition analysis of Tris-like inhibitors, the pH effect of Tris inhibition and Tris protection of 1-(3-dimethylaminopropyl)-3-ethylcarbodi-imide inactivation permitted characterization and location of a separate site containing a carboxy group for Tris binding, which may also be the binding region. On the basis of these findings, a possible structure for the active site of trehalase is proposed. PMID:3426558

  5. Active Site and Remote Contributions to Catalysis in Methylthioadenosine Nucleosidases

    PubMed Central

    Thomas, Keisha; Cameron, Scott A.; Almo, Steven C.; Burgos, Emmanuel S.; Gulab, Shivali A.; Schramm, Vern L.

    2015-01-01

    5′-Methylthioadenosine/S-adenosyl-L-homocysteine nucleosidases (MTANs) catalyze the hydrolysis of 5′-methylthioadenosine to adenine and 5-methylthioribose. The amino acid sequences of the MTANs from Vibrio cholerae (VcMTAN) and Escherichia coli (EcMTAN) are 60% identical and 75% similar. Protein structure folds and kinetic properties are similar. However, binding of transition-state analogues is dominated by favorable entropy in VcMTAN and by enthalpy in EcMTAN. Catalytic sites of VcMTAN and EcMTAN in contact with reactants differ by two residues; Ala113 and Val153 in VcMTAN are Pro113 and Ile152, respectively, in EcMTAN. We mutated the VcMTAN catalytic site residues to match those of EcMTAN in anticipation of altering its properties toward EcMTAN. Inhibition of VcMTAN by transition-state analogues required filling both active sites of the homodimer. However, in the Val153Ile mutant or double mutants, transition-state analogue binding at one site caused complete inhibition. Therefore, a single amino acid, Val153, alters the catalytic site cooperativity in VcMTAN. The transition-state analogue affinity and thermodynamics in mutant VcMTAN became even more unlike those of EcMTAN, the opposite of expectations from catalytic site similarity; thus, catalytic site contacts in VcMTAN are unable to recapitulate the properties of EcMTAN. X-ray crystal structures of EcMTAN, VcMTAN, and a multiple-site mutant of VcMTAN most closely resembling EcMTAN in catalytic site contacts show no major protein conformational differences. The overall protein architectures of these closely related proteins are implicated in contributing to the catalytic site differences. PMID:25806409

  6. Synthesis and anti-inflammatory activity of 5-(6-methyl-2-substituted 4-pyrimidinyloxymethyl)-1,3,4-oxadiazole-2-thiones and their 3-morpholinomethyl derivatives.

    PubMed

    Jakubkiene, Virginija; Burbuliene, Milda Malvina; Mekuskiene, Giedrute; Udrenaite, Emilija; Gaidelis, Povilas; Vainilavicius, Povilas

    2003-04-01

    The synthesis of 5-(6-methyl-2-substituted 4-pyrimidinyloxymethyl)-2,3-dihydro-1,3,4-oxadiazole-2-thiones and their 3-morpholinomethyl derivatives and the results of anti-inflammatory activity in vivo are described. Most of the tested compounds exhibited anti-inflammatory activity and some of them were more active than acetylsalicylic acid. PMID:12727542

  7. Resonant active sites in catalytic ammonia synthesis: A structural model

    NASA Astrophysics Data System (ADS)

    Cholach, Alexander R.; Bryliakova, Anna A.; Matveev, Andrey V.; Bulgakov, Nikolai N.

    2016-03-01

    Adsorption sites Mn consisted of n adjacent atoms M, each bound to the adsorbed species, are considered within a realistic model. The sum of bonds Σ lost by atoms in a site in comparison with the bulk atoms was used for evaluation of the local surface imperfection, while the reaction enthalpy at that site was used as a measure of activity. The comparative study of Mn sites (n = 1-5) at basal planes of Pt, Rh, Ir, Fe, Re and Ru with respect to heat of N2 dissociative adsorption QN and heat of Nad + Had → NHad reaction QNH was performed using semi-empirical calculations. Linear QN(Σ) increase and QNH(Σ) decrease allowed to specify the resonant Σ for each surface in catalytic ammonia synthesis at equilibrium Nad coverage. Optimal Σ are realizable for Ru2, Re2 and Ir4 only, whereas other centers meet steric inhibition or unreal crystal structure. Relative activity of the most active sites in proportion 5.0 × 10- 5: 4.5 × 10- 3: 1: 2.5: 3.0: 1080: 2270 for a sequence of Pt4, Rh4, Fe4(fcc), Ir4, Fe2-5(bcc), Ru2, Re2, respectively, is in agreement with relevant experimental data. Similar approach can be applied to other adsorption or catalytic processes exhibiting structure sensitivity.

  8. Binding of Mn-deoxyribonucleoside Triphosphates to the Active Site of the DNA Polymerase of Bacteriophage T7

    SciTech Connect

    B Akabayov; C Richardson

    2011-12-31

    Divalent metal ions are crucial as cofactors for a variety of intracellular enzymatic activities. Mg{sup 2+}, as an example, mediates binding of deoxyribonucleoside 5'-triphosphates followed by their hydrolysis in the active site of DNA polymerase. It is difficult to study the binding of Mg{sup 2+} to an active site because Mg{sup 2+} is spectroscopically silent and Mg{sup 2+} binds with low affinity to the active site of an enzyme. Therefore, we substituted Mg{sup 2+} with Mn{sup 2+}:Mn{sup 2+} that is not only visible spectroscopically but also provides full activity of the DNA polymerase of bacteriophage T7. In order to demonstrate that the majority of Mn{sup 2+} is bound to the enzyme, we have applied site-directed titration analysis of T7 DNA polymerase using X-ray near edge spectroscopy. Here we show how X-ray near edge spectroscopy can be used to distinguish between signal originating from Mn{sup 2+} that is free in solution and Mn{sup 2+} bound to the active site of T7 DNA polymerase. This method can be applied to other enzymes that use divalent metal ions as a cofactor.

  9. The measured and calculated affinity of methyl and methoxy substituted benzoquinones for the QA site of bacterial reaction centers

    PubMed Central

    Zheng, Zhong; Dutton, P. Leslie; Gunner, M. R.

    2010-01-01

    Quinones play important roles in mitochondrial and photosynthetic energy conversion acting as intramembrane, mobile electron and proton carriers between catalytic sites in various electron transfer proteins. They display different affinity, selectivity, functionality and exchange dynamics in different binding sites. The computational analysis of quinone binding sheds light on the requirements for quinone affinity and specificity. The affinities of ten oxidized, neutral benzoquinones (BQs) were measured for the high affinity QA site in the detergent solubilized Rhodobacter sphaeroides bacterial photosynthetic reaction center. Multi-Conformation Continuum Electrostatics (MCCE) was then used to calculate their relative binding free energies by Grand Canonical Monte Carlo sampling with a rigid protein backbone, flexible ligand and side chain positions and protonation states. Van der Waals and torsion energies, Poisson-Boltzmann continuum electrostatics and accessible surface area dependent ligand-solvent interactions are considered. An initial, single cycle of GROMACS backbone optimization improves the match with experiment as do coupled ligand and side chain motions. The calculations match experiment with an RMSD of 2.29 and a slope of 1.28. The affinities are dominated by favorable protein-ligand van der Waals rather than electrostatic interactions. Each quinone appears in a closely clustered set of positions. Methyl and methoxy groups move into the same positions as found for the native quinone. Difficulties putting methyls into methoxy sites are observed. Calculations using an SAS dependent implicit van der Waals interaction smoothed out small clashes, providing a better match to experiment with a RMSD of 0.77 and a slope of 0.97. PMID:20607696

  10. Water in the Active Site of Ketosteroid Isomerase

    PubMed Central

    Hanoian, Philip; Hammes-Schiffer, Sharon

    2011-01-01

    Classical molecular dynamics simulations were utilized to investigate the structural and dynamical properties of water in the active site of ketosteroid isomerase (KSI) to provide insight into the role of these water molecules in the enzyme-catalyzed reaction. This reaction is thought to proceed via a dienolate intermediate that is stabilized by hydrogen bonding with residues Tyr16 and Asp103. A comparative study was performed for the wild-type (WT) KSI and the Y16F, Y16S, and Y16F/Y32F/Y57F (FFF) mutants. These systems were studied with three different bound ligands: equilenin, which is an intermediate analog, and the intermediate states of two steroid substrates. Several distinct water occupation sites were identified in the active site of KSI for the WT and mutant systems. Three additional sites were identified in the Y16S mutant that were not occupied in WT KSI or the other mutants studied. The number of water molecules directly hydrogen bonded to the ligand oxygen was approximately two waters in the Y16S mutant, one water in the Y16F and FFF mutants, and intermittent hydrogen bonding of one water molecule in WT KSI. The molecular dynamics trajectories of the Y16F and FFF mutants reproduced the small conformational changes of residue 16 observed in the crystal structures of these two mutants. Quantum mechanical/molecular mechanical calculations of 1H NMR chemical shifts of the protons in the active site hydrogen-bonding network suggest that the presence of water in the active site does not prevent the formation of short hydrogen bonds with far-downfield chemical shifts. The molecular dynamics simulations indicate that the active site water molecules exchange much more frequently for WT KSI and the FFF mutant than for the Y16F and Y16S mutants. This difference is most likely due to the hydrogen-bonding interaction between Tyr57 and an active site water molecule that is persistent in the Y16F and Y16S mutants but absent in the FFF mutant and significantly less

  11. CDK1 substitutes for mTOR kinase to activate mitotic cap-dependent protein translation

    PubMed Central

    Shuda, Masahiro; Velásquez, Celestino; Cheng, Erdong; Cordek, Daniel G.; Kwun, Hyun Jin; Chang, Yuan; Moore, Patrick S.

    2015-01-01

    Mitosis is commonly thought to be associated with reduced cap-dependent protein translation. Here we show an alternative control mechanism for maintaining cap-dependent translation during mitosis revealed by a viral oncoprotein, Merkel cell polyomavirus small T (MCV sT). We find MCV sT to be a promiscuous E3 ligase inhibitor targeting the anaphase-promoting complex, which increases cell mitogenesis. MCV sT binds through its Large T stabilization domain region to cell division cycle protein 20 (Cdc20) and, possibly, cdc20 homolog 1 (Cdh1) E3 ligase adapters. This activates cyclin-dependent kinase 1/cyclin B1 (CDK1/CYCB1) to directly hyperphosphorylate eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP1) at authentic sites, generating a mitosis-specific, mechanistic target of rapamycin (mTOR) inhibitor-resistant δ phospho-isoform not present in G1-arrested cells. Recombinant 4E-BP1 inhibits capped mRNA reticulocyte translation, which is partially reversed by CDK1/CYCB1 phosphorylation of 4E-BP1. eIF4G binding to the eIF4E–m7GTP cap complex is resistant to mTOR inhibition during mitosis but sensitive during interphase. Flow cytometry, with and without sT, reveals an orthogonal pH3S10+ mitotic cell population having higher inactive p4E-BP1T37/T46+ saturation levels than pH3S10– interphase cells. Using a Click-iT flow cytometric assay to directly measure mitotic protein synthesis, we find that most new protein synthesis during mitosis is cap-dependent, a result confirmed using the eIF4E/4G inhibitor drug 4E1RCat. For most cell lines tested, cap-dependent translation levels were generally similar between mitotic and interphase cells, and the majority of new mitotic protein synthesis was cap-dependent. These findings suggest that mitotic cap-dependent translation is generally sustained during mitosis by CDK1 phosphorylation of 4E-BP1 even under conditions of reduced mTOR signaling. PMID:25883264

  12. Structure-activity relationships for novel drug precursor N-substituted-6-acylbenzothiazolon derivatives: A theoretical approach

    NASA Astrophysics Data System (ADS)

    Sıdır, Yadigar Gülseven; Sıdır, İsa

    2013-08-01

    In this study, the twelve new modeled N-substituted-6-acylbenzothiazolon derivatives having analgesic analog structure have been investigated by quantum chemical methods using a lot of electronic parameters and structure-activity properties; such as molecular polarizability (α), dipole moment (μ), EHOMO, ELUMO, q-, qH+, molecular volume (Vm), ionization potential (IP), electron affinity (EA), electronegativity (χ), molecular hardness (η), molecular softness (S), electrophilic index (ω), heat of formation (HOF), molar refractivity (MR), octanol-water partition coefficient (log P), thermochemical properties (entropy (S), capacity of heat (Cv)); as to investigate activity relationships with molecular structure. The correlations of log P with Vm, MR, ω, EA, EHOMO - ELUMO (ΔE), HOF in aqueous phase, χ, μ, S, η parameters, respectively are obtained, while the linear relation of log P with IP, Cv, HOF in gas phase are not observed. The log P parameter is obtained to be depending on different properties of compounds due to their complexity.

  13. Energy transfer at the active sites of heme proteins

    SciTech Connect

    Dlott, D.D.; Hill, J.R.

    1995-12-31

    Experiments using a picosecond pump-probe apparatus at the Picosecond Free-electron Laser Center at Stanford University, were performed to investigate the relaxation of carbon monoxide bound to the active sites of heme proteins. The significance of these experiments is two-fold: (1) they provide detailed information about molecular dynamics occurring at the active sites of proteins; and (2) they provide insight into the nature of vibrational relaxation processes in condensed matter. Molecular engineering is used to construct various molecular systems which are studied with the FEL. We have studied native proteins, mainly myoglobin obtained from different species, mutant proteins produced by genetic engineering using recombinant DNA techniques, and a variety of model systems which mimic the structures of the active sites of native proteins, which are produced using molecular synthesis. Use of these different systems permits us to investigate how specific molecular structural changes affect dynamical processes occurring at the active sites. This research provides insight into the problems of how different species needs are fulfilled by heme proteins which have greatly different functionality, which is induced by rather small structural changes.

  14. Chemical Modification of Papain and Subtilisin: An Active Site Comparison

    ERIC Educational Resources Information Center

    St-Vincent, Mireille; Dickman, Michael

    2004-01-01

    An experiment using methyle methanethiosulfonate (MMTS) and phenylmethylsulfonyl flouride (PMSF) to specifically modify the cysteine and serine residues in the active sites of papain and subtilism respectively is demonstrated. The covalent modification of these enzymes and subsequent rescue of papain shows the beginning biochemist that proteins…

  15. Triazolines 26: 1-Aryl-5-amido-1,2,3-triazolines, a new group of triazoline anticonvulsants. Effect of 5-substitution on anticonvulsant activity.

    PubMed

    Kadaba, P K

    1996-01-01

    Studies in our laboratories have led to the discovery of the delta 2-1,2,3-triazolines as a unique family of anticonvulsant agents hitherto unknown. The anticonvulsant activity of 1,5-diaryl- and 1-aryl-5-pyridyltriazolines was previously reported; this paper describes the evaluation of two series of 1-aryl-5-amido-1,2,3-triazolines, A and B, where the 5-amido groups are (2-oxo-1-pyrrolidino)- (1-8) and (N-methyl-N-acetamido)- (9-15), respectively. The 1-aryl-5-(2-oxo-1-pyrrolidino)-1,2,3-triazolines of the A series, which are uniquely substituted with the pyrrolidinone lactam ring, a cyclic gamma-aminobutyric acid (GABA) structure, seem to function by enhancing inhibitory GABAergic mechanisms. Radioligand binding studies for the two most active triazolines 2 and 7, indicate that both compounds strongly inhibit the specific binding of [3H]GABA to GABAB receptor sites, with Ki = 1.7 and 0.91 microM respectively. The anticonvulsant activity among the various groups of triazolines studied so far appears to be dependent on the 5-substituent groups: 4-pyridyl- > 2-oxo-1-pyrrolidino- > N-methyl-N-acetamido- > 3-pyridyl > or = aryl approximately 2-pyridyl > 2-quinolyl. PMID:8881374

  16. Changes in active site histidine hydrogen bonding trigger cryptochrome activation.

    PubMed

    Ganguly, Abir; Manahan, Craig C; Top, Deniz; Yee, Estella F; Lin, Changfan; Young, Michael W; Thiel, Walter; Crane, Brian R

    2016-09-01

    Cryptochrome (CRY) is the principal light sensor of the insect circadian clock. Photoreduction of the Drosophila CRY (dCRY) flavin cofactor to the anionic semiquinone (ASQ) restructures a C-terminal tail helix (CTT) that otherwise inhibits interactions with targets that include the clock protein Timeless (TIM). All-atom molecular dynamics (MD) simulations indicate that flavin reduction destabilizes the CTT, which undergoes large-scale conformational changes (the CTT release) on short (25 ns) timescales. The CTT release correlates with the conformation and protonation state of conserved His378, which resides between the CTT and the flavin cofactor. Poisson-Boltzmann calculations indicate that flavin reduction substantially increases the His378 pKa Consistent with coupling between ASQ formation and His378 protonation, dCRY displays reduced photoreduction rates with increasing pH; however, His378Asn/Arg variants show no such pH dependence. Replica-exchange MD simulations also support CTT release mediated by changes in His378 hydrogen bonding and verify other responsive regions of the protein previously identified by proteolytic sensitivity assays. His378 dCRY variants show varying abilities to light-activate TIM and undergo self-degradation in cellular assays. Surprisingly, His378Arg/Lys variants do not degrade in light despite maintaining reactivity toward TIM, thereby implicating different conformational responses in these two functions. Thus, the dCRY photosensory mechanism involves flavin photoreduction coupled to protonation of His378, whose perturbed hydrogen-bonding pattern alters the CTT and surrounding regions. PMID:27551082

  17. Active sites environmental monitoring program. Annual report FY 1992

    SciTech Connect

    Morrissey, C.M.; Ashwood, T.L.; Hicks, D.S.

    1994-04-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) at ORNL from October 1991 through September 1992. Solid Waste Operations and the Environmental Sciences Division established ASEMP in 1989 to provide early detection and performance monitoring at active low-level waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 as required by Chapter 2 and 3 of US Department of Energy Order 5820.2A. The Interim Waste Management Facility (IWMF) began operation in December 1991. Monitoring results from the tumulus and IWMF disposal pads continue to indicate that no LLW is leaching from the storage vaults. Storm water falling on the IWMF active pad was collected and transported to the Process Waste Treatment Plant while operators awaited approval of the National Pollutant Discharge Elimination System (NPDES) permit. Several of the recent samples collected from the active IWMF pad had pH levels above the NPDES limit of 9.0 because of alkali leached from the concrete. The increase in gross beta activity has been slight; only 1 of the 21 samples collected contained activity above the 5.0 Bq/L action level. Automated sample-collection and flow-measurement equipment has been installed at IWMF and is being tested. The flume designed to electronically measure flow from the IWMF pads and underpads is too large to be of practical value for measuring most flows at this site. Modification of this system will be necessary. A CO{sub 2} bubbler system designed to reduce the pH of water from the pads is being tested at IWMF.

  18. Effect of Mg content on the bioactivity and biocompatibility of Mg-substituted fluorapatite nanopowders fabricated via mechanical activation.

    PubMed

    Kheradmandfard, M; Fathi, M H; Ansari, F; Ahmadi, T

    2016-11-01

    The aim of this work was preparation, characterization, bioactivity and biocompatibility evaluation of Mg-substituted fluorapatite (Mg-FA) nanopowders. Mg-FA nanopowders with a chemical composition of Ca10-xMgx(PO4)6F2, with x=0, 0.5, 1, and 2 were prepared by mechanically activated method. The in vitro bioactivity was investigated by soaking the powders in simulated body fluid (SBF) for various time periods to analyze the nucleation and growth of bone-like apatite on the surface of the samples. Cell viability and cell attachment were studied by MTT assay. Results indicated that the bioactivity of all of samples with different Mg content was improved compared with the pure FA. However, the mechanism of bioactivity is different and depends on the amount of Mg substitution. Finally, cell culture suggested that the addition of Mg(2+) has no adverse effect and Mg-FA samples have good biocompatibility. The Mg-FA material shows potential in satisfying the requirements of biomedical applications. PMID:27524005

  19. High-mobility thin film transistors with neodymium-substituted indium oxide active layer

    SciTech Connect

    Lin, Zhenguo; Lan, Linfeng Xiao, Peng; Sun, Sheng; Li, Yuzhi; Song, Wei; Gao, Peixiong; Wang, Lei; Ning, Honglong; Peng, Junbiao

    2015-09-14

    Thin-film transistors (TFTs) with neodymium-substituted indium oxide (InNdO) channel layer were demonstrated. The structural properties of the InNdO films as a function of annealing temperature have been analyzed using X-ray diffraction and transmission electron microscopy. The InNdO thin films showed polycrystalline nature when annealed at 450 °C with a lattice parameter (cubic cell) of 10.255 Å, which is larger than the cubic In{sub 2}O{sub 3} film (10.117 Å). The high-resolution transmission electron microscopy and energy dispersive X-ray spectroscopy showed that no Nd{sub 2}O{sub 3} clusters were found in the InNdO film, implying that Nd was incorporated into the In{sub 2}O{sub 3} lattice. The InNdO TFTs annealed at 450 °C exhibited more excellent electrical properties with a high mobility of 20.4 cm{sup 2} V{sup −1} s{sup −1} and better electric bias stability compared to those annealed at 300 °C, which was attributed to the reduction of the scattering centers and/or charge traps due to the decrease of the |Nd3d{sub 5/2}{sup 5}4f{sup 4}O2p{sup −1}〉 electron configuration.

  20. Ferrocenyl-substituted dinuclear Cu(II) complex: Synthesis, spectroscopy, electrochemistry, DFT calculations and catecholase activity

    NASA Astrophysics Data System (ADS)

    Emirik, Mustafa; Karaoğlu, Kaan; Serbest, Kerim; Menteşe, Emre; Yilmaz, Ismail

    2016-02-01

    A new ferrocenyl-substituted heterocyclic hydrazide ligand and its Cu(II) complex were prepared. The DFT calculations were performed to determine the electronic and molecular structures of the title compounds. The electronic spectra were calculated by using time-dependent DFT method, and the transitions were correlated with the molecular orbitals of the compounds. The bands assignments of IR spectra were achieved in the light of the theoretical vibrational spectral data and total energy distribution values calculated at DFT/B3LYP/6-311++G(d,p) level. The redox behaviors of the ferrocene derivatives were investigated by cyclic voltammetry. The compounds show reversible redox couple assignable to Fc+/Fc couple. The copper(II) complex behaves as an effective catalyst towards oxidation of 3,5-di-tert-butylcatechol to its corresponding quinone derivative in DMF saturated with O2. The reaction follows Michaelis-Menten enzymatic reaction kinetics with turnover numbers 2.32 × 103.

  1. Electro-Optically Active Monomers: Synthesis and Characterization of Thin Films of Liquid Crystalline Substituted Polyacetylenes

    NASA Technical Reports Server (NTRS)

    Duran, R. S.

    1995-01-01

    The overall objective of this study was the description of the behavior of mesogen substituted acetylene monomers and polymers in monolayer films at the air/water interface and as multilayer films including the formation of such films. Fundamental knowledge to be gained would include the effect of balancing hydrophilic and hydrophobic tendencies in a molecule more complex than the classical fatty acids or lipids. The effect of molecular shape on the packing and thus the ultimate stability of monolayers formed from these new molecules was explored. The work takes on the challenge of preorienting monomers in well-ordered arrays prior to attempting polymerization with the hope that order would be preserved in any resulting polymer. New knowledge gained with regard to the acetylenic monomers includes processing of the acetylene monomer into multi-layer films, followed by the design and synthesis of a second generation of improved monomer structure for superior LBK film transfer properties. A third generation of acetylenic monomer was synthesized which approaches more closely the goal of solid state polymerization of these materials. A parallel study took a different approach. The materials are pre-formed poly(phenylene-acetylene) polymers so questions about reactivity are mute. The materials are a variation on the well-known hairy-rod polymers with regard to their Langmuir film-forming properties. Overall, the goal was to demonstrate that these polymers could be processed into NLO materials with novel polar order.

  2. Design, synthesis, crystal structure, insecticidal activity, molecular docking, and QSAR studies of novel N3-substituted imidacloprid derivatives.

    PubMed

    Wang, Mei-Juan; Zhao, Xiao-Bo; Wu, Dan; Liu, Ying-Qian; Zhang, Yan; Nan, Xiang; Liu, Huanxiang; Yu, Hai-Tao; Hu, Guan-Fang; Yan, Li-Ting

    2014-06-18

    Three novel series of N3-substituted imidacloprid derivatives were designed and synthesized, and their structures were identified on the basis of satisfactory analytical and spectral ((1)H NMR, (13)C NMR, MS, elemental analysis, and X-ray) data. Preliminary bioassays indicated that all of the derivatives exhibited significant insecticidal activities against Aphis craccivora, with LC50 values ranging from 0.00895 to 0.49947 mmol/L, and the insecticidal activities of some of them were comparable to those of the control imidacloprid. Some key structural features related to their insecticidal activities were identified, and the binding modes between target compounds and nAChR model were also further explored by molecular docking. By comparing the interaction features of imidacloprid and compound 26 with highest insecticidal activity, the origin of the high insecticidal activity of compound 26 was identified. On the basis of the conformations generated by molecular docking, a satisfactory 2D-QSAR model with six selected descriptors was built using genetic algorithm-multiple linear regression (GA-MLR) method. The analysis of the built model showed the molecular size, shape, and the ability to form hydrogen bond were important for insecticidal potency. The information obtained in the study will be very helpful for the design of new derivatives with high insecticidal activities. PMID:24834971

  3. The divalent metal ion in the active site of uteroferrin modulates substrate binding and catalysis

    PubMed Central

    Mitić, Nataša; Hadler, Kieran S.; Gahan, Lawrence R; Hengge, Alvan C.; Schenk, Gerhard

    2010-01-01

    The purple acid phosphatases (PAP) are binuclear metallohydrolases that catalyze the hydrolysis of a broad range of phosphomonoester substrates. The mode of substrate binding during catalysis and the identity of the nucleophile is subject to debate. Here, we used native Fe3+-Fe2+ pig PAP (uteroferrin; Uf) and its Fe3+-Mn2+ derivative to investigate the effect of metal ion substitution on the mechanism of catalysis. Replacement of the Fe2+ by Mn2+ lowers the reactivity of Uf. However, using stopped-flow measurements it could be shown that this replacement facilitates approximately a ten-fold faster reaction between both substrate and inorganic phosphate with the chromophoric Fe3+ site. These data also indicate that in both metal forms of Uf, phenyl phosphate hydrolysis occurs faster than formation of a μ-1,3 phosphate complex. The slower rate of interaction between substrate and the Fe3+ site relative to catalysis suggests that the substrate is hydrolyzed while coordinated only to the divalent metal ion. The likely nucleophile is a water molecule in the second coordination sphere, activated by a hydroxide terminally coordinated to Fe3+. The faster rates of interaction with the Fe3+ site in the Fe3+-Mn2+ derivative than the native Fe3+-Fe2+ form are likely mediated via a hydrogen bond network connecting the first and second coordination spheres, and illustrate how the selection of metal ions may be important in fine-tuning the function of this enzyme. PMID:20433174

  4. Increased activity of antagonists of growth hormone-releasing hormone substituted at positions 8, 9, and 10

    PubMed Central

    Varga, Jozsef L.; Schally, Andrew V.; Horvath, Judit E.; Kovacs, Magdolna; Halmos, Gabor; Groot, Kate; Toller, Gabor L.; Rekasi, Zoltan; Zarandi, Marta

    2004-01-01

    Antagonists of human growth hormone-releasing hormone (hGHRH) with increased potency and improved enzymatic and chemical stability are needed for potential clinical applications. We synthesized 21 antagonistic analogs of hGHRH(1-29)NH2, substituted at positions 8, 9, and 10 of the common core sequence {phenylacetyl-Tyr1, d-Arg2,28, para-chloro-phenylalanine 6, Arg9/homoarginine 9, Tyr10/O-methyltyrosine 10, α-aminobutyric acid 15, norleucine 27, Har29} hGHRH(1-29)NH2. Inhibitory effects on hGHRH-induced GH release were evaluated in vitro in a superfused rat pituitary system, as well as in vivo after i.v. injection into rats. The binding affinities of the peptides to pituitary GHRH receptors were also determined. Introduction of para-amidinophenylalanine 10 yielded antagonists JV-1-62 and -63 with the highest activities in vitro and lowest receptor dissociation constants (Ki = 0.057-0.062 nM). Antagonists JV-1-62 and -63 also exhibited the strongest effect in vivo, significantly (P < 0.05-0.001) inhibiting hGHRH-induced GH release for at least 1 h. Para-aminophenylalanine 10 and O-ethyltyrosine 10 substitutions yielded antagonists potent in vitro, but His10, 3,3′-diphenylalanine 10, 2-naphthylalanine 10, and cyclohexylalanine 10 modifications were detrimental. Antagonists containing citrulline 9 (in MZ-J-7-72), amidinophenylalanine 9 (in JV-1-65), His9, d-Arg9, citrulline 8, Ala8, d-Ala8, or α-aminobutyric acid 8 substituents also had high activity and receptor affinity in vitro. However, in vitro potencies of analogs with substitution in position 9 correlated poorly with acute endocrine effects in vivo, as exemplified by the weak and/or short inhibitory actions of antagonists JV-1-65 and MZ-J-7-72 on GH release in vivo. Nevertheless, antagonist JV-1-65 was more potent than JV-1-63 in tests on inhibition of the growth of human prostatic and lung cancer lines xenografted into nude mice. This indicates that oncological activity may be based on several mechanisms

  5. Docking Studies, Synthesis, Characterization and Evaluation of Their Antioxidant and Cytotoxic Activities of Some Novel Isoxazole-Substituted 9-Anilinoacridine Derivatives

    PubMed Central

    Kalirajan, R.; Rafick, M. H. Mohammed; Sankar, S.; Jubie, S.

    2012-01-01

    A convenient synthesis of novel isoxazole-substituted 9-anilinoacridine derivatives 5a–j was reported. The compounds were confirmed by physical and analytical data and screened for in vitro antioxidant activity by DPPH method, reducing power assay and total antioxidant capacity method. The cytotoxic activity of the compounds was also studied in HEp-2 cell line. The docking studies of the synthesized compounds were performed towards the key nucleoside dsDNA by using AutoDock vina 4.0 programme. All the isoxazole-substituted compounds have significant activities. PMID:22593663

  6. Skin Substitutes

    PubMed Central

    Howe, Nicole; Cohen, George

    2014-01-01

    In a relatively short timespan, a wealth of new skin substitutes made of synthetic and biologically derived materials have arisen for the purpose of wound healing of various etiologies. This review article focuses on providing an overview of skin substitutes including their indications, contraindications, benefits, and limitations. The result of this overview was an appreciation of the vast array of options available for clinicians, many of which did not exist a short time ago. Yet, despite the rapid expansion this field has undergone, no ideal skin substitute is currently available. More research in the field of skin substitutes and wound healing is required not only for the development of new products made of increasingly complex biomolecular material, but also to compare the existing skin substitutes. PMID:25371771

  7. The active site of yeast phosphatidylinositol synthase Pis1 is facing the cytosol.

    PubMed

    Bochud, Arlette; Conzelmann, Andreas

    2015-05-01

    Five yeast enzymes synthesizing various glycerophospholipids belong to the CDP-alcohol phosphatidyltransferase (CAPT) superfamily. They only share the so-called CAPT motif, which forms the active site of all these enzymes. Bioinformatic tools predict the CAPT motif of phosphatidylinositol synthase Pis1 as either ER luminal or cytosolic. To investigate the membrane topology of Pis1, unique cysteine residues were introduced into either native or a Cys-free form of Pis1 and their accessibility to the small, membrane permeating alkylating reagent N-ethylmaleimide (NEM) and mass tagged, non-permeating maleimides, in the presence and absence of non-denaturing detergents, was monitored. The results clearly point to a cytosolic location of the CAPT motif. Pis1 is highly sensitive to non-denaturing detergent, and low concentrations (0.05%) of dodecylmaltoside change the accessibility of single substituted Cys in the active site of an otherwise cysteine free version of Pis1. Slightly higher detergent concentrations inactivate the enzyme. Removal of the ER retrieval sequence from (wt) Pis1 enhances its activity, again suggesting an influence of the lipid environment. The central 84% of the Pis1 sequence can be aligned and fitted onto the 6 transmembrane helices of two recently crystallized archaeal members of the CAPT family. Results delineate the accessibility of different parts of Pis1 in their natural context and allow to critically evaluate the performance of different cysteine accessibility methods. Overall the results show that cytosolically made inositol and CDP-diacylglycerol can access the active site of the yeast PI synthase Pis1 from the cytosolic side and that Pis1 structure is strongly affected by mild detergents. PMID:25687304

  8. Exploring the size adaptability of the B ring binding zone of the colchicine site of tubulin with para-nitrogen substituted isocombretastatins.

    PubMed

    Jiménez, Carmen; Ellahioui, Younes; Álvarez, Raquel; Aramburu, Laura; Riesco, Alejandra; González, Myriam; Vicente, Alba; Dahdouh, Abdelaziz; Ibn Mansour, Ahmed; Jiménez, Carlos; Martín, Diego; Sarmiento, Rogelio G; Medarde, Manuel; Caballero, Esther; Peláez, Rafael

    2015-07-15

    We have synthesized and assayed dimethylaminophenyl, pyrrolidin-1-ylphenyl and carbazole containing phenstatins and isocombretastatins as analogues of the highly potent indoleisocombretastatins with extended or reduced ring sizes. This is an attempt to explore beyond the structural constraints of the X-ray crystal structures the zone of the colchicine site where the tropolone ring of colchicine binds to tubulin (zone 1). The isocombretastatins display up to 30 fold increased water solubility when compared with combretastatin A-4, potent inhibition of tubulin polymerization, and nanomolar cytotoxicities against several human cancer cell lines irrespective of the size of the B ring. On the other hand, substitutions ortho to the nitrogen cause an important reduction in potency. We have also shown that representative compounds inhibit autophagy. These results show that zone 1 can adapt to systems of different size as far as they stay in a common plane, but does not tolerate substituents protruding above or below it. These results can help in the understanding of the binding modes of structures with similar systems and in the design of new colchicine site ligands. PMID:26092446

  9. Tuning Metal-Organic Frameworks with Open-Metal Sites and Its Origin for Enhancing CO2 Affinity by Metal Substitution.

    PubMed

    Park, Joonho; Kim, Heejin; Han, Sang Soo; Jung, Yousung

    2012-04-01

    Reducing anthropogenic carbon emission is a problem that requires immediate attention. Metal-organic frameworks (MOFs) have emerged as a promising new materials platform for carbon capture, of which Mg-MOF-74 offers chemospecific affinity toward CO2 because of the open Mg sites. Here we tune the binding affinity of CO2 for M-MOF-74 by metal substitution (M = Mg, Ca, and the first transition metal elements) and show that Ti- and V-MOF-74 can have an enhanced affinity compared to Mg-MOF-74 by 6-9 kJ/mol. Electronic structure calculations suggest that the origin of the major affinity trend is the local electric field effect of the open metal site that stabilizes CO2, but forward donation from the lone-pair electrons of CO2 to the empty d-levels of transition metals as in a weak coordination bond makes Ti and V have an even higher binding strength than Mg, Ca, and Sc. PMID:26286404

  10. Surface-active ionic liquids in micellar catalysis: impact of anion selection on reaction rates in nucleophilic substitutions.

    PubMed

    Cognigni, Alice; Gaertner, Peter; Zirbs, Ronald; Peterlik, Herwig; Prochazka, Katharina; Schröder, Christian; Bica, Katharina

    2016-05-21

    A series of surface-active ionic liquids based on the 1-dodecyl-3-methylimidazolium cation and different anions such as halides and alkylsulfates was synthesized. The aggregation behavior of these ionic liquids in water was characterized by surface tension, conductivity measurements and UV-Vis spectroscopy in order to determine the critical micelle concentration (CMC) and to provide aggregation parameters. The determination of surface activity and aggregation properties of amphiphilic ionic liquids was accompanied by SAXS studies on selected surface-active ionic liquids. The application of these surface-active ionic liquids with different anions was tested in nucleophilic substitution reactions for the degradation of organophosphorus compounds. Kinetic studies via UV-Vis spectrophotometry showed a strong acceleration of the reaction in the micellar system compared to pure water. In addition, an influence of the anion was observed, resulting in a correlation between the anion binding to the micelle and the reaction rate constants, indicating that the careful choice of the surface-active ionic liquid can considerably affect the outcome of reactions. PMID:27121134

  11. Computer-aided identification of novel 3,5-substituted rhodanine derivatives with activity against Staphylococcus aureus DNA gyrase.

    PubMed

    Werner, Malela M; Li, Zhiyu; Zauhar, Randy J

    2014-04-01

    Methicillin resistant Staphylococcus aureus (MRSA) is among the major drug resistant bacteria that persist in both the community and clinical settings due to resistance to commonly used antimicrobials. This continues to fuel the need for novel compounds that are active against this organism. For this purpose we have targeted the type IIA bacterial topoisomerase, DNA gyrase, an essential enzyme involved in bacterial replication, through the ATP-dependent supercoiling of DNA. The virtual screening tool Shape Signatures was applied to screen a large database for agents with shape similar to Novobiocin, a known gyrase B inhibitor. The binding energetics of the top hits from this initial screen were further validated by molecular docking. Compounds with the highest score on available crystal structure of homologous DNA gyrase from Thermus thermophilus were selected. From this initial set of compounds, several rhodanine-substituted derivatives had the highest antimicrobial activity against S. aureus, as determined by minimal inhibitory concentration assays, with Novobiocin as the positive control. Further activity validation of the rhodanine compounds through biochemical assays confirmed their inhibition of both the supercoiling and the ATPase activity of DNA gyrase. Subsequent docking and molecular dynamics on the crystal structure of DNA gyrase from S. aureus when it became available, provides further rationalization of the observed biochemical activity and understanding of the receptor-ligand interactions. A regression model for MIC prediction against S. aureus is generated based on the current molecules studied as well as other rhodanines derivatives found in the literature. PMID:24629449

  12. Mechanistic and Bioinformatic Investigation of a Conserved Active Site Helix in α-Isopropylmalate Synthase from Mycobacterium tuberculosis, a Member of the DRE-TIM Metallolyase Superfamily

    PubMed Central

    2015-01-01

    The characterization of functionally diverse enzyme superfamilies provides the opportunity to identify evolutionarily conserved catalytic strategies, as well as amino acid substitutions responsible for the evolution of new functions or specificities. Isopropylmalate synthase (IPMS) belongs to the DRE-TIM metallolyase superfamily. Members of this superfamily share common active site elements, including a conserved active site helix and an HXH divalent metal binding motif, associated with stabilization of a common enolate anion intermediate. These common elements are overlaid by variations in active site architecture resulting in the evolution of a diverse set of reactions that include condensation, lyase/aldolase, and carboxyl transfer activities. Here, using IPMS, an integrated biochemical and bioinformatics approach has been utilized to investigate the catalytic role of residues on an active site helix that is conserved across the superfamily. The construction of a sequence similarity network for the DRE-TIM metallolyase superfamily allows for the biochemical results obtained with IPMS variants to be compared across superfamily members and within other condensation-catalyzing enzymes related to IPMS. A comparison of our results with previous biochemical data indicates an active site arginine residue (R80 in IPMS) is strictly required for activity across the superfamily, suggesting that it plays a key role in catalysis, most likely through enolate stabilization. In contrast, differential results obtained from substitution of the C-terminal residue of the helix (Q84 in IPMS) suggest that this residue plays a role in reaction specificity within the superfamily. PMID:24720347

  13. Development of Novel Sugar Isomerases by Optimization of Active Sites in Phosphosugar Isomerases for Monosaccharides

    PubMed Central

    Yeom, Soo-Jin; Kim, Yeong-Su

    2013-01-01

    Phosphosugar isomerases can catalyze the isomerization of not only phosphosugar but also of monosaccharides, suggesting that the phosphosugar isomerases can be used as sugar isomerases that do not exist in nature. Determination of active-site residues of phosphosugar isomerases, including ribose-5-phosphate isomerase from Clostridium difficile (CDRPI), mannose-6-phosphate isomerase from Bacillus subtilis (BSMPI), and glucose-6-phosphate isomerase from Pyrococcus furiosus (PFGPI), was accomplished by docking of monosaccharides onto the structure models of the isomerases. The determinant residues, including Arg133 of CDRPI, Arg192 of BSMPI, and Thr85 of PFGPI, were subjected to alanine substitutions and found to act as phosphate-binding sites. R133D of CDRPI, R192 of BSMPI, and T85Q of PFGPI displayed the highest catalytic efficiencies for monosaccharides at each position. These residues exhibited 1.8-, 3.5-, and 4.9-fold higher catalytic efficiencies, respectively, for the monosaccharides than the wild-type enzyme. However, the activities of these 3 variant enzymes for phosphosugars as the original substrates disappeared. Thus, R133D of CDRPI, R192 of BSMPI, and T85Q of PFGPI are no longer phosphosugar isomerases; instead, they are changed to a d-ribose isomerase, an l-ribose isomerase, and an l-talose isomerase, respectively. In this study, we used substrate-tailored optimization to develop novel sugar isomerases which are not found in nature based on phosphosugar isomerases. PMID:23204422

  14. The effect of Ca-substitution in La-site on the magnetic properties of La{sub 2}CoMnO{sub 6}

    SciTech Connect

    Li, Qiu-hang; Li, Na; Hu, Jian-zhong; Han, Qi; Ma, Qing-shan; Ge, Lei; Xiao, Biao; Xu, Ming-xiang

    2014-07-21

    Double perovskite La{sub 2-x}Ca{sub x}CoMnO{sub 6} (x = 0, 0.1, 0.2, 0.5) ceramics samples were synthesized by the standard solid-state reaction method. The crystal structures have been confirmed to be monoclinic perovskite structure with space group of P2{sub 1}/n at room temperature by the powder x-ray diffraction (Cu-Kα). The grains of the samples are relatively dense and distribute in random directions from the scanning electron microscope patterns. Holes are doped into La{sub 2}CoMnO{sub 6} with the substitution of the Ca{sup 2+} ions in La-site, and further lead to the valences of Mn ions and Co ions transforming from Mn{sup 4+} and Co{sup 2+} to Mn{sup 3+} and Co{sup 3+}, respectively. Ferromagnetic coupling of Mn{sup 4+}-O{sup 2–}-Co{sup 2+} and antiferromagnetic coupling of Mn{sup 3+}-O{sup 2–}-Co{sup 3+} are coexistent in La{sub 2-x}Ca{sub x}CoMnO{sub 6} system, which correspond to two transitions existed in field-cooling magnetization versus temperature curves. Curie temperature of La{sub 2-x}Ca{sub x}CoMnO{sub 6} system decreases with the increasing of Ca-substitution amount. The coexistence of different magnetic phases leads to the exchange bias effect.

  15. The T210M Substitution in the HLA-a*02:01 gp100 Epitope Strongly Affects Overall Proteasomal Cleavage Site Usage and Antigen Processing.

    PubMed

    Textoris-Taube, Kathrin; Keller, Christin; Liepe, Juliane; Henklein, Petra; Sidney, John; Sette, Alessandro; Kloetzel, Peter M; Mishto, Michele

    2015-12-18

    MHC class I-restricted epitopes, which carry a tumor-specific mutation resulting in improved MHC binding affinity, are preferred T cell receptor targets in innovative adoptive T cell therapies. However, T cell therapy requires efficient generation of the selected epitope. How such mutations may affect proteasome-mediated antigen processing has so far not been studied. Therefore, we analyzed by in vitro experiments the effect on antigen processing and recognition of a T210M exchange, which previously had been introduced into the melanoma gp100209-217 tumor epitope to improve the HLA-A*02:01 binding and its immunogenicity. A quantitative analysis of the main steps of antigen processing shows that the T210M exchange affects proteasomal cleavage site usage within the mutgp100201-230 polypeptide, leading to the generation of an unique set of cleavage products. The T210M substitution qualitatively affects the proteasome-catalyzed generation of spliced and non-spliced peptides predicted to bind HLA-A or -B complexes. The T210M substitution also induces an enhanced production of the mutgp100209-217 epitope and its N-terminally extended peptides. The T210M exchange revealed no effect on ERAP1-mediated N-terminal trimming of the precursor peptides. However, mutant N-terminally extended peptides exhibited significantly increased HLA-A*02:01 binding affinity and elicited CD8(+) T cell stimulation in vitro similar to the wtgp100209-217 epitope. Thus, our experiments demonstrate that amino acid exchanges within an epitope can result in the generation of an altered peptide pool with new antigenic peptides and in a wider CD8(+) T cell response also towards N-terminally extended versions of the minimal epitope. PMID:26507656

  16. Active-Site-Accessible, Porphyrinic Metal;#8722;Organic Framework Materials

    SciTech Connect

    Farha, Omar K.; Shultz, Abraham M.; Sarjeant, Amy A.; Nguyen, SonBinh T.; Hupp, Joseph T.

    2012-02-06

    On account of their structural similarity to cofactors found in many metallo-enzymes, metalloporphyrins are obvious potential building blocks for catalytically active, metal-organic framework (MOF) materials. While numerous porphyrin-based MOFs have already been described, versions featuring highly accessible active sites and permanent microporosity are remarkably scarce. Indeed, of the more than 70 previously reported porphyrinic MOFs, only one has been shown to be both permanently microporous and contain internally accessible active sites for chemical catalysis. Attempts to generalize the design approach used in this single successful case have failed. Reported here, however, is the synthesis of an extended family of MOFs that directly incorporate a variety of metalloporphyrins (specifically Al{sup 3+}, Zn{sup 2+}, Pd{sup 2+}, Mn{sup 3+}, and Fe{sup 3+} complexes). These robust porphyrinic materials (RPMs) feature large channels and readily accessible active sites. As an illustrative example, one of the manganese-containing RPMs is shown to be catalytically competent for the oxidation of alkenes and alkanes.

  17. Nest predation increases with parental activity: Separating nest site and parental activity effects

    USGS Publications Warehouse

    Martin, T.E.; Scott, J.; Menge, C.

    2000-01-01

    Alexander Skutch hypothesized that increased parental activity can increase the risk of nest predation. We tested this hypothesis using ten open-nesting bird species in Arizona, USA. Parental activity was greater during the nestling than incubation stage because parents visited the nest frequently to feed their young during the nestling stage. However, nest predation did not generally increase with parental activity between nesting stages across the ten study species. Previous investigators have found similar results. We tested whether nest site effects might yield higher predation during incubation because the most obvious sites are depredated most rapidly. We conducted experiments using nest sites from the previous year to remove parental activity. Our results showed that nest sites have highly repeatable effects on nest predation risk; poor nest sites incurred rapid predation and caused predation rates to be greater during the incubation than nestling stage. This pattern also was exhibited in a bird species with similar (i.e. controlled) parental activity between nesting stages. Once nest site effects are taken into account, nest predation shows a strong proximate increase with parental activity during the nestling stage within and across species. Parental activity and nest sites exert antagonistic influences on current estimates of nest predation between nesting stages and both must be considered in order to understand current patterns of nest predation, which is an important source of natural selection.

  18. Nest predation increases with parental activity: separating nest site and parental activity effects.

    PubMed Central

    Martin, T E; Scott, J; Menge, C

    2000-01-01

    Alexander Skutch hypothesized that increased parental activity can increase the risk of nest predation. We tested this hypothesis using ten open-nesting bird species in Arizona, USA. Parental activity was greater during the nestling than incubation stage because parents visited the nest frequently to feed their young during the nestling stage. However, nest predation did not generally increase with parental activity between nesting stages across the ten study species. Previous investigators have found similar results. We tested whether nest site effects might yield higher predation during incubation because the most obvious sites are depredated most rapidly. We conducted experiments using nest sites from the previous year to remove parental activity. Our results showed that nest sites have highly repeatable effects on nest predation risk; poor nest sites incurred rapid predation and caused predation rates to be greater during the incubation than nestling stage. This pattern also was exhibited in a bird species with similar (i.e. controlled) parental activity between nesting stages. Once nest site effects are taken into account, nest predation shows a strong proximate increase with parental activity during the nestling stage within and across species. Parental activity and nest sites exert antagonistic influences on current estimates of nest predation between nesting stages and both must be considered in order to understand current patterns of nest predation, which is an important source of natural selection. PMID:11413645

  19. Design, Synthesis, and Evaluation of in Vitro and in Vivo Anticancer Activity of 4-Substituted Coumarins: A Novel Class of Potent Tubulin Polymerization Inhibitors.

    PubMed

    Cao, Dong; Liu, Yibin; Yan, Wei; Wang, Chunyu; Bai, Peng; Wang, Taijin; Tang, Minghai; Wang, Xiaoyan; Yang, Zhuang; Ma, Buyun; Ma, Liang; Lei, Lei; Wang, Fang; Xu, Bixue; Zhou, Yuanyuan; Yang, Tao; Chen, Lijuan

    2016-06-23

    In this paper, a series of novel 4-substituted coumarin derivatives were synthesized. Among these compounds 34, 39, 40, 43, 62, 65, and 67 exhibited significant antiproliferative activity toward a panel of tumor cell lines at subnanomolar IC50 values. Compound 65 showed potent antiproliferative ability (IC50 values of 7-47 nM) and retained full activity in multidrug resistant cancer cells. Compound 65 caused G2/M phase arrest and interacted with the colchicine-binding site in tubulin, as confirmed by immune-fluorescence staining, microtubule dynamics assays, and competition assays with N,N'-ethylene-bis(iodoacetamide). Compound 65 reduced the cell migration and disrupted capillary-like tube formation in HUVEC cells. Importantly, compound 65 significantly and dose-dependently reduced tumor growth in four xenografts models including paclitaxel sensitive and resistant ovarian tumors (A2780s and A2780/T), adrmicycin sensitive and resistant breast tumors (MCF-7 and MCF-7/ADR), suggesting that compound 65 is a promising novel antimitotic compound for the potential treatment of cancer. PMID:27213819

  20. Oxidative stability and antioxidant enzyme activities of dry-cured bacons as affected by the partial substitution of NaCl with KCl.

    PubMed

    Wu, Haizhou; Yan, Wenjing; Zhuang, Hong; Huang, Mingming; Zhao, Jianying; Zhang, Jianhao

    2016-06-15

    This study investigated the influence of partial substitution of NaCl with KCl on protein and lipid oxidation as well as antioxidant enzyme activities in dry-cured bacons during processing. The partial substitution was 0% KCl (I), 40% KCl (II), and 70% KCl (III). Compared with 0% KCl (I), the substitution of 40% NaCl with KCl did not significantly influence the protein and lipid oxidation and antioxidant enzyme activities. The bacons that were treated with 70% KCl treatment (III) showed increased lipid oxidation and antioxidant enzyme GSH-Px activity, whereas samples treated with formulas I and II showed higher protein oxidation and antioxidant enzyme catalase activity. These results demonstrate that the substitution of NaCl with KCl by more than 40% may significantly affect protein and lipid oxidation and that for the substitution of NaCl in further processed meat products with other chloride salts, salt content is very important for control of protein and lipid biochemical changes in finished products. PMID:26868571

  1. Identification of Ice Nucleation Active Sites on Silicate Dust Particles

    NASA Astrophysics Data System (ADS)

    Zolles, Tobias; Burkart, Julia; Häusler, Thomas; Pummer, Bernhard; Hitzenberger, Regina; Grothe, Hinrich

    2015-04-01

    Mineral dusts originating from Earth's crust are known to be important atmospheric ice nuclei. In agreement with earlier studies, feldspar was found as the most active of the tested natural mineral dusts [1-3]. Nevertheless, among those structures K-feldspar showed by far the highest ice nucleation activity. In this study, the reasons for its activity and the difference in the activity of the different feldspars were investigated in closer details. Conclusions are drawn from scanning electron microscopy, X-ray powder diffraction, infrared spectroscopy, and oil-immersion freezing experiments. We give a potential explanation of the increased ice nucleation activity of K-feldspar. The ice nucleating sites are very much dependent on the alkali ion present by altering the water structure and the feldspar surface. The higher activity of K-feldspar can be attributed to the presence of potassium ions on the surface and surface bilayer. The alkali-ions have different hydration shells and thus an influence on the ice nucleation activity of feldspar. Chaotropic behavior of Calcium and Sodium ions are lowering the ice nucleation potential of the other feldspars, while kosmotropic Potassium has a neutral or even positive effect. Furthermore we investigated the influence of milling onto the ice nucleation of quartz particles. The ice nucleation activity can be increased by mechanical milling, by introducing more molecular, nucleation active defects to the particle surface. This effect is larger than expected by plane surface increase. [1] Atkinson et al. The Importance of Feldspar for Ice Nucleation by Mineral Dust in Mixed-Phase Clouds. Nature 2013, 498, 355-358. [2] Yakobi-Hancock et al.. Feldspar Minerals as Efficient Deposition Ice Nuclei. Atmos. Chem. Phys. 2013, 13, 11175-11185. [3] Zolles et al. Identification of Ice Nucleation Active Sites on Feldspar Dust Particles. J. Phys. Chem. A 2015 accepted.

  2. Process, Goal and Social Interaction Differences in Recreation: What Makes an Activity Substitutable.

    ERIC Educational Resources Information Center

    Baumgartner, Robert; Heberlein, Thomas A.

    Two recreational activities, deer hunting and goose hunting, both similar in form, are compared. It was hypothesized that the activity for which participants rated the process, the goal, and the social interaction as most important to the experience and for which participants showed the strongest family ties and social support for participation…

  3. 5-Alkyl-2-urea-Substituted Pyridines: Identification of Efficacious Glucokinase Activators with Improved Properties.

    PubMed

    Kohn, Todd J; Du, Xiaohui; Lai, SuJen; Xiong, YuMei; Komorowski, Renee; Veniant, Murielle; Fu, Zice; Jiao, Xianyun; Pattaropong, Vatee; Chow, David; Cardozo, Mario; Jin, Lixia; Conn, Marion; DeWolf, Walter E; Kraser, Christopher F; Hinklin, Ronald J; Boys, Mark L; Medina, Julio C; Houze, Jonathan; Dransfield, Paul; Coward, Peter

    2016-07-14

    Two 1-(4-aryl-5-alkyl-pyridin-2-yl)-3-methylurea glucokinase activators were identified with robust in vivo efficacy. These two compounds possessed higher solubilities than the previously identified triaryl compounds (i.e., AM-2394). Structure-activity relationship studies are presented along with relevant pharmacokinetic and in vivo data. PMID:27437074

  4. Active Sites Environmental Monitoring Program. FY 1993: Annual report

    SciTech Connect

    Morrissey, C.M.; Ashwood, T.L.; Hicks, D.S.; Marsh, J.D.

    1994-08-01

    This report continues a series of annual and semiannual reports that present the results of the Active Sites Environmental Monitoring Program (ASEMP) monitoring activities. The report details monitoring data for fiscal year (FY) 1993 and is divided into three major areas: SWSA 6 [including tumulus pads, Interim Waste Management Facility (IWMF), and other sites], the low-level Liquid-Waste Solidification Project (LWSP), and TRU-waste storage facilities in SWSA 5 N. The detailed monitoring methodology is described in the second revision of the ASEMP program plan. This report also presents a summary of the methodology used to gather data for each major area along with the results obtained during FY 1993.

  5. Removal potential of toxic 2378-substituted PCDD/F from incinerator flue gases by waste-derived activated carbons.

    PubMed

    Hajizadeh, Yaghoub; Onwudili, Jude A; Williams, Paul T

    2011-06-01

    The application of activated carbons has become a commonly used emission control protocol for the removal or adsorption of persistent organic pollutants from the flue gas streams of waste incinerators. In this study, the 2378-substituted PCDD/F removal efficiency of three types of activated carbons derived from the pyrolysis of refuse derived fuel, textile waste and scrap tyre was investigated and compared with that of a commercial carbon. Experiments were carried out in a laboratory scale fixed-bed reactor under a simulated flue gas at 275°C with a reaction period of four days. The PCDD/F in the solid matrices and exhaust gas, were analyzed using gas chromatography coupled with a triple quadrupole mass spectrometer. In the absence of activated carbon adsorbent, there was a significant increase in the concentration of toxic PCDD/F produced in the reacted flyash, reaching up to 6.6 times higher than in the raw flyash. In addition, there was a substantial release of PCDD/F into the gas phase, which was found in the flue gas trapping system. By application of the different commercial, refuse derived fuel, textile and tyre activated carbons the total PCDD/F toxic equivalent removal efficiencies in the exhaust gas stream were 58%, 57%, 64% and 52%, respectively. In general, the removal of the PCDDs was much higher with an average of 85% compared to PCDFs at 41%. Analysis of the reacted activated carbons showed that there was some formation of PCDD/F, for instance, a total of 60.6 μg I-TEQ kg(-1) toxic PCDD/F was formed in the refuse derived fuel activated carbon compared to 34 μg I-TEQ kg(-1) in the commercial activated carbon. The activated carbons derived from the pyrolysis of waste, therefore, showed good potential as a control material for PCDD/F emissions in waste incinerator flue gases. PMID:21334872

  6. Removal potential of toxic 2378-substituted PCDD/F from incinerator flue gases by waste-derived activated carbons

    SciTech Connect

    Hajizadeh, Yaghoub; Onwudili, Jude A.; Williams, Paul T.

    2011-06-15

    The application of activated carbons has become a commonly used emission control protocol for the removal or adsorption of persistent organic pollutants from the flue gas streams of waste incinerators. In this study, the 2378-substituted PCDD/F removal efficiency of three types of activated carbons derived from the pyrolysis of refuse derived fuel, textile waste and scrap tyre was investigated and compared with that of a commercial carbon. Experiments were carried out in a laboratory scale fixed-bed reactor under a simulated flue gas at 275 deg. C with a reaction period of four days. The PCDD/F in the solid matrices and exhaust gas, were analyzed using gas chromatography coupled with a triple quadrupole mass spectrometer. In the absence of activated carbon adsorbent, there was a significant increase in the concentration of toxic PCDD/F produced in the reacted flyash, reaching up to 6.6 times higher than in the raw flyash. In addition, there was a substantial release of PCDD/F into the gas phase, which was found in the flue gas trapping system. By application of the different commercial, refuse derived fuel, textile and tyre activated carbons the total PCDD/F toxic equivalent removal efficiencies in the exhaust gas stream were 58%, 57%, 64% and 52%, respectively. In general, the removal of the PCDDs was much higher with an average of 85% compared to PCDFs at 41%. Analysis of the reacted activated carbons showed that there was some formation of PCDD/F, for instance, a total of 60.6 {mu}g I-TEQ kg{sup -1} toxic PCDD/F was formed in the refuse derived fuel activated carbon compared to 34 {mu}g I-TEQ kg{sup -1} in the commercial activated carbon. The activated carbons derived from the pyrolysis of waste, therefore, showed good potential as a control material for PCDD/F emissions in waste incinerator flue gases.

  7. A comprehensive search for calcium binding sites critical for TMEM16A calcium-activated chloride channel activity.

    PubMed

    Tien, Jason; Peters, Christian J; Wong, Xiu Ming; Cheng, Tong; Jan, Yuh Nung; Jan, Lily Yeh; Yang, Huanghe

    2014-01-01

    TMEM16A forms calcium-activated chloride channels (CaCCs) that regulate physiological processes such as the secretions of airway epithelia and exocrine glands, the contraction of smooth muscles, and the excitability of neurons. Notwithstanding intense interest in the mechanism behind TMEM16A-CaCC calcium-dependent gating, comprehensive surveys to identify and characterize potential calcium sensors of this channel are still lacking. By aligning distantly related calcium-activated ion channels in the TMEM16 family and conducting systematic mutagenesis of all conserved acidic residues thought to be exposed to the cytoplasm, we identify four acidic amino acids as putative calcium-binding residues. Alterations of the charge, polarity, and size of amino acid side chains at these sites alter the ability of different divalent cations to activate the channel. Furthermore, TMEM16A mutant channels containing double cysteine substitutions at these residues are sensitive to the redox potential of the internal solution, providing evidence for their physical proximity and solvent accessibility. PMID:24980701

  8. Room Temperature Synthesis and Antibacterial Activity of New Sulfonamides Containing N,N-Diethyl-Substituted Amido Moieties

    PubMed Central

    Ajani, Olayinka O.; Familoni, Oluwole B.; Wu, Feipeng; Echeme, Johnbull O.; Sujiang, Zheng

    2012-01-01

    Sulfonamide drugs which have brought about an antibiotic revolution in medicine are associated with a wide range of biological activities. We have synthesized a series of α-tolylsulfonamide, 1–11 and their substituted N,N-diethyl-2-(phenylmethylsulfonamido) alkanamide derivatives, 12–22 in improved and excellent yields in aqueous medium at room temperature through highly economical synthetic routes. The chemical structures of the synthesized compounds 1–22 were confirmed by analytical and spectral data such as IR, 1H- and 13C-NMR, and mass spectra. The in vitro antibacterial activity of these compounds along with standard clinical reference, streptomycin, was investigated on two key targeted organisms. It was observed that 1-(benzylsulfonyl)pyrrolidine-2-carboxylic acid, 2 emerged as the most active compound against Staphylococcus aureus at MIC value of 1.8 μg/mL while 4-(3-(diethylamino)-3-oxo-2-(phenylmethylsulfonamido) propyl)phenyl phenylmethanesulfonate, 22 was the most active sulfonamide scaffold on Escherichia coli at MIC value of 12.5 μg/mL. PMID:25374686

  9. Active sites in char gasification: Final technical report

    SciTech Connect

    Wojtowicz, M.; Lilly, W.D.; Perkins, M.T.; Hradil, G.; Calo, J.M.; Suuberg, E.M.

    1987-09-01

    Among the key variables in the design of gasifiers and combustors is the reactivity of the chars which must be gasified or combusted. Significant loss of unburned char is unacceptable in virtually any process; the provision of sufficient residence time for complete conversion is essential. A very wide range of reactivities are observed, depending upon the nature of the char in a process. The current work focuses on furthering the understanding of gasification reactivities of chars. It has been well established that the reactivity of char to gasification generally depends upon three principal factors: (1) the concentration of ''active sites'' in the char; (2) mass transfer within the char; and (3) the type and concentration of catalytic impurities in the char. The present study primarily addresses the first factor. The subject of this research is the origin, nature, and fate of active sites in chars derived from parent hydrocarbons with coal-like structure. The nature and number of the active sites and their reactivity towards oxygen are examined in ''model'' chars derived from phenol-formaldehyde type resins. How the active sites are lost by the process of thermal annealing during heat treatment of chars are studied, and actual rate for the annealing process is derived. Since intrinsic char reactivities are of primary interest in the present study, a fair amount of attention was given to the model char synthesis and handling so that the effect of catalytic impurities and oxygen-containing functional groups in the chemical structure of the material were minimized, if not completely eliminated. The project would not be considered complete without comparing characteristic features of synthetic chars with kinetic behavior exhibited by natural chars, including coal chars.

  10. Enantioselective synthesis and antioxidant activity of 3,4,5-substituted piperidine derivatives.

    PubMed

    Kim, Jin Ho; Shyam, Pranab K; Kim, Mi Jeong; Lee, Hwa-Jung; Lee, Jeong Tae; Jang, Hye-Young

    2016-07-01

    In this study, 3,4,5-trisubstituted piperidines were synthesized enantioselectively, and their antioxidant activity was evaluated. The 3,4,5-trisubstituted piperidines containing TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) and a spatially proximal hydroxy group showed good antioxidant activity. Some of these compounds showed IC50 values in a nanomolar range, comparable to that of TEMPO. Probably the TEMPO generated from the homolysis of the CON bond of 3,4,5-trisubstituted piperidines functions as a radical-scavenging entity, and the hydroxy group of piperidines has a synergistic effect to the antioxidant activity. PMID:27177825

  11. Identification of catalytically important residues in the active site of Escherichia coli transaldolase.

    PubMed

    Schörken, U; Thorell, S; Schürmann, M; Jia, J; Sprenger, G A; Schneider, G

    2001-04-01

    The roles of invariant residues at the active site of transaldolase B from Escherichia coli have been probed by site-directed mutagenesis. The mutant enzymes D17A, N35A, E96A, T156A, and S176A were purified from a talB-deficient host and analyzed with respect to their 3D structure and kinetic behavior. X-ray analysis showed that side chain replacement did not induce unanticipated structural changes in the mutant enzymes. Three mutations, N35A, E96A, and T156A resulted mainly in an effect on apparent kcat, with little changes in apparent Km values for the substrates. Residues N35 and T156 are involved in the positioning of a catalytic water molecule at the active site and the side chain of E96 participates in concert with this water molecule in proton transfer during catalysis. Substitution of Ser176 by alanine resulted in a mutant enzyme with 2.5% residual activity. The apparent Km value for the donor substrate, fructose 6-phosphate, was increased nearly fivefold while the apparent Km value for the acceptor substrate, erythrose 4-phosphate remained unchanged, consistent with a function for S176 in the binding of the C1 hydroxyl group of the donor substrate. The mutant D17A showed a 300-fold decrease in kcat, and a fivefold increase in the apparent Km value for the acceptor substrate erythrose 4-phosphate, suggesting a role of this residue in carbon-carbon bond cleavage and stabilization of the carbanion/enamine intermediate. PMID:11298760

  12. Potential sites of CFTR activation by tyrosine kinases.

    PubMed

    Billet, Arnaud; Jia, Yanlin; Jensen, Timothy J; Hou, Yue-Xian; Chang, Xiu-Bao; Riordan, John R; Hanrahan, John W

    2016-05-01

    The CFTR chloride channel is tightly regulated by phosphorylation at multiple serine residues. Recently it has been proposed that its activity is also regulated by tyrosine kinases, however the tyrosine phosphorylation sites remain to be identified. In this study we examined 2 candidate tyrosine residues near the boundary between the first nucleotide binding domain and the R domain, a region which is important for channel function but devoid of PKA consensus sequences. Mutating tyrosines at positions 625 and 627 dramatically reduced responses to Src or Pyk2 without altering the activation by PKA, suggesting they may contribute to CFTR regulation. PMID:26645934

  13. Brownian aggregation rate of colloid particles with several active sites

    SciTech Connect

    Nekrasov, Vyacheslav M.; Yurkin, Maxim A.; Chernyshev, Andrei V.; Polshchitsin, Alexey A.; Yakovleva, Galina E.; Maltsev, Valeri P.

    2014-08-14

    We theoretically analyze the aggregation kinetics of colloid particles with several active sites. Such particles (so-called “patchy particles”) are well known as chemically anisotropic reactants, but the corresponding rate constant of their aggregation has not yet been established in a convenient analytical form. Using kinematic approximation for the diffusion problem, we derived an analytical formula for the diffusion-controlled reaction rate constant between two colloid particles (or clusters) with several small active sites under the following assumptions: the relative translational motion is Brownian diffusion, and the isotropic stochastic reorientation of each particle is Markovian and arbitrarily correlated. This formula was shown to produce accurate results in comparison with more sophisticated approaches. Also, to account for the case of a low number of active sites per particle we used Monte Carlo stochastic algorithm based on Gillespie method. Simulations showed that such discrete model is required when this number is less than 10. Finally, we applied the developed approach to the simulation of immunoagglutination, assuming that the formed clusters have fractal structure.

  14. Influence of metallocene substitution on the antibacterial activity of multivalent peptide conjugates.

    PubMed

    Hoffknecht, Barbara C; Prochnow, Pascal; Bandow, Julia E; Metzler-Nolte, Nils

    2016-07-01

    Peptide dendrimers and derivatisation of peptides with metallocenes showed promising results in the search for new antibacterial agents. The two concepts are combined in this work leading to multivalent, metallocene-containing peptide derivates. These new peptides were synthesised utilising microwave assisted, copper(I) catalyzed alkyne-azide cycloaddition (CuAAC, "click" chemistry). Twelve new peptide conjugates, containing either a ferrocenoyl group or a ruthenocenoyl group on so-called ultrashort (i.e. < 5 amino acids) peptides, and ranging from monovalent to trivalent conjugates, were synthesised and their antibacterial activity was investigated by minimal inhibitory concentration (MIC) assays on five different bacterial strains. The antibacterial activity was compared to the same peptide conjugates without metallocenes. The resulting MIC values showed a significant enhancement of the antibacterial activity of these peptide conjugates against Gram-positive bacteria by the metallocenoyl groups. Additionally, the compounds with two metallocenoyl groups presented the best antibacterial activities overall. PMID:26988572

  15. Antimicrobial Activity of Novel C2-Substituted 1,4-Dihydropyridine Analogues

    PubMed Central

    Olejníková, Petra; Švorc, L’ubomír; Olšovská, Denisa; Panáková, Anna; Vihonská, Zuzana; Kovaryová, Katarína; Marchalín, Štefan

    2014-01-01

    The antimicrobial activity of 3-methyl-5-isopropyl (or ethyl) 6-methyl-4-nitrophenyl-1,4-dihydropyridine-3,5-dicarboxylate derivatives was evaluated. Prokaryotes (bacteria) appeared to be more sensitive to their antimicrobial activity than were eukaryotes (filamentous fungi). The best antibacterial activity was shown by derivative 33, which was able to inhibit the growth of Mycobacterium smegmatis (MIC33 = 9 μg.ml−1), Staphylococcus aureus (MIC33 = 25 μg.ml−1), and Escherichia coli (MIC33 = 100 μg.ml−1). In addition, derivative 4 demonstrated its antibacterial power on the acid-fast bacterial species M. smegmatis and on Gram-positive S. aureus. Focusing on the structure-activity relationship, it appears that the increase in the substituent bulk at the C2 position improved the antibacterial activity of the set of compounds studied. Derivatives 33 and 4, carrying 2-cyano-3-oxo-3-phenylprop-1-en-1-yl and allyliminomethyl groups, respectively, showed significantly higher inhibition activities on all tested microorganisms in comparison with the rest of the derivatives. This enhancement was also in good correlation with different log P values (lipophilicity parameter). PMID:24959401

  16. Structure-activity relationships of novel substituted naphthalene diimides as anticancer agents.

    PubMed

    Milelli, Andrea; Tumiatti, Vincenzo; Micco, Marialuisa; Rosini, Michela; Zuccari, Guendalina; Raffaghello, Lizzia; Bianchi, Giovanna; Pistoia, Vito; Fernando Díaz, J; Pera, Benet; Trigili, Chiara; Barasoain, Isabel; Musetti, Caterina; Toniolo, Marianna; Sissi, Claudia; Alcaro, Stefano; Moraca, Federica; Zini, Maddalena; Stefanelli, Claudio; Minarini, Anna

    2012-11-01

    Novel 1,4,5,8-naphthalenetetracarboxylic diimide (NDI) derivatives were synthesized and evaluated for their antiproliferative activity on a wide number of different tumor cell lines. The prototypes of the present series were derivatives 1 and 2 characterized by interesting biological profiles as anticancer agents. The present investigation expands on the study of structure-activity relationships of prototypes 1 and 2, namely, the influence of the different substituents of the phenyl rings on the biological activity. Derivatives 3-22, characterized by a different substituent on the aromatic rings and/or a different chain length varying from two to three carbon units, were synthesized and evaluated for their cytostatic and cytotoxic activities. The most interesting compound was 20, characterized by a linker of three methylene units and a 2,3,4-trimethoxy substituent on the two aromatic rings. It displayed antiproliferative activity in the submicromolar range, especially against some different cell lines, the ability to inhibit Taq polymerase and telomerase, to trigger caspase activation by a possible oxidative mechanism, to downregulate ERK 2 protein and to inhibit ERKs phosphorylation, without acting directly on microtubules and tubuline. Its theoretical recognition against duplex and quadruplex DNA structures have been compared to experimental thermodynamic measurements and by molecular modeling investigation leading to putative binding modes. Taken together these findings contribute to define this compound as potential Multitarget-Directed Ligands interacting simultaneously with different biological targets. PMID:22819507

  17. Ligand Displacement Reaction Paths in a Diiron Hydrogenase Active Site Model Complex.

    PubMed

    Blank, Jan H; Moncho, Salvador; Lunsford, Allen M; Brothers, Edward N; Darensbourg, Marcetta Y; Bengali, Ashfaq A

    2016-08-26

    The mechanism and energetics of CO, 1-hexene, and 1-hexyne substitution from the complexes (SBenz)2 [Fe2 (CO)6 ] (SBenz=SCH2 Ph) (1-CO), (SBenz)2 [Fe2 (CO)5 (η(2) -1-hexene)] (1-(η(2) -1-hexene)), and (SBenz)2 [Fe2 (CO)5 (η(2) -1-hexyne)] (1-(η(2) -1-hexyne)) were studied by using time-resolved infrared spectroscopy. Exchange of both CO and 1-hexyne by P(OEt)3 and pyridine, respectively, proceeds by a bimolecular mechanism. As similar activation enthalpies are obtained for both reactions, the rate-determining step in both cases is assumed to be the rotation of the Fe(CO)2 L (L=CO or 1-hexyne) unit to accommodate the incoming ligand. The kinetic profile for the displacement of 1-hexene is quite different than that for the alkyne and, in this case, both reaction channels, that is, dissociative (SN 1) and associative (SN 2), were found to be competitive. Because DFT calculations predict similar binding enthalpies of alkene and alkyne to the iron center, the results indicate that the bimolecular pathway in the case of the alkyne is lower in free energy than that of the alkene. In complexes of this type, subtle changes in the departing ligand characteristics and the nature of the mercapto bridge can influence the exchange mechanism, such that more than one reaction pathway is available for ligand substitution. The difference between this and the analogous study of (μ-pdt)[Fe(CO)3 ]2 (pdt=S(CH2 )3 S) underscores the unique characteristics of a three-atom S-S linker in the active site of diiron hydrogenases. PMID:27482938

  18. The T----C substitution at -198 of the A gamma-globin gene associated with the British form of HPFH generates overlapping recognition sites for two DNA-binding proteins.

    PubMed Central

    Fischer, K D; Nowock, J

    1990-01-01

    Defects in the developmental changes of human hemoglobin production characterized by the continued expression of fetal globin during adult life are classified as hereditary persistence of fetal hemoglobin (HPFH). Among the various molecular lesions associated with this phenotype, the non-deletion forms with point mutations in the promoter region are thought to provide mechanistic clues for gamma-globin gene regulation. The natural occurrence of four different base substitutions mapping within six nucleotides of a homopurine.homopyrimidine motif in the upstream promoter region demarcate a potential control element. To assess its importance for transcriptional activity, we compared the -202 (C----G), -198 (T----C) and -196 (C----T) HPFH mutations with the normal sequence in binding studies with nuclear proteins from erythroid and non-erythroid cells. Wildtype DNA and HPFH mutations at -202 or -196 showed only a weak protein interaction of unclear functional significance. In contrast, -198 (T----C) generated overlapping, high-affinity binding sites for two ubiquitous nuclear proteins. One cognate protein was identified as the transcription factor Sp1. The second one was termed NF-G.C as it interacted strongly with the homopolymer poly(dG).poly(dC). The generation of additional recognition sites for trans-acting factors by the -198 HPFH mutation correlated with a modest increase in promoter activity in vitro specifically with nuclear extracts from erythroid cells. The activation appears to be mediated by binding of Sp1, but it requires interaction with an erythroid-specific factor, most likely GF-1. Templates containing the -196 HPFH mutation showed a transcriptional activity identical to wildtype. This suggests that despite the topological proximity of the mutations, the HPFH phenotype may be established by different mechanisms. Images PMID:1699206

  19. Synthesis and biological activity of substituted-4,5,6,7-tetrahydrothieno pyridines: a review.

    PubMed

    Sangshetti, Jaiprakash N; Zambare, Abhay S; Khan, Firoz A Kalam; Gonjari, Indrajeet; Zaheer, Zahid

    2014-01-01

    4,5,6,7-Tetrahydrothieno pyridine is an important class of heterocyclic nucleus. Various 4,5,6,7-tetrahydrothieno pyridine derivatives have been synthesized and evaluated for various biological activities in different models with desired findings. Some analogs have shown potent biological activities and may be considered as lead molecule for the development of future drugs. Number of drug molecules are available in the market and many molecules are in clinical development containing 4,5,6,7-tetrahydrothieno pyridine nucleus as an important core. This review is an attempt to organize the chemical and biological aspects of 4,5,6,7-tetrahydrothieno pyridine analogs reported in last 20 year to till date. Review mainly focuses on the important role of the core in synthesis of drug or drug intermediates giving emphasis on synthetic schemes and biological activities of the different 4,5,6,7-tetrahydrothieno pyridine analogs. PMID:25373848

  20. Synthesis and Anticonvulsant Activity of Substituted-1,3-diazaspiro[4.5]decan-4-ones.

    PubMed

    Aboul-Enein, Mohamed Nabil; El-Azzouny, Aida Abdel Sattar; Saleh, Ola Ahmed; Amin, Kamilia Mahmoud; Maklad, Yousreya Ali; Hassan, Rasha Mohamed

    2015-08-01

    A series of novel spiroimidazolidinone derivatives 6a-d and 8a-x were synthesized and biologically evaluated for their anticonvulsant activity in the maximal electroshock seizure (MES) assay and the subcutaneous pentylenetetrazole (scPTZ) screening test. Compound 8w was the most active derivative in the scPTZ screening test with an ED50 value by about 5- and 83.6-fold lower than those of phenobarbital and ethosuximide as reference drugs, respectively. Most of the tested compounds exhibited moderate to weak activity in the MES screen test, except for 8a which displayed 100% protection at 0.09 mmol/kg. Moreover, all the test compounds did not show any minimal motor impairment in the neurotoxicity test. PMID:26032619

  1. Synthesis, in vitro antibacterial activities of a series of 3-N-substituted canthin-6-ones.

    PubMed

    Dai, Jiang-Kun; Dan, Wen-Jia; Li, Na; Du, Hong-Tao; Zhang, Ji-Wen; Wang, Jun-Ru

    2016-01-15

    An improved synthetic route of canthin-6-one was accomplished. To further enhance the antibacterial potency and improve water solubility, a series of 3-N-alkylated and 3-N-benzylated canthin-6-ones were designed and synthesized, and their in vitro antibacterial activities were evaluated. A clear structure-activity relationship with peak minimal inhibitory concentration (MIC) values of 0.98 (μg·mL(-)(1)) was investigated. Particularly, compounds 6i-r and 6t were found to be the most potent compounds with minimal inhibitory concentration (MIC) values lower than 1.95 (μg·mL(-)(1)) against Staphylococcus aureus. PMID:26681509

  2. A High-Conduction Ge Substituted Li3AsS4 Solid Electrolyte with Exceptional Low Activation Energy

    SciTech Connect

    Sahu, Gayatri; Rangasamy, Ezhiylmurugan; Li, Juchuan; Chen, Yan; An, Ke; Dudney, Nancy J; Liang, Chengdu

    2014-01-01

    Lithium-ion conducting solid electrolytes show potential to enable high-energy-density secondary batteries and offer distinctive safety features as an advantage over traditional liquid electrolytes. Achieving the combination of high ionic conductivity, low activation energy, and outstanding electrochemical stability in crystalline solid electrolytes is a challenge for the synthesis of novel solid electrolytes. Herein we report an exceptionally low activation energy (Ea) and high room temperature superionic conductivity via facile aliovalent substitution of Li3AsS4 by Ge, which increased the conductivity by two orders of magnitude as compared to the parent compound. The composition Li3.334Ge0.334As0.666S4 has a high ionic conductivity of 1.12 mScm-1 at 27oC. Local Li+ hopping in this material is accompanied by distinctive low activation energy Ea of 0.17 eV being the lowest of Li+ solid conductors. Furthermore, this study demonstrates the efficacy of surface passivation of solid electrolyte to achieve compatibility with metallic lithium electrodes.

  3. Catalysis of hydrolysis and nucleophilic substitution at the P-N bond of phosphoimidazolide-activated nucleotides in phosphate buffers

    NASA Technical Reports Server (NTRS)

    Kanavarioti, A.; Rosenbach, M. T.

    1991-01-01

    Phosphoimidazolide-activated derivatives of guanosine and cytidine 5'-monophosphates, henceforth called ImpN's, exhibit enhanced rates of degradation in the presence of aqueous inorganic phosphate in the range 4.0 < or = pH < or = 8.6. This degradation is been attributed to (i) nucleophilic substitution of the imidazolide and (ii) catalysis of the P-N bond hydrolysis by phosphate. The first reaction results in the formation of nucleoside 5'-diphosphate and the second in nucleoside 5'-monophosphate. Analysis of the observed rates as well as the product ratios as a function of pH and phosphate concentration allow distinction between various mechanistic possibilities. The results show that both H2PO4- and HPO4(2-) participate in both hydrolysis and nucleophilic substitution. Statistically corrected biomolecular rate constants indicate that the dianion is 4 times more effective as a general base than the monoanion, and 8 times more effective as nucleophile. The low Bronsted value beta = 0.15 calculated for these phosphate species, presumed to act as general bases in facilitating water attack, is consistent with the fact that catalysis of the hydrolysis of the P-N bond in ImpN's has not been detected before. The beta nuc = 0.35 calculated for water, H2PO4-, HPO4(2-), and hydroxide acting as nucleophiles indicates a more associative transition state for nucleotidyl (O2POR- with R = nucleoside) transfers than that observed for phosphoryl (PO3(2-)) transfers (beta nuc = 0.25). With respect to the stability/reactivity of ImpN's under prebiotic conditions, our study shows that these materials would not suffer additional degradation due to inorganic phosphate, assuming the concentrations of phosphate, Pi, on prebiotic Earth were similar to those in the present oceans ([Pi] approximately 2.25 micromoles).

  4. Synthesis, Antitubercular Activity and Docking Study of Novel Cyclic Azole Substituted Diphenyl Ether Derivatives

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tuberculosis is a global health problem. New of heterocyclic diphenyl ether derivatives were tested against the Mycobacterium in laboratory settings. All ten compounds inhibited the growth of the mycobacterium at concentrations as low as 1 ug/mL. This level of activity was found comparable to refe...

  5. [FeFe]-Hydrogenase with Chalcogenide Substitutions at the H-Cluster Maintains Full H2 Evolution Activity.

    PubMed

    Noth, Jens; Esselborn, Julian; Güldenhaupt, Jörn; Brünje, Annika; Sawyer, Anne; Apfel, Ulf-Peter; Gerwert, Klaus; Hofmann, Eckhard; Winkler, Martin; Happe, Thomas

    2016-07-11

    The [FeFe]-hydrogenase HYDA1 from Chlamydomonas reinhardtii is particularly amenable to biochemical and biophysical characterization because the H-cluster in the active site is the only inorganic cofactor present. Herein, we present the complete chemical incorporation of the H-cluster into the HYDA1-apoprotein scaffold and, furthermore, the successful replacement of sulfur in the native [4FeH ] cluster with selenium. The crystal structure of the reconstituted pre-mature HYDA1[4Fe4Se]H protein was determined, and a catalytically intact artificial H-cluster variant was generated upon in vitro maturation. Full hydrogen evolution activity as well as native-like composition and behavior of the redesigned enzyme were verified through kinetic assays, FTIR spectroscopy, and X-ray structure analysis. These findings reveal that even a bioinorganic active site with exceptional complexity can exhibit a surprising level of compositional plasticity. PMID:27214763

  6. Highly Ordered Mesoporous Cobalt-Containing Oxides: Structure, Catalytic Properties, and Active Sites in Oxidation of Carbon Monoxide.

    PubMed

    Gu, Dong; Jia, Chun-Jiang; Weidenthaler, Claudia; Bongard, Hans-Josef; Spliethoff, Bernd; Schmidt, Wolfgang; Schüth, Ferdi

    2015-09-01

    Co3O4 with a spinel structure is a very active oxide catalyst for the oxidation of CO. In such catalysts, octahedrally coordinated Co(3+) is considered to be the active site, while tetrahedrally coordinated Co(2+) is assumed to be basically inactive. In this study, a highly ordered mesoporous CoO has been prepared by H2 reduction of nanocast Co3O4 at low temperature (250 °C). The as-prepared CoO material, which has a rock-salt structure with a single Co(2+) octahedrally coordinated by lattice oxygen in Fm3̅m symmetry, exhibited unexpectedly high activity for CO oxidation. Careful investigation of the catalytic behavior of mesoporous CoO catalyst led to the conclusion that the oxidation of surface Co(2+) to Co(3+) causes the high activity. Other mesoporous spinels (CuCo2O4, CoCr2O4, and CoFe2O4) with different Co species substituted with non/low-active metal ions were also synthesized to investigate the catalytically active site of cobalt-based catalysts. The results show that not only is the octahedrally coordinated Co(3+) highly active but also the octahedrally coordinated Co(2+) species in CoFe2O4 with an inverse spinel structure shows some activity. These results suggest that the octahedrally coordinated Co(2+) species is easily oxidized and shows high catalytic activity for CO oxidation. PMID:26301797

  7. Structure-activity studies of dicationically substituted bis-benzimidazoles against Giardia lamblia: correlation of antigiardial activity with DNA binding affinity and giardial topoisomerase II inhibition.

    PubMed Central

    Bell, C A; Dykstra, C C; Naiman, N A; Cory, M; Fairley, T A; Tidwell, R R

    1993-01-01

    Nine dicationically substituted bis-benzimidazoles were examined for their in vitro activities against Giardia lamblia WB (ATCC 30957). The potential mechanisms of action of these compounds were evaluated by investigating the relationship among in vitro antigiardial activity and the affinity of the molecules for DNA and their ability to inhibit the activity of giardial topoisomerase II. Each compound demonstrated antigiardial activity, as measured by assessing the incorporation of [methyl-3H]thymidine by giardial trophozoites exposed to the test agents. Three compounds exhibited excellent in vitro antigiardial activities, with 50% inhibitory concentrations which compared very favorably with those of two currently used drugs, quinacrine HCl and metronidazole. Putative mechanisms of action for these compounds were suggested by the strong correlation observed among in vitro antigiardial activity and the affinity of the molecules for natural and synthetic DNA and their ability to inhibit the relaxation activity of giardial topoisomerase II. A strong correlation between the DNA binding affinity of these compounds and their inhibition of giardial topoisomerase II activity was also observed. Images PMID:8109934

  8. Current activities handbook: formerly utilized sites remedial action program

    SciTech Connect

    1981-02-27

    This volume is one of a series produced under contract with the DOE, by Politech Corporation to develop a legislative and regulatory data base to assist the FUSRAP management in addressing the institutional and socioeconomic issues involved in carrying out the Formerly Utilized Sites Remedial Action Program. This Information Handbook series contains information about all relevant government agencies at the Federal and state levels, the pertinent programs they administer, each affected state legislature, and current Federal and state legislative and regulatory initiatives. This volume is a compilation of information about the activities each of the thirteen state legislatures potentially affected by the Formerly Utilized Sites Remedial Action Program. It contains a description of the state legislative procedural rules and a schedule of each legislative session; a summary of pending relevant legislation; the name and telephone number of legislative and state agency contacts; and the full text of all bills identified.

  9. Combinatorial active-site variants confer sustained clavulanate resistance in BlaC β-lactamase from Mycobacterium tuberculosis

    PubMed Central

    Egesborg, Philippe; Carlettini, Hélène; Volpato, Jordan P; Doucet, Nicolas

    2015-01-01

    Bacterial resistance to β-lactam antibiotics is a global issue threatening the success of infectious disease treatments worldwide. Mycobacterium tuberculosis has been particularly resilient to β-lactam treatment, primarily due to the chromosomally encoded BlaC β-lactamase, a broad-spectrum hydrolase that renders ineffective the vast majority of relevant β-lactam compounds currently in use. Recent laboratory and clinical studies have nevertheless shown that specific β-lactam–BlaC inhibitor combinations can be used to inhibit the growth of extensively drug-resistant strains of M. tuberculosis, effectively offering new tools for combined treatment regimens against resistant strains. In the present work, we performed combinatorial active-site replacements in BlaC to demonstrate that specific inhibitor-resistant (IRT) substitutions at positions 69, 130, 220, and/or 234 can act synergistically to yield active-site variants with several thousand fold greater in vitro resistance to clavulanate, the most common clinical β-lactamase inhibitor. While most single and double variants remain sensitive to clavulanate, double mutants R220S-K234R and S130G-K234R are substantially less affected by time-dependent clavulanate inactivation, showing residual β-lactam hydrolytic activities of 46% and 83% after 24 h incubation with a clinically relevant inhibitor concentration (5 μg/ml, 25 µM). These results demonstrate that active-site alterations in BlaC yield resistant variants that remain active and stable over prolonged bacterial generation times compatible with mycobacterial proliferation. These results also emphasize the formidable adaptive potential of inhibitor-resistant substitutions in β-lactamases, potentially casting a shadow on specific β-lactam–BlaC inhibitor combination treatments against M. tuberculosis. PMID:25492589

  10. Site-directed mutagenesis of the human DNA repair enzyme HAP1: identification of residues important for AP endonuclease and RNase H activity.

    PubMed

    Barzilay, G; Walker, L J; Robson, C N; Hickson, I D

    1995-05-11

    HAP1 protein, the major apurinic/apyrimidinic (AP) endonuclease in human cells, is a member of a homologous family of multifunctional DNA repair enzymes including the Escherichia coli exonuclease III and Drosophila Rrp1 proteins. The most extensively characterised member of this family, exonuclease III, exhibits both DNA- and RNA-specific nuclease activities. Here, we show that the RNase H activity characteristic of exonuclease III has been conserved in the human homologue, although the products resulting from RNA cleavage are dissimilar. To identify residues important for enzymatic activity, five mutant HAP1 proteins containing single amino acid substitutions were purified and analysed in vitro. The substitutions were made at sites of conserved amino acids and targeted either acidic or histidine residues because of their known participation in the active sites of hydrolytic nucleases. One of the mutant proteins (replacement of Asp-219 by alanine) showed a markedly reduced enzymatic activity, consistent with a greatly diminished capacity to bind DNA and RNA. In contrast, replacement of Asp-90, Asp-308 or Glu-96 by alanine led to a reduction in enzymatic activity without significantly compromising nucleic acid binding. Replacement of His-255 by alanine led to only a very small reduction in enzymatic activity. Our data are consistent with the presence of a single catalytic active site for the DNA- and RNA-specific nuclease activities of the HAP1 protein. PMID:7784208

  11. Synthesis of Substituted Pyrroles via Copper-Catalyzed Cyclization of Ethyl Allenoates with Activated Isocyanides.

    PubMed

    Lu, Kui; Ding, Fang; Qin, Long; Jia, Xiaoliang; Xu, Chuanming; Zhao, Xia; Yao, Qingwei; Yu, Peng

    2016-08-01

    A new method for the synthesis of di- and trisubstituted pyrroles via copper-catalyzed cyclization of ethyl allenoates with activated isocyanides has been developed. In contrast to related annulation reactions previously reported, this new process features a skeletal rearrangement in which the aryl sulfonyl moiety, which functions as the electron-withdrawing group in the α-carbon of the isocyanide, was found to migrate to the γ-carbon of the starting allenoate in the final product for the first time. PMID:27348611

  12. On the nature and formation of the active sites in Re[sub 2]O[sub 7] metathesis catalysts supported on borated alumina

    SciTech Connect

    Sibeijn, M.; Bliek, A. ); Veen, J.A.R. van ); Moulijn, J.A. )

    1994-02-01

    Re[sub 2]O[sub 7] catalysts on borated aluminas have been investigated with a view to correlating the structure of the active site and its activity in the metathesis of methyl oleate. Modification of alumina with boria results in much more active metathesis catalysts. Infrared spectroscopy was used for the characterization, pyridine adsorption measurements for determining the Lewis acid and Bronsted acid sites, and temperature-programmed IR measurements to follow the reactions occurring during calcination of the supports and catalysts. Boria binds to the surface via the alumina hydroxyls. Upon Re[sub 2]O[sub 7] loading of nonborated alumina, the ReO[sub 4] groups react first with Lewis acid sites, onto which they are strongly bonded. Above a Re[sub 2]O[sub 7] loading of 3 wt% surface hydroxyls are also substituted by Re[sub 2]O[sub 7] groups, resulting in an increase in catalytic activity. When the borated supports are loaded with Re[sub 2]O[sub 7], the ReO[sub 4] groups are also first bonded to the Lewis acid sites. During calcination these ReO[sub 4] groups substitute surface hydroxyls preferably on alumina hydroxyls. The substitution of the boron hydroxyls only takes place at a calcination time of at least 2 h at 823 K. At high borate loadings (>10 wt%) the reaction of ReO[sub 4] groups with boron hydroxyls competes with the condensation reaction of two neighbouring boron hydroxyls. Taking into account that a ReO[sub 4] group which has substituted in acidic OH group on the support is the precursor of an active site, the increase in activity of Re[sub 2]O[sub 7] catalysts by modification of the alumina support with boria can be ascribed to two effects, namely, the reduction of the bonding strength of Lewis acid sites with ReO[sub 4], making the ReO[sub 4]-OH substitution reaction possible during calcination even at low rhenium loadings, and the formation of acidic surface hydroxyls. 16 refs., 11 figs., 3 tabs.

  13. Enhanced OCT4 transcriptional activity substitutes for exogenous SOX2 in cellular reprogramming

    PubMed Central

    Marthaler, Adele G.; Adachi, Kenjiro; Tiemann, Ulf; Wu, Guangming; Sabour, Davood; Velychko, Sergiy; Kleiter, Ingo; Schöler, Hans R.; Tapia, Natalia

    2016-01-01

    Adenoviral early region 1A (E1A) is a viral gene that can promote cellular proliferation and de-differentiation in mammalian cells, features required for the reprogramming of somatic cells to a pluripotent state. E1A has been shown to interact with OCT4, and as a consequence, to increase OCT4 transcriptional activity. Indeed, E1A and OCT4 are sufficient to revert neuroepithelial hybrids to pluripotency, as demonstrated in previous cell fusion experiments. However, the role that E1A might play in the generation of induced pluripotent stem cells (iPSCs) has not been investigated yet. In this report, we show that E1A can generate iPSCs in combination with OCT4 and KLF4, thus replacing exogenous SOX2. The generated iPSCs are bona fide pluripotent cells as shown by in vitro and in vivo tests. Overall, our study suggests that E1A might replace SOX2 through enhancing OCT4 transcriptional activity at the early stages of reprogramming. PMID:26762895

  14. Effects of 7-O substitutions on estrogenic and anti-estrogenic activities of daidzein analogues in MCF-7 breast cancer cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Daidzein (1) is a natural estrogenic isoflavone. We report here that 1 can be transformed into an antiestrogenic ligand by simple alkyl substitutions of the 7-hydroxyl hydrogen. To test the effect of such structural modifications on the hormonal activities of the resulting compounds, a series of dai...

  15. Structure-activity relationship study of 4-substituted piperidines at Leu26 moiety of novel p53-hDM2 inhibitors.

    PubMed

    Tian, Yuan; Ma, Yao; Gibeau, Craig R; Lahue, Brian R; Shipps, Gerald W; Strickland, Corey; Bogen, Stéphane L

    2016-06-01

    Led by the structural information of the screening hit with mDM2 protein, a structure modification of Leu26 moiety of the novel p53-hDM2 inhibitors was conducted. A structure-activity relationship study of 4-substituted piperidines revealed compound 20t with good potencies and excellent CYP450 profiles. PMID:27080185

  16. Discovery of molluscicidal and cercaricidal activities of 3-substituted quinazolinone derivatives by a scaffold hopping approach using a pseudo-ring based on the intramolecular hydrogen bond formation.

    PubMed

    Guo, Wei; Zheng, Lv-Yin; Li, Yong-Dong; Wu, Ren-Miao; Chen, Qiang; Yang, Ding-Qiao; Fan, Xiao-Lin

    2016-06-10

    Discovery of novel topological agents against Oncomelania hupensis snails and cercariae remains a significant challenge in current Schistosomiasis control. A pseudo-ring formed from salicylanilide by an intramolecular hydrogen bond led to the discovery of 3-substituted quinazolinone derivatives which showed a potent molluscicidal and cercaricidal activities. PMID:27017555

  17. Identification of BRCA1 missense substitutions that confer partial functional activity: potential moderate risk variants?

    PubMed Central

    Lovelock, Paul K; Spurdle, Amanda B; Mok, Myth TS; Farrugia, Daniel J; Lakhani, Sunil R; Healey, Sue; Arnold, Stephen; Buchanan, Daniel; Investigators, kConFab; Couch, Fergus J; Henderson, Beric R; Goldgar, David E; Tavtigian, Sean V; Chenevix-Trench, Georgia; Brown, Melissa A

    2007-01-01

    Introduction Many of the DNA sequence variants identified in the breast cancer susceptibility gene BRCA1 remain unclassified in terms of their potential pathogenicity. Both multifactorial likelihood analysis and functional approaches have been proposed as a means to elucidate likely clinical significance of such variants, but analysis of the comparative value of these methods for classifying all sequence variants has been limited. Methods We have compared the results from multifactorial likelihood analysis with those from several functional analyses for the four BRCA1 sequence variants A1708E, G1738R, R1699Q, and A1708V. Results Our results show that multifactorial likelihood analysis, which incorporates sequence conservation, co-inheritance, segregation, and tumour immunohistochemical analysis, may improve classification of variants. For A1708E, previously shown to be functionally compromised, analysis of oestrogen receptor, cytokeratin 5/6, and cytokeratin 14 tumour expression data significantly strengthened the prediction of pathogenicity, giving a posterior probability of pathogenicity of 99%. For G1738R, shown to be functionally defective in this study, immunohistochemistry analysis confirmed previous findings of inconsistent 'BRCA1-like' phenotypes for the two tumours studied, and the posterior probability for this variant was 96%. The posterior probabilities of R1699Q and A1708V were 54% and 69%, respectively, only moderately suggestive of increased risk. Interestingly, results from functional analyses suggest that both of these variants have only partial functional activity. R1699Q was defective in foci formation in response to DNA damage and displayed intermediate transcriptional transactivation activity but showed no evidence for centrosome amplification. In contrast, A1708V displayed an intermediate transcriptional transactivation activity and a normal foci formation response in response to DNA damage but induced centrosome amplification. Conclusion

  18. Identification of covalent active site inhibitors of dengue virus protease

    PubMed Central

    Koh-Stenta, Xiaoying; Joy, Joma; Wang, Si Fang; Kwek, Perlyn Zekui; Wee, John Liang Kuan; Wan, Kah Fei; Gayen, Shovanlal; Chen, Angela Shuyi; Kang, CongBao; Lee, May Ann; Poulsen, Anders; Vasudevan, Subhash G; Hill, Jeffrey; Nacro, Kassoum

    2015-01-01

    Dengue virus (DENV) protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described. PMID:26677315

  19. C2-substituted aromatic cytokinin sugar conjugates delay the onset of senescence by maintaining the activity of the photosynthetic apparatus.

    PubMed

    Vylíčilová, Hana; Husičková, Alexandra; Spíchal, Lukáš; Srovnal, Josef; Doležal, Karel; Plíhal, Ondřej; Plíhalová, Lucie

    2016-02-01

    Cytokinins are plant hormones with biological functions ranging from coordination of plant growth and development to the regulation of senescence. A series of 2-chloro-N(6)-(halogenobenzylamino)purine ribosides was prepared and tested for cytokinin activity in detached wheat leaf senescence, tobacco callus and Amaranthus bioassays. The synthetic compounds showed significant activity, especially in delaying senescence in detached wheat leaves. They were also tested in bacterial receptor bioassays using both monocot and dicot members of the cytokinin receptor family. Most of the derivatives did not trigger cytokinin signaling via the AHK3 and AHK4 receptors from Arabidopsis thaliana in the bacterial assay, but some of them specifically activated the ZmHK1 receptor from Zea mays and were also more active than the aromatic cytokinin BAP in an ARR5::GUS cytokinin bioassay using transgenic Arabidopsis plants. Whole transcript expression analysis was performed using an Arabidopsis model to gather information about the reprogramming of gene transcription when senescent leaves were treated with selected C2-substituted aromatic cytokinin ribosides. Genome-wide expression profiling revealed that the synthetic halogenated derivatives induced the expression of genes related to cytokinin signaling and metabolism. They also prompted both up- and down-regulation of a unique combination of genes coding for components of the photosystem II (PSII) reaction center, light-harvesting complex II (LHCII), and the oxygen-evolving complex, as well as several stress factors responsible for regulating photosynthesis and chlorophyll degradation. Chlorophyll content and fluorescence analyses demonstrated that treatment with the halogenated derivatives increased the efficiency of PSII photochemistry and the abundance of LHCII relative to DMSO- and BAP-treated controls. These findings demonstrate that it is possible to manipulate and fine-tune leaf longevity using synthetic aromatic cytokinin

  20. Zwitterionic Cellulose Carbamate with Regioselective Substitution Pattern: A Coating Material Possessing Antimicrobial Activity.

    PubMed

    Elschner, Thomas; Lüdecke, Claudia; Kalden, Diana; Roth, Martin; Löffler, Bettina; Jandt, Klaus D; Heinze, Thomas

    2016-04-01

    A polyzwitterion is synthesized by regioselective functionalization of cellulose possessing a uniform charge distribution. The positively charged ammonium group is present at position 6, while the negative charge of carboxylate is located at positions 2 and 3 of the repeating unit. The molecular structure of the biopolymer derivative is proved by NMR spectroscopy. This cellulose-based zwitterion is applied to several support materials by spin-coating and characterized by means of atomic force microscope, contact angle measurements, ellipsometry, and X-ray photoelectron spectroscopy. The coatings possess antimicrobial activity depending on the support materials (glass, titanium, tissue culture poly(styrene)) as revealed by confocal laser scanning microscopy and live/dead staining. PMID:26632022

  1. Membrane anchoring of diacylglycerol-lactones substituted with rigid hydrophobic acyl domains correlates with biological activities

    PubMed Central

    Raifman, Or; Kolusheva, Sofiya; Comin, Maria J.; Kedei, Noemi; Lewin, Nancy E.; Blumberg, Peter M.; Marquez, Victor E.; Jelinek, Raz

    2009-01-01

    Summary Synthetic diacylglycerol lactones (DAG-lactones) are effective modulators of critical cellular signaling pathways, downstream of the lipophilic second messenger diacylglycerol, that activate a host of protein kinase C (PKC) isozymes as well as other non-kinase proteins that share with PKC similar C1 membrane-targeting domains. A fundamental determinant of the biological activity of these amphiphilic molecules is the nature of their interactions with cellular membranes. This study characterizes the membrane interactions and bilayer anchoring of a series of DAG-lactones in which the hydrophobic moiety is a “molecular rod”, namely a rigid 4-[2-(R-phenyl)ethynyl]benzoate moiety in the acyl position. Application of assays employing chromatic biomimetic vesicles and biophysical techniques reveals that the mode of membrane anchoring of the DAG-lactone derivatives was markedly affected by the presence of the hydrophobic diphenyl rod and by the size of the functional unit displayed at the terminus of the rod. Two primary mechanisms of interaction were observed: surface binding of the DAG-lactones at the lipid/water interface and deep insertion of the ligands into the alkyl core of the lipid bilayer. These membrane-insertion properties could explain the different patterns of PKC translocation from cytosol to membranes induced by the molecular-rod DAG-lactones. This investigation emphasizes that the side-residues of DAG-lactones, rather than simply conferring hydrophobicity, profoundly influence membrane interactions and in that fashion may further contribute to the diversity of biological actions of these synthetic biomimetic ligands. PMID:19961537

  2. Conformational flexibility related to enzyme activity: evidence for a dynamic active-site gatekeeper function of Tyr(215) in Aerococcus viridans lactate oxidase.

    PubMed

    Stoisser, Thomas; Brunsteiner, Michael; Wilson, David K; Nidetzky, Bernd

    2016-01-01

    L-Lactate oxidase (LOX) belongs to a large family of flavoenzymes that catalyze oxidation of α-hydroxy acids. How in these enzymes the protein structure controls reactivity presents an important but elusive problem. LOX contains a prominent tyrosine in the substrate binding pocket (Tyr(215) in Aerococcus viridans LOX) that is partially responsible for securing a flexible loop which sequesters the active site. To characterize the role of Tyr(215), effects of substitutions of the tyrosine (Y215F, Y215H) were analyzed kinetically, crystallographically and by molecular dynamics simulations. Enzyme variants showed slowed flavin reduction and oxidation by up to 33-fold. Pyruvate release was also decelerated and in Y215F, it was the slowest step overall. A 2.6-Å crystal structure of Y215F in complex with pyruvate shows the hydrogen bond between the phenolic hydroxyl and the keto oxygen in pyruvate is replaced with a potentially stronger hydrophobic interaction between the phenylalanine and the methyl group of pyruvate. Residues 200 through 215 or 216 appear to be disordered in two of the eight monomers in the asymmetric unit suggesting that they function as a lid controlling substrate entry and product exit from the active site. Substitutions of Tyr(215) can thus lead to a kinetic bottleneck in product release. PMID:27302031

  3. Conformational flexibility related to enzyme activity: evidence for a dynamic active-site gatekeeper function of Tyr215 in Aerococcus viridans lactate oxidase

    PubMed Central

    Stoisser, Thomas; Brunsteiner, Michael; Wilson, David K.; Nidetzky, Bernd

    2016-01-01

    L-Lactate oxidase (LOX) belongs to a large family of flavoenzymes that catalyze oxidation of α-hydroxy acids. How in these enzymes the protein structure controls reactivity presents an important but elusive problem. LOX contains a prominent tyrosine in the substrate binding pocket (Tyr215 in Aerococcus viridans LOX) that is partially responsible for securing a flexible loop which sequesters the active site. To characterize the role of Tyr215, effects of substitutions of the tyrosine (Y215F, Y215H) were analyzed kinetically, crystallographically and by molecular dynamics simulations. Enzyme variants showed slowed flavin reduction and oxidation by up to 33-fold. Pyruvate release was also decelerated and in Y215F, it was the slowest step overall. A 2.6-Å crystal structure of Y215F in complex with pyruvate shows the hydrogen bond between the phenolic hydroxyl and the keto oxygen in pyruvate is replaced with a potentially stronger hydrophobic interaction between the phenylalanine and the methyl group of pyruvate. Residues 200 through 215 or 216 appear to be disordered in two of the eight monomers in the asymmetric unit suggesting that they function as a lid controlling substrate entry and product exit from the active site. Substitutions of Tyr215 can thus lead to a kinetic bottleneck in product release. PMID:27302031

  4. Polarizability of the active site of cytochrome c reduces the activation barrier for electron transfer.

    PubMed

    Dinpajooh, Mohammadhasan; Martin, Daniel R; Matyushov, Dmitry V

    2016-01-01

    Enzymes in biology's energy chains operate with low energy input distributed through multiple electron transfer steps between protein active sites. The general challenge of biological design is how to lower the activation barrier without sacrificing a large negative reaction free energy. We show that this goal is achieved through a large polarizability of the active site. It is polarized by allowing a large number of excited states, which are populated quantum mechanically by electrostatic fluctuations of the protein and hydration water shells. This perspective is achieved by extensive mixed quantum mechanical/molecular dynamics simulations of the half reaction of reduction of cytochrome c. The barrier for electron transfer is consistently lowered by increasing the number of excited states included in the Hamiltonian of the active site diagonalized along the classical trajectory. We suggest that molecular polarizability, in addition to much studied electrostatics of permanent charges, is a key parameter to consider in order to understand how enzymes work. PMID:27306204

  5. Polarizability of the active site of cytochrome c reduces the activation barrier for electron transfer

    NASA Astrophysics Data System (ADS)

    Dinpajooh, Mohammadhasan; Martin, Daniel R.; Matyushov, Dmitry V.

    2016-06-01

    Enzymes in biology’s energy chains operate with low energy input distributed through multiple electron transfer steps between protein active sites. The general challenge of biological design is how to lower the activation barrier without sacrificing a large negative reaction free energy. We show that this goal is achieved through a large polarizability of the active site. It is polarized by allowing a large number of excited states, which are populated quantum mechanically by electrostatic fluctuations of the protein and hydration water shells. This perspective is achieved by extensive mixed quantum mechanical/molecular dynamics simulations of the half reaction of reduction of cytochrome c. The barrier for electron transfer is consistently lowered by increasing the number of excited states included in the Hamiltonian of the active site diagonalized along the classical trajectory. We suggest that molecular polarizability, in addition to much studied electrostatics of permanent charges, is a key parameter to consider in order to understand how enzymes work.

  6. Polarizability of the active site of cytochrome c reduces the activation barrier for electron transfer

    PubMed Central

    Dinpajooh, Mohammadhasan; Martin, Daniel R.; Matyushov, Dmitry V.

    2016-01-01

    Enzymes in biology’s energy chains operate with low energy input distributed through multiple electron transfer steps between protein active sites. The general challenge of biological design is how to lower the activation barrier without sacrificing a large negative reaction free energy. We show that this goal is achieved through a large polarizability of the active site. It is polarized by allowing a large number of excited states, which are populated quantum mechanically by electrostatic fluctuations of the protein and hydration water shells. This perspective is achieved by extensive mixed quantum mechanical/molecular dynamics simulations of the half reaction of reduction of cytochrome c. The barrier for electron transfer is consistently lowered by increasing the number of excited states included in the Hamiltonian of the active site diagonalized along the classical trajectory. We suggest that molecular polarizability, in addition to much studied electrostatics of permanent charges, is a key parameter to consider in order to understand how enzymes work. PMID:27306204

  7. The copper active site of CBM33 polysaccharide oxygenases.

    PubMed

    Hemsworth, Glyn R; Taylor, Edward J; Kim, Robbert Q; Gregory, Rebecca C; Lewis, Sally J; Turkenburg, Johan P; Parkin, Alison; Davies, Gideon J; Walton, Paul H

    2013-04-24

    The capacity of metal-dependent fungal and bacterial polysaccharide oxygenases, termed GH61 and CBM33, respectively, to potentiate the enzymatic degradation of cellulose opens new possibilities for the conversion of recalcitrant biomass to biofuels. GH61s have already been shown to be unique metalloenzymes containing an active site with a mononuclear copper ion coordinated by two histidines, one of which is an unusual τ-N-methylated N-terminal histidine. We now report the structural and spectroscopic characterization of the corresponding copper CBM33 enzymes. CBM33 binds copper with high affinity at a mononuclear site, significantly stabilizing the enzyme. X-band EPR spectroscopy of Cu(II)-CBM33 shows a mononuclear type 2 copper site with the copper ion in a distorted axial coordination sphere, into which azide will coordinate as evidenced by the concomitant formation of a new absorption band in the UV/vis spectrum at 390 nm. The enzyme's three-dimensional structure contains copper, which has been photoreduced to Cu(I) by the incident X-rays, confirmed by X-ray absorption/fluorescence studies of both aqueous solution and intact crystals of Cu-CBM33. The single copper(I) ion is ligated in a T-shaped configuration by three nitrogen atoms from two histidine side chains and the amino terminus, similar to the endogenous copper coordination geometry found in fungal GH61. PMID:23540833

  8. The Copper Active Site of CBM33 Polysaccharide Oxygenases

    PubMed Central

    2013-01-01

    The capacity of metal-dependent fungal and bacterial polysaccharide oxygenases, termed GH61 and CBM33, respectively, to potentiate the enzymatic degradation of cellulose opens new possibilities for the conversion of recalcitrant biomass to biofuels. GH61s have already been shown to be unique metalloenzymes containing an active site with a mononuclear copper ion coordinated by two histidines, one of which is an unusual τ-N-methylated N-terminal histidine. We now report the structural and spectroscopic characterization of the corresponding copper CBM33 enzymes. CBM33 binds copper with high affinity at a mononuclear site, significantly stabilizing the enzyme. X-band EPR spectroscopy of Cu(II)-CBM33 shows a mononuclear type 2 copper site with the copper ion in a distorted axial coordination sphere, into which azide will coordinate as evidenced by the concomitant formation of a new absorption band in the UV/vis spectrum at 390 nm. The enzyme’s three-dimensional structure contains copper, which has been photoreduced to Cu(I) by the incident X-rays, confirmed by X-ray absorption/fluorescence studies of both aqueous solution and intact crystals of Cu-CBM33. The single copper(I) ion is ligated in a T-shaped configuration by three nitrogen atoms from two histidine side chains and the amino terminus, similar to the endogenous copper coordination geometry found in fungal GH61. PMID:23540833

  9. Novel substituted 1-iminoisoindoline derivatives: Synthesis, structure determination and antiproliferative activity

    NASA Astrophysics Data System (ADS)

    Sović, Irena; Stilinović, Vladimir; Kaitner, Branko; Kraljević-Pavelić, Sandra; Bujak, Maro; Čuljak, Katarina; Novak, Predrag; Karminski-Zamola, Grace

    2011-12-01

    Novel derivatives of isoindoline, N-phenyl-1-iminophenylisoindoline 6, N-(4-methylphenyl)-1-imino-(4-methylphenyl)-isoindoline 7, N-(pyridin-2-yl)-1-imino-(pyridin-2-yl)-isoindoline 8 and N-(5-methylpyridin-2-yl)-1-imino(5-methylpyridin-2-yl)-isoindoline 9 were prepared by the reaction of condensation of phthalaldehyde and corresponding amines. Structures of all compounds have been studied using one- and two-dimensional 1H and 13C NMR, IR, MS and UV/Vis spectroscopy. The crystal and molecular structures of 7, 8 and 9 were determined by X-ray diffraction on single crystals. In all three molecules the isoindoline system and its N-substituent are approximately coplanar. The crystal structures comprise of discrete molecules linked only by weak C-H⋯N interactions in the case of 8 and 9. NMR analysis showed that conformations of compounds 6 and 7 differ from those of 8 and 9 in solution. Differences between solution and solid state structures were also noticed. All prepared compounds were tested on their antiproliferative activity in vitro. Compound 7 exerted the strongest non-specific antiproliferative effect on all cell lines and compounds 8 and 9 showed selective antiproliferative effect on HepG2 cell line.

  10. Synthesis and biological screening for cytotoxic activity of N-substituted indolines and morpholines.

    PubMed

    Doan, Phuong; Karjalainen, Anzhelika; Chandraseelan, Jerome G; Sandberg, Ossi; Yli-Harja, Olli; Rosholm, Tomi; Franzen, Robert; Candeias, Nuno R; Kandhavelu, Meenakshisundaram

    2016-09-14

    Development of novel anticancer drugs is inevitable to improve treatment of cancers. In this study, novel derivatives of indoline and morpholine were synthesized and tested for their cytotoxic effects on osteosarcoma and Human Embryonic Kidney cells. To characterize cytotoxicity and the mechanism of cell death, we used cytotoxicity, migration, apoptosis markers and mitochondrial calcium assays. Among the compounds tested, the indoline derivatives, generally, produced a higher cytotoxic effect compared to the morpholine derivatives, in osteosarcoma cells. Specifically, new indoline derivative N-(2-hydroxy-5-nitrophenyl(4'-methylphenyl)methyl)indoline exhibited effective cytotoxic activity, with an IC50 of ∼74 μM. The same molecule induced cell death by apoptosis and inhibited migration of the cells. Further, analysis of mitochondrial calcium levels revealed the existence of calcium dependent cell death mechanisms in different cell types. Therefore, N-(2-hydroxy-5-nitrophenyl(4'-methylphenyl)methyl)indoline can be considered as a potential drug-lead compound towards the discovery of new anti-cancer agents. PMID:27214140

  11. An Active Site Water Network in the Plasminogen Activator Pla from Yersinia pestis

    SciTech Connect

    Eren, Elif; Murphy, Megan; Goguen, Jon; van den Berg, Bert

    2010-08-13

    The plasminogen activator Pla from Yersinia pestis is an outer membrane protease (omptin) that is important for the virulence of plague. Here, we present the high-resolution crystal structure of wild-type, enzymatically active Pla at 1.9 {angstrom}. The structure shows a water molecule located between active site residues D84 and H208, which likely corresponds to the nucleophilic water. A number of other water molecules are present in the active site, linking residues important for enzymatic activity. The R211 sidechain in loop L4 is close to the nucleophilic water and possibly involved in the stabilization of the oxyanion intermediate. Subtle conformational changes of H208 result from the binding of lipopolysaccharide to the outside of the barrel, explaining the unusual dependence of omptins on lipopolysaccharide for activity. The Pla structure suggests a model for the interaction with plasminogen substrate and provides a more detailed understanding of the catalytic mechanism of omptin proteases.

  12. From Alcohol Dehydrogenase to a “One-way” Carbonyl Reductase by Active-site Redesign

    PubMed Central

    Klimacek, Mario; Nidetzky, Bernd

    2010-01-01

    Directional preference in catalysis is often used to distinguish alcohol dehydrogenases from carbonyl reductases. However, the mechanistic basis underpinning this discrimination is weak. In mannitol 2-dehydrogenase from Pseudomonas fluorescens, stabilization of (partial) negative charge on the substrate oxyanion by the side chains of Asn-191 and Asn-300 is a key feature of catalysis in the direction of alcohol oxidation. We have disrupted this ability through individual and combined substitutions of the two asparagines by aspartic acid. Kinetic data and their thermodynamic analysis show that the internal equilibrium of enzyme-NADH-fructose and enzyme-NAD+-mannitol (Kint) was altered dramatically (104- to 105-fold) from being balanced in the wild-type enzyme (Kint ≈ 3) to favoring enzyme-NAD+-mannitol in the single site mutants, N191D and N300D. The change in Kint reflects a selective slowing down of the mannitol oxidation rate, resulting because Asn → Asp replacement (i) disfavors partial abstraction of alcohol proton by Lys-295 in a step preceding catalytic hydride transfer, and (ii) causes stabilization of a nonproductive enzyme-NAD+-mannitol complex. N191D and N300D appear to lose fructose binding affinity due to deprotonation of the respective Asp above apparent pK values of 5.3 ± 0.1 and 6.3 ± 0.2, respectively. The mutant incorporating both Asn→Asp substitutions behaved as a slow “fructose reductase” at pH 5.2, lacking measurable activity for mannitol oxidation in the pH range 6.8–10. A mechanism is suggested in which polarization of the substrate carbonyl by a doubly protonated diad of Asp and Lys-295 facilitates NADH-dependent reduction of fructose by N191D and N300D under optimum pH conditions. Creation of an effectively “one-way” reductase by active-site redesign of a parent dehydrogenase has not been previously reported and holds promise in the development of carbonyl reductases for application in organic synthesis. PMID:20639204

  13. Large steric effect in the substitution reaction of amines with phosphoimidazolide-activated nucleosides

    NASA Technical Reports Server (NTRS)

    Kanavarioti, A.; Stronach, M. W.; Ketner, R. J.; Hurley, T. B.

    1995-01-01

    Aliphatic amines react with phosphoimidazolide-activated derivatives of guanosine and cytidine (ImpN) by replacing the imidazole group. The kinetics of reaction of guanosine 5'-phospho-2-methylimidazolide (2-MeImpG) with glycine ethyl ester, glycinamide, 2-methoxyethylamine, n-butylamine, morpholine, dimethylamine (Me2NH), ethylmethylamine (EtNHMe), diethylamine (Et2NH), pyrrolidine, and piperidine were determined in water at 37 degrees C. With primary amines, a plot of the logarithm of the rate constant for attack by the amine on the protonated substrate, log kSH(A), versus the pKa of the amine exhibits a good linear correlation with a Bronsted slope, beta nuc = 0.48. Most of the secondary amines tested react with slightly higher reactivity than primary amines of similar pKa. Interestingly, some secondary amines show substantially lower reactivity than might be expected: EtNHMe reacts about eight times, and Et2NH at least 100 times, more slowly than Me2NH although all three amines are of similar basicity. For comparison, the kinetics of reaction of guanosine 5'-phosphoimidazolide (ImpG) and cytidine 5'-phosphoimidazolide (ImpC) were determined with Me2NH, EtNHMe, and Et2NH, and similar results were obtained. These results establish that the increased steric hindrance observed with the successive addition of ethyl groups are not due to any special steric requirements imposed by the guanosine or the methyl on the 2-methylimidazole leaving group of 2-MeImpG. It is concluded that addition of ethyl and, perhaps, groups larger than ethyl dramatically increases the kinetic barrier for addition of aliphatic secondary amines to the P-N bond of ImpN. This study supports the observation that the primary amino groups on the natural polyamines are at least 2 orders of magnitude more reactive than the secondary amino groups in the reaction with ImpN.

  14. Target-classification approach applied to active UXO sites

    NASA Astrophysics Data System (ADS)

    Shubitidze, F.; Fernández, J. P.; Shamatava, Irma; Barrowes, B. E.; O'Neill, K.

    2013-06-01

    This study is designed to illustrate the discrimination performance at two UXO active sites (Oklahoma's Fort Sill and the Massachusetts Military Reservation) of a set of advanced electromagnetic induction (EMI) inversion/discrimination models which include the orthonormalized volume magnetic source (ONVMS), joint diagonalization (JD), and differential evolution (DE) approaches and whose power and flexibility greatly exceed those of the simple dipole model. The Fort Sill site is highly contaminated by a mix of the following types of munitions: 37-mm target practice tracers, 60-mm illumination mortars, 75-mm and 4.5'' projectiles, 3.5'', 2.36'', and LAAW rockets, antitank mine fuzes with and without hex nuts, practice MK2 and M67 grenades, 2.5'' ballistic windshields, M2A1-mines with/without bases, M19-14 time fuzes, and 40-mm practice grenades with/without cartridges. The site at the MMR site contains targets of yet different sizes. In this work we apply our models to EMI data collected using the MetalMapper (MM) and 2 × 2 TEMTADS sensors. The data for each anomaly are inverted to extract estimates of the extrinsic and intrinsic parameters associated with each buried target. (The latter include the total volume magnetic source or NVMS, which relates to size, shape, and material properties; the former includes location, depth, and orientation). The estimated intrinsic parameters are then used for classification performed via library matching and the use of statistical classification algorithms; this process yielded prioritized dig-lists that were submitted to the Institute for Defense Analyses (IDA) for independent scoring. The models' classification performance is illustrated and assessed based on these independent evaluations.

  15. Spectroscopic Definition of the Ferroxidase Site in M Ferritin: Comparison of Binuclear Substrate vs. Cofactor Active Sites

    PubMed Central

    Schwartz, Jennifer K.; Liu, Xiaofeng S.; Tosha, Takehiko; Theil, Elizabeth C.; Solomon, Edward I.

    2008-01-01

    Maxi ferritins, 24 subunit protein nanocages, are essential in humans, plants, bacteria, and other animals for the concentration and storage of iron as hydrated ferric oxide, while minimizing free radical generation or use by pathogens. Formation of the precursors to these ferric oxides is catalyzed at a non-heme biferrous substrate site, which has some parallels with the cofactor sites in other biferrous enzymes. A combination of circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature, variable-field MCD (VTVH MCD) has been used to probe Fe(II) binding to the substrate active site in frog M ferritin. These data determined that the active site within each subunit consists of two inequivalent five-coordinate (5C) ferrous centers that are weakly anti-ferromagnetically coupled, consistent with a μ-1,3 carboxylate bridge. The active site ligand set is unusual and likely includes a terminal water bound to each Fe(II) center. The Fe(II) ions bind to the active sites in a concerted manner, and cooperativity among the sites in each subunit is observed, potentially providing a mechanism for the control of ferritin iron loading. Differences in geometric and electronic structure – including a weak ligand field, availability of two water ligands at the biferrous substrate site, and the single carboxylate bridge in ferritin – coincide with the divergent reaction pathways observed between this substrate site and the previously studied cofactor active sites. PMID:18576633

  16. Metal active site elasticity linked to activation of homocysteine in methionine synthases

    SciTech Connect

    Koutmos, Markos; Pejchal, Robert; Bomer, Theresa M.; Matthews, Rowena G.; Smith, Janet L.; Ludwig, Martha L.

    2008-04-02

    Enzymes possessing catalytic zinc centers perform a variety of fundamental processes in nature, including methyl transfer to thiols. Cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthases are two such enzyme families. Although they perform the same net reaction, transfer of a methyl group from methyltetrahydrofolate to homocysteine (Hcy) to form methionine, they display markedly different catalytic strategies, modular organization, and active site zinc centers. Here we report crystal structures of zinc-replete MetE and MetH, both in the presence and absence of Hcy. Structural investigation of the catalytic zinc sites of these two methyltransferases reveals an unexpected inversion of zinc geometry upon binding of Hcy and displacement of an endogenous ligand in both enzymes. In both cases a significant movement of the zinc relative to the protein scaffold accompanies inversion. These structures provide new information on the activation of thiols by zinc-containing enzymes and have led us to propose a paradigm for the mechanism of action of the catalytic zinc sites in these and related methyltransferases. Specifically, zinc is mobile in the active sites of MetE and MetH, and its dynamic nature helps facilitate the active site conformational changes necessary for thiol activation and methyl transfer.

  17. Evidence for segmental mobility in the active site of pepsin

    SciTech Connect

    Pohl, J.; Strop, P.; Senn, H.; Foundling, S.; Kostka, V.

    1986-05-01

    The low hydrolytic activity (k/sub cat/ < 0.001 s/sup -1/) of chicken pepsin (CP) towards tri- and tetrapeptides is enhanced at least 100 times by modification of its single sulfhydryl group of Cys-115, with little effect on K/sub m/-values. Modification thus simulates the effect of secondary substrate binding on pepsin catalysis. The rate of Cys-115 modification is substantially decreased in the presence of some competitive inhibitors, suggesting its active site location. Experiments with CP alkylated at Cys-115 with Acrylodan as a fluorescent probe or with N-iodoacetyl-(4-fluoro)-aniline as a /sup 19/F-nmr probe suggest conformation change around Cys-115 to occur on substrate or substrate analog binding. The difference /sup 1/H-nmr spectra (500 MHz) of unmodified free and inhibitor-complexed CP reveal chemical shifts almost exclusively in the aromatic region. The effects of Cu/sup + +/ on /sup 19/F- and /sup 1/H-nmr spectra have been studied. Examination of a computer graphics model of CP based on E. parasitica pepsin-inhibitor complex X-ray coordinates suggests that Cys-115 is located near the S/sub 3//S/sub 5/ binding site. The results are interpreted in favor of segmental mobility of this region important for pepsin substrate binding and catalysis.

  18. Perchlorate Reductase Is Distinguished by Active Site Aromatic Gate Residues.

    PubMed

    Youngblut, Matthew D; Tsai, Chi-Lin; Clark, Iain C; Carlson, Hans K; Maglaqui, Adrian P; Gau-Pan, Phonchien S; Redford, Steven A; Wong, Alan; Tainer, John A; Coates, John D

    2016-04-22

    Perchlorate is an important ion on both Earth and Mars. Perchlorate reductase (PcrAB), a specialized member of the dimethylsulfoxide reductase superfamily, catalyzes the first step of microbial perchlorate respiration, but little is known about the biochemistry, specificity, structure, and mechanism of PcrAB. Here we characterize the biophysics and phylogeny of this enzyme and report the 1.86-Å resolution PcrAB complex crystal structure. Biochemical analysis revealed a relatively high perchlorate affinity (Km = 6 μm) and a characteristic substrate inhibition compared with the highly similar respiratory nitrate reductase NarGHI, which has a relatively much lower affinity for perchlorate (Km = 1.1 mm) and no substrate inhibition. Structural analysis of oxidized and reduced PcrAB with and without the substrate analog SeO3 (2-) bound to the active site identified key residues in the positively charged and funnel-shaped substrate access tunnel that gated substrate entrance and product release while trapping transiently produced chlorate. The structures suggest gating was associated with shifts of a Phe residue between open and closed conformations plus an Asp residue carboxylate shift between monodentate and bidentate coordination to the active site molybdenum atom. Taken together, structural and mutational analyses of gate residues suggest key roles of these gate residues for substrate entrance and product release. Our combined results provide the first detailed structural insight into the mechanism of biological perchlorate reduction, a critical component of the chlorine redox cycle on Earth. PMID:26940877

  19. Eel calcitonin binding site distribution and antinociceptive activity in rats

    SciTech Connect

    Guidobono, F.; Netti, C.; Sibilia, V.; Villa, I.; Zamboni, A.; Pecile, A.

    1986-03-01

    The distribution of binding site for (/sup 125/I)-eel-calcitonin (ECT) to rat central nervous system, studied by an autoradiographic technique, showed concentrations of binding in the diencephalon, the brain stem and the spinal cord. Large accumulations of grains were seen in the hypothalamus, the amygdala, in the fasciculus medialis prosencephali, in the fasciculus longitudinalis medialis, in the ventrolateral part of the periventricular gray matter, in the lemniscus medialis and in the raphe nuclei. The density of grains in the reticular formation and in the nucleus tractus spinalis nervi trigemini was more moderate. In the spinal cord, grains were scattered throughout the dorsal horns. Binding of the ligand was displaced equally by cold ECT and by salmon CT(sCT), indicating that both peptides bind to the same receptors. Human CT was much weaker than sCT in displacing (/sup 125/I)-ECT binding. The administration of ECT into the brain ventricles of rats dose-dependently induced a significant and long-lasting enhancement of hot-plate latencies comparable with that obtained with sCT. The antinociceptive activity induced by ECT is compatible with the topographical distribution of binding sites for the peptide and is a further indication that fish CTs are active in the mammalian brain.

  20. A base substitution in the promoter associated with the human haptoglobin 2-1 modified phenotype decreases transcriptional activity and responsiveness to interleukin-6 in human hepatoma cells

    SciTech Connect

    Grant, D.J.; Maeda, N. )

    1993-05-01

    An A-to-C base substitution at nucleotide position -61 in the promoter region of the human haptoglobin gene (Hp) has been shown to be strongly associated with the haptoglobin 2-1 modified (Hp2-1mod) phenotype. In order to investigate whether this base substitution is the cause of reduced expression of the Hp[sup 2] allele relative to the Hp[sup 1] allele in individuals with the Hp2-1mod phenotype, the authors used the chloramphenicol acetyl transferase (CAT) expression system to evaluate promoter function. In HepG2 cells, which normally express their endogenous haptoglobin genes, CAT plasmid constructs with the -61C base change in the promoter had about 10-fold-lower transcriptional activity after transfection than did the Hp control construct. The -61C substitution also rendered the construct unresponsive to treatment by interleukin-6 after transfection into Hep3B2 cells, which normally do not express haptoglobin but do so in response to stimulation by acute-phase reactants. In addition, two base substitutions, T to A and A to G, at positions -104 and -55G, respectively, in the promoter region of the Hp[sup 1] allele, are also associated with the Hp2-1mod phenotype. CAT constructs with both substitutions (-104A-55G) and with one substitution (-55G) showed activity similar to that in the Hp control when transfected into both HepG2 and Hep3B2 cells, although interleukin-6 induction was less than with the Hp control construct. These results further support the hypothesis that the Hp2-1mod phenotype results, in part, from the -61C mutation in the promoter region of the Hp[sup 2] gene.

  1. Synthesis and anticancer activities of 4-(4-substituted piperazin)-5,6,7-trialkoxy quinazoline derivatives.

    PubMed

    Zhang, Ying; Huang, Yin-Jiu; Xiang, Hong-Mei; Wang, Pei-Yi; Hu, De-Yu; Xue, Wei; Song, Bao-An; Yang, Song

    2014-05-01

    A series of 4-(4-substituted piperazin)-5,6,7-trialkoxy quinazoline was prepared by conventional heating methods. Among these compounds, the crystal structure of compound 10o (CCDC: 916922) was determined by X-ray crystallography. Bioassay results showed that most target compounds had certain inhibition activities against proliferation of tumor cells, and some compounds even had good broad-spectrum inhibition activities. The ethoxyl series of compounds possessed higher inhibition activities against tumor cells than the methoxyl series of compounds. Bioactivity tests showed that the IC50 values of compound 10s against PC3, MGC803, A375, and A549 cells were 1.8, 2.8, 1.3, and 2.9 μΜ, respectively, which were much higher than those of commercial gefitinib (7.2, 7.6, 7.2, and 9.8 μM, respectively). Conversely, the IC50 values of compound 10s were very low against NH3T3, indicating only weak effect on normal cells as also proven by lactate dehydrogenase and acridine orange/ethidium bromide staining. Analyses of cell configuration and cell cycle revealed that compound 10s possibly caused cells to remain at G0/G1 phase by inhibiting cell proliferation for 24 h. Compound 10s also inhibited the phosphorylation of ERK1/2 and P38 with obvious concentration dependence. Thus, these compounds can inhibit the proliferation of A549 cells through the interruption of ERK1/2 and P38signaling pathways. PMID:24675177

  2. Synthesis and Antibacterial Activity of 3-(Substituted)-2-(4-oxo-2-phenylquinazolin-3(4H)-ylamino)quinazolin-4(3H)-one.

    PubMed

    Appani, Ramgopal; Bhukya, Baburao; Gangarapu, Kiran

    2016-01-01

    A series of novel 3-(substituted)-2-(substituted quinazolinylamino)quinazolin-4(3H)-ones were synthesized by the reaction of 3-(substituted)-2-hydrazino-quinazoline-4(3H)-ones with 2-phenyl-3,1-benzoxazin-4-one. The starting materials 3-(substituted)-2-hydrazino-quinazolin-4(3H)-ones were synthesized from various primary amines by a multistep synthesis. All the title compounds were tested for their antibacterial activity using ciprofloxacin as reference standard. Compounds 3-(4-fluorophenyl)-2-(4-oxo-2-phenylquinazolin-3(4H)-ylamino)quinazolin-4(3H)-one (9a) and 3-(4-chlorophenyl)-2-(4-oxo-2-phenylquinazolin-3(4H)-ylamino)quinazolin-4(3H)-one (9h) emerged as the most active compounds of the series. These compounds have shown most potent antibacterial activity against the tested organisms of Proteus vulgaris and Bacillus subtilis having zone of inhibition values of 1.1 cm and 1.4 cm for compound 9a 1.2 cm and 1.0 cm for compound 9h, respectively. PMID:27190676

  3. Synthesis and Antibacterial Activity of 3-(Substituted)-2-(4-oxo-2-phenylquinazolin-3(4H)-ylamino)quinazolin-4(3H)-one

    PubMed Central

    Appani, Ramgopal; Bhukya, Baburao; Gangarapu, Kiran

    2016-01-01

    A series of novel 3-(substituted)-2-(substituted quinazolinylamino)quinazolin-4(3H)-ones were synthesized by the reaction of 3-(substituted)-2-hydrazino-quinazoline-4(3H)-ones with 2-phenyl-3,1-benzoxazin-4-one. The starting materials 3-(substituted)-2-hydrazino-quinazolin-4(3H)-ones were synthesized from various primary amines by a multistep synthesis. All the title compounds were tested for their antibacterial activity using ciprofloxacin as reference standard. Compounds 3-(4-fluorophenyl)-2-(4-oxo-2-phenylquinazolin-3(4H)-ylamino)quinazolin-4(3H)-one (9a) and 3-(4-chlorophenyl)-2-(4-oxo-2-phenylquinazolin-3(4H)-ylamino)quinazolin-4(3H)-one (9h) emerged as the most active compounds of the series. These compounds have shown most potent antibacterial activity against the tested organisms of Proteus vulgaris and Bacillus subtilis having zone of inhibition values of 1.1 cm and 1.4 cm for compound 9a 1.2 cm and 1.0 cm for compound 9h, respectively. PMID:27190676

  4. Short-term effects of mineral particle sizes on cellular degradation activity after implantation of injectable calcium phosphate biomaterials and the consequences for bone substitution.

    PubMed

    Gauthier, O; Bouler, J M; Weiss, P; Bosco, J; Aguado, E; Daculsi, G

    1999-08-01

    This in vivo study investigated the influence of two calcium phosphate particle sizes (40-80 microm and 200-500 microm) on the cellular degradation activity associated with the bone substitution process of two injectable bone substitutes (IBS). The tested biomaterials were obtained by associating a biphasic calcium phosphate (BCP) ceramic mineral phase and a 3% aqueous solution of a cellulosic polymer (hydroxypropylmethylcellulose). Both were injected into osseous defects at the distal end of rabbit femurs for 2- and 3-week periods. Quantitative results for tartrate-resistant acid phosphatase (TRAP) cellular activity, new bone formation, and ceramic resorption were studied for statistical purposes. Positive TRAP-stained degradation cells were significantly more numerous for IBS 40-80 than IBS 200-500, regardless of implantation time. BCP degradation was quite marked during the first 2 weeks for IBS 40-80, and bone colonization occurred more extensively for IBS 40-80 than for IBS 200-500. The resorption-bone substitution process occurred earlier and faster for IBS 40-80 than IBS 200-500. Both tested IBS displayed similar biological efficiency, with conserved in vivo bioactivity and bone-filling ability. Differences in calcium phosphate particle sizes influenced cellular degradation activity and ceramic resorption but were compatible with efficient bone substitution. PMID:10458280

  5. Synthesis, characterization and catalytic activity of indium substituted nanocrystalline Mobil Five (MFI) zeolite

    SciTech Connect

    Shah, Kishor Kr.; Nandi, Mithun; Talukdar, Anup K.

    2015-06-15

    Highlights: • In situ modification of the MFI zeolite by incorporation of indium. • The samples were characterized by XRD, FTIR, TGA, UV–vis (DRS), SAA, EDX and SEM. • The incorporation of indium was confirmed by XRD, FT-IR, UV–vis (DRS), EDX and TGA. • Hydroxylation of phenol reaction was studied on the synthesized catalysts. - Abstract: A series of indium doped Mobil Five (MFI) zeolite were synthesized hydrothermally with silicon to aluminium and indium molar ratio of 100 and with aluminium to indium molar ratios of 1:1, 2:1 and 3:1. The MFI zeolite phase was identified by XRD and FT-IR analysis. In XRD analysis the prominent peaks were observed at 2θ values of around 6.5° and 23° with a few additional shoulder peaks in case of all the indium incorporated samples suggesting formation of pure phase of the MFI zeolite. All the samples under the present investigation were found to exhibit high crystallinity (∼92%). The crystallite sizes of the samples were found to vary from about 49 to 55 nm. IR results confirmed the formation of MFI zeolite in all cases showing distinct absorbance bands near 1080, 790, 540, 450 and 990 cm{sup −1}. TG analysis of In-MFI zeolites showed mass losses in three different steps which are attributed to the loss due to adsorbed water molecules and the two types TPA{sup +} cations. Further, the UV–vis (DRS) studies reflected the position of the indium metal in the zeolite framework. Surface area analysis of the synthesized samples was carried out to characterize the synthesized samples The analysis showed that the specific surface area ranged from ∼357 to ∼361 m{sup 2} g{sup −1} and the pore volume of the synthesized samples ranged from 0.177 to 0.182 cm{sup 3} g{sup −1}. The scanning electron microscopy studies showed the structure of the samples to be rectangular and twinned rectangular shaped. The EDX analysis was carried out for confirmation of Si, Al and In in zeolite frame work. The catalytic activities of

  6. Probing the Active Center of Benzaldehyde Lyase with Substitutions and the Pseudosubstrate Analogue Benzoylphosphonic Acid Methyl Ester

    SciTech Connect

    Brandt, Gabriel S.; Nemeria, Natalia; Chakraborty, Sumit; McLeish, Michael J.; Yep, Alejandra; Kenyon, George L.; Petsko, Gregory A.; Jordan, Frank; Ringe, Dagmar

    2008-07-28

    Benzaldehyde lyase (BAL) catalyzes the reversible cleavage of (R)-benzoin to benzaldehyde utilizing thiamin diphosphate and Mg{sup 2+} as cofactors. The enzyme is important for the chemoenzymatic synthesis of a wide range of compounds via its carboligation reaction mechanism. In addition to its principal functions, BAL can slowly decarboxylate aromatic amino acids such as benzoylformic acid. It is also intriguing mechanistically due to the paucity of acid-base residues at the active center that can participate in proton transfer steps thought to be necessary for these types of reactions. Here methyl benzoylphosphonate, an excellent electrostatic analogue of benzoylformic acid, is used to probe the mechanism of benzaldehyde lyase. The structure of benzaldehyde lyase in its covalent complex with methyl benzoylphosphonate was determined to 2.49 {angstrom} (Protein Data Bank entry 3D7K) and represents the first structure of this enzyme with a compound bound in the active site. No large structural reorganization was detected compared to the complex of the enzyme with thiamin diphosphate. The configuration of the predecarboxylation thiamin-bound intermediate was clarified by the structure. Both spectroscopic and X-ray structural studies are consistent with inhibition resulting from the binding of MBP to the thiamin diphosphate in the active centers. We also delineated the role of His29 (the sole potential acid-base catalyst in the active site other than the highly conserved Glu50) and Trp163 in cofactor activation and catalysis by benzaldehyde lyase.

  7. Probing the active center of benzaldehyde lyase with substitutions and the pseudo-substrate analog benzoylphosphonic acid methyl ester

    PubMed Central

    Brandt, Gabriel S.; Nemeria, Natalia; Chakraborty, Sumit; McLeish, Michael J.; Yep, Alejandra; Kenyon, George L.; Petsko, Gregory A.; Jordan, Frank; Ringe, Dagmar

    2009-01-01

    Benzaldehyde lyase (BAL) catalyzes the reversible cleavage of (R)-benzoin to benzaldehyde utilizing thiamin diphosphate and Mg2+ as cofactors. The enzyme is important for the chemoenzymatic synthesis of a wide range of compounds via its carboligation reaction mechanism. In addition to its principal functions, BAL can slowly decarboxylate aromatic amino acids such as benzoylformic acid. It is also intriguing mechanistically due to the paucity of acid-base residues at the active center that can participate in proton transfer steps thought to be necessary for these type of reactions. Here methyl benzoylphosphonate, an excellent electrostatic analog of benzoylformic acid, is used to probe the mechanism of benzaldehyde lyase. The structure of benzaldehyde lyase in its covalent complex with methyl benzoylphosphonate was determined to 2.49 Å (PDB ID: 3D7K) and represents the first structure of this enzyme with a compound bound in the active site. No large structural reorganization was detected compared to the complex of the enzyme with thiamin diphosphate. The configuration of the predecarboxylation thiamin-bound intermediate was clarified by the structure. Both spectroscopic and X-ray structural studies are consistent with inhibition resulting from the binding of MBP to the thiamin diphosphate in the active centers. We also delineated the role of His29 (the sole potential acid-base catalyst in the active site other than the highly conserved Glu50) and Trp163 in cofactor activation and catalysis by benzaldehyde lyase. PMID:18570438

  8. Synthesis, anticonvulsant, antioxidant and binding interaction of novel N-substituted methylquinazoline-2,4(1H, 3H)-dione derivatives to bovine serum albumin: A structure-activity relationship study

    NASA Astrophysics Data System (ADS)

    Prashanth, M. K.; Madaiah, M.; Revanasiddappa, H. D.; Veeresh, B.

    2013-06-01

    A novel class of N-substituted glycosmicine derivatives was synthesized, and their anticonvulsant, antioxidant activity and interaction with bovine serum albumin (BSA) were evaluated. The synthesized compounds 4a-j were examined for anticonvulsant activity by maximal electroshock induced seizures (MESs) test and their neurotoxic effects were determined by rotorod test in mice. The structure-activity relationships (SARs) of these compounds were also investigated. Compounds 4d, 4g, 4i and 4j were found to have good protective effect from seizure. The in vitro antioxidant activity was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide radical scavenging assay. The interaction between novel N-substituted methylquinazoline-2,4(1H, 3H)-dione (NMQ) and BSA was analyzed by fluorescence and ultraviolet spectroscopy at 304 K under simulative physiological conditions. BSA fluorescence quenched by NMQ is discussed according to the Stern-Volmer equation. The binding constant and binding sites of NMQ with BSA were calculated. According to Forster non-radiation energy transfer theory, the binding distance (r) between NMQ and BSA was calculated.

  9. Structural Mutations that Probe the Interactions between the Catalytic and Dianion Activation Sites at Triosephosphate Isomerase‡

    PubMed Central

    Zhai, Xiang; Amyes, Tina L.; Wierenga, Rik K.; Loria, J. Patrick; Richard, John P.

    2013-01-01

    Triosephosphate isomerase (TIM) catalyzes the isomerization of dihydroxyacetone phosphate to form D-glyceraldehyde 3-phosphate. The effects of two structural mutations at TIM on the kinetic parameters for catalysis of the reaction of the truncated substrate glycolaldehyde (GA) and the activation of this reaction by phosphite dianion are reported. The P168A mutation results in similar 50-fold and 80-fold decreases, respectively, in (kcat/Km)E and (kcat/Km)E•HPi for deprotonation of GA catalyzed by free TIM and by the TIM•HPO32− complex. The mutation has little effect on the observed and intrinsic phosphite dianion binding energy, or on the magnitude of phosphite dianion activation of TIM for catalysis of deprotonation of GA. A loop 7 replacement mutant (L7RM) of TIM from chicken muscle was prepared by substitution of the archaeal sequence 208-TGAG for 208-YGGS. The L7RM exhibits a 25-fold decrease in (kcat/Km)E and a larger 170-fold decrease in (kcat/Km)E•HPi for reactions of GA. The mutation has little effect on the observed and intrinsic phosphodianion binding energy, and only a modest effect on phosphite dianion activation of TIM. The observation that both the P168A and loop 7 replacement mutations affect mainly the kinetic parameters for TIM-catalyzed deprotonation, but result in much smaller changes in the parameters for enzyme activation by phosphite dianion provide support for the conclusion that catalysis of proton transfer and dianion activation of TIM take place at separate, weakly interacting, sites in the protein catalyst. PMID:23909928

  10. Identification and Structural Analysis of Amino Acid Substitutions that Increase the Stability and Activity of Aspergillus niger Glucose Oxidase

    PubMed Central

    Marín-Navarro, Julia; Roupain, Nicole; Talens-Perales, David; Polaina, Julio

    2015-01-01

    Glucose oxidase is one of the most conspicuous commercial enzymes due to its many different applications in diverse industries such as food, chemical, energy and textile. Among these applications, the most remarkable is the manufacture of glucose biosensors and in particular sensor strips used to measure glucose levels in serum. The generation of ameliorated versions of glucose oxidase is therefore a significant biotechnological objective. We have used a strategy that combined random and rational approaches to isolate uncharacterized mutations of Aspergillus niger glucose oxidase with improved properties. As a result, we have identified two changes that increase significantly the enzyme's thermal stability. One (T554M) generates a sulfur-pi interaction and the other (Q90R/Y509E) introduces a new salt bridge near the interphase of the dimeric protein structure. An additional double substitution (Q124R/L569E) has no significant effect on stability but causes a twofold increase of the enzyme's specific activity. Our results disclose structural motifs of the protein which are critical for its stability. The combination of mutations in the Q90R/Y509E/T554M triple mutant yielded a version of A. niger glucose oxidase with higher stability than those previously described. PMID:26642312

  11. Active urea transport by the skin of Bufo viridis: Amiloride- and phloretin-sensitive transport sites

    SciTech Connect

    Rapoport, J.; Abuful, A.; Chaimovitz, C.; Noeh, Z.; Hays, R.M. Albert Einstein College of Medicine, New York, NY )

    1988-09-01

    Urea is actively transported inwardly (J{sub i}) across the skin of the green toad Bufo viridis. J{sub i} is markedly enhanced in toads adapted to hypertonic saline. The authors studied urea transport across the skin of Bufo viridis under a variety of experimental conditions, including treatment with amiloride and phloretin, agents that inhibit urea permeability in the bladder of Bufo marinus. Amiloride (10{sup {minus}4} M) significantly inhibited J{sub i} in both adapted and unadapted animals and was unaffected by removal of sodium from the external medium. Phloretin (10{sup {minus}4} M) significantly inhibited J{sub i} in adapted animals by 23-46%; there was also a reduction in J{sub i} in unadapted toads at 10{sup {minus}4} and 5 {times} 10{sup {minus}4} M phloretin. A dose-response study revealed that the concentration of phloretin causing half-maximal inhibition (K{sub {1/2}}) was 5 {times} 10{sup {minus}4} M for adapted animals. J{sub i} was unaffected by the substitution of sucrose for Ringer solution or by ouabain. They conclude (1) the process of adaptation appears to involve an increase in the number of amiloride- and phloretin-inhibitable urea transport sites in the skin, with a possible increase in the affinity of the sites for phloretin; (2) the adapted skin resembles the Bufo marinus urinary bladder with respect to amiloride and phloretin-inhibitable sites; (3) they confirm earlier observations that J{sub i} is independent of sodium transport.

  12. Active Sites Environmental Monitoring Program: Program plan. Revision 1

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1992-02-01

    The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of transuranic (TRU) waste and active low-level waste (LLW) facilities at Oak Ridge National Laboratory (ORNL) in accordance with US Department of Energy (DOE) Order 5820.2A. Active LLW facilities in Solid Waste Storage Area (SWSA) 6 include Tumulus I and Tumulus II, the Interim Waste Management Facility (IWMF), LLW silos, high-range wells, asbestos silos, and fissile wells. The tumulus pads and IWMF are aboveground, high-strength concrete pads on which concrete vaults containing metal boxes of LLW are placed; the void space between the boxes and vaults is filled with grout. Eventually, these pads and vaults will be covered by an engineered multilayered cap. All other LLW facilities in SWSA 6 are below ground. In addition, this plan includes monitoring of the Hillcut Disposal Test Facility (HDTF) in SWSA 6, even though this facility was completed prior to the data of the DOE order. In SWSA 5 North, the TRU facilities include below-grade engineered caves, high-range wells, and unlined trenches. All samples from SWSA 6 are screened for alpha and beta activity, counted for gamma-emitting isotopes, and analyzed for tritium. In addition to these analytes, samples from SWSA 5 North are analyzed for specific transuranic elements.

  13. Antitumor agents. 2. Synthesis, structure-activity relationships, and biological evaluation of substituted 5H-pyridophenoxazin-5-ones with potent antiproliferative activity.

    PubMed

    Bolognese, Adele; Correale, Gaetano; Manfra, Michele; Lavecchia, Antonio; Mazzoni, Orazio; Novellino, Ettore; Barone, Vincenzo; La Colla, Paolo; Loddo, Roberta

    2002-11-21

    New antiproliferative compounds, 5H-pyrido[3,2-a]phenoxazin-5-ones (1-10), 5H-benzophenoxazin-5-one (11), 5H-pyrido[2,3-a]phenoxazin-5-one (12), 5H-pyrido[3,4-a]phenoxazin-5-one (13), and 5H-pyrido[4,3-a]phenoxazin-5-one (14), were synthesized and evaluated against representative human neoplastic cell lines. The excellent cytotoxic activity of these polycyclic phenoxazinones, structurally related to the actinomycin chromophore, is discussed in terms of structural changes made to rings A and D (Chart 1). Electron-withdrawing or electron-donating substituents were introduced at different positions of ring A to probe the electronic and positional effects of the substitution. A nitro group in R(2) or in R(1) increases the cytotoxic activity, whereas electron-donating methyl groups in any position lead to 10- to 100-fold decreasing of the activity. The low antiproliferative activity of benzophenoxazinone 11 and pyridophenoxazinones 13 and 14 confirms the crucial role of pyridine nitrogen in the W position of ring D in DNA binding. The unexpected high activity exhibited by 12, which has the nitrogen in the X position, could be ascribed to a different mechanism of action, which needs further investigation. PMID:12431049

  14. Structural Dissection of the Active Site of Thermotoga maritima β-Galactosidase Identifies Key Residues for Transglycosylating Activity.

    PubMed

    Talens-Perales, David; Polaina, Julio; Marín-Navarro, Julia

    2016-04-13

    Glycoside hydrolases, specifically β-galactosidases, can be used to synthesize galacto-oligosaccharides (GOS) due to the transglycosylating (secondary) activity of these enzymes. Site-directed mutagenesis of a thermoresistant β-galactosidase from Thermotoga maritima has been carried out to study the structural basis of transgalactosylation and to obtain enzymatic variants with better performance for GOS biosynthesis. Rational design of mutations was based on homologous sequence analysis and structural modeling. Analysis of mutant enzymes indicated that residue W959, or an alternative aromatic residue at this position, is critical for the synthesis of β-3'-galactosyl-lactose, the major GOS obtained with the wild-type enzyme. Mutants W959A and W959C, but not W959F, showed an 80% reduced synthesis of this GOS. Other substitutions, N574S, N574A, and F571L, increased the synthesis of β-3'-galactosyl-lactose about 40%. Double mutants F571L/N574S and F571L/N574A showed an increase of about 2-fold. PMID:26998654

  15. Differences in frontal cortical activation by a working memory task after substitution of risperidone for typical antipsychotic drugs in patients with schizophrenia

    PubMed Central

    Honey, Garry D.; Bullmore, Edward T.; Soni, William; Varatheesan, Malini; Williams, Steve C. R.; Sharma, Tonmoy

    1999-01-01

    Antipsychotic drug treatment of schizophrenia may be complicated by side effects of widespread dopaminergic antagonism, including exacerbation of negative and cognitive symptoms due to frontal cortical hypodopaminergia. Atypical antipsychotics have been shown to enhance frontal dopaminergic activity in animal models. We predicted that substitution of risperidone for typical antipsychotic drugs in the treatment of schizophrenia would be associated with enhanced functional activation of frontal cortex. We measured cerebral blood oxygenation changes during periodic performance of a verbal working memory task, using functional MRI, on two occasions (baseline and 6 weeks later) in two cohorts of schizophrenic patients. One cohort (n = 10) was treated with typical antipsychotic drugs throughout the study. Risperidone was substituted for typical antipsychotics after baseline assessment in the second cohort (n = 10). A matched group of healthy volunteers (n = 10) was also studied on a single occasion. A network comprising bilateral dorsolateral prefrontal and lateral premotor cortex, the supplementary motor area, and posterior parietal cortex was activated by working memory task performance in both the patients and comparison subjects. A two-way analysis of covariance was used to estimate the effect of substituting risperidone for typical antipsychotics on power of functional response in the patient group. Substitution of risperidone increased functional activation in right prefrontal cortex, supplementary motor area, and posterior parietal cortex at both voxel and regional levels of analysis. This study provides direct evidence for significantly enhanced frontal function in schizophrenic patients after substitution of risperidone for typical antipsychotic drugs, and it indicates the potential value of functional MRI as a tool for longitudinal assessment of psychopharmacological effects on cerebral physiology. PMID:10557338

  16. Dioxygen and water activation processes on multi-Ru-substituted polyoxometalates: comparison with the "blue-dimer" water oxidation catalyst.

    PubMed

    Kuznetsov, Aleksey E; Geletii, Yurii V; Hill, Craig L; Morokuma, Keiji; Musaev, Djamaladdin G

    2009-05-20

    Dioxygen and water activation on multi-Ru-substituted polyoxometalates were studied using the B3LYP density functional method. It was shown that the reaction of the Ru(2)-substituted gamma-Keggin polyoxotungstate {gamma-[(H(2)O)Ru(III)-(mu-OH)(2)-Ru(III)(H(2)O)][SiW(10)O(36)]}(4-), I(H(2)O), with O(2) is a 4-electron highly exothermic [DeltaE(gas) = 62.5 (DeltaE(gas) + DeltaG(solv(water)) = 24.6) kcal/mol] process and leads to formation of (H(2)O){gamma-[(O)Ru-(mu-OH)(2)-Ru(O)](H(2)O)[SiW(10)O(36)]}(4-), IV(H(2)O). Both the stepwise (or dissociative) and the concerted (or associative) pathways of this reaction occurring with and without water dissociation, respectively, were examined, and the latter has been found to be kinetically more favorable. It was shown that the first 1e-oxidation is achieved by the H(2)O-to-O(2) substitution, which might occur with a maximum of 23.1 (10.5) kcal/mol barrier and leads to the formation of {gamma-[(OO)Ru-(mu-OH)(2)-Ru(H(2)O)](H(2)O)[SiW(10)O(36)]}(4-), II(H(2)O). The second 1e-oxidation is initiated by the proton transfer from the coordinated water molecule to the superoxide (OO(-)) ligand in II(H(2)O) and is completed upon formation of hydroperoxo-hydroxo intermediate {gamma-[(OOH)Ru-(mu-OH)(2)-Ru(OH)](H(2)O)[SiW(10)O(36)]}(4-), III-1(H(2)O). The final 2e-oxidation occurs upon the proton transfer from the terminal OH-ligand to the Ru-coordinated OOH fragment and is completed at the formation of (H(2)O)...{gamma-[(O)Ru-(mu-OH)(2)-Ru(O)](H(2)O)[SiW(10)O(36)]}(4-), IV(H(2)O), with two Ru=O bonds. Each step in the associative pathway is exothermic and occurs with small energy barriers. During the process, the oxidation state of Ru centers increases from +3 to +4. The resulting IV(H(2)O) with a {Ru(O)-(mu-OH)(2)-Ru(O)} core should be formulated to have the Ru(IV)=O(*) units, rather than the Ru(V)=O groups. The reverse reaction, water oxidation by IV(H(2)O), is found to be highly endothermic and cannot occur; this finding is

  17. Active Site and Laminarin Binding in Glycoside Hydrolase Family 55*

    PubMed Central

    Bianchetti, Christopher M.; Takasuka, Taichi E.; Deutsch, Sam; Udell, Hannah S.; Yik, Eric J.; Bergeman, Lai F.; Fox, Brian G.

    2015-01-01

    The Carbohydrate Active Enzyme (CAZy) database indicates that glycoside hydrolase family 55 (GH55) contains both endo- and exo-β-1,3-glucanases. The founding structure in the GH55 is PcLam55A from the white rot fungus Phanerochaete chrysosporium (Ishida, T., Fushinobu, S., Kawai, R., Kitaoka, M., Igarashi, K., and Samejima, M. (2009) Crystal structure of glycoside hydrolase family 55 β-1,3-glucanase from the basidiomycete Phanerochaete chrysosporium. J. Biol. Chem. 284, 10100–10109). Here, we present high resolution crystal structures of bacterial SacteLam55A from the highly cellulolytic Streptomyces sp. SirexAA-E with bound substrates and product. These structures, along with mutagenesis and kinetic studies, implicate Glu-502 as the catalytic acid (as proposed earlier for Glu-663 in PcLam55A) and a proton relay network of four residues in activating water as the nucleophile. Further, a set of conserved aromatic residues that define the active site apparently enforce an exo-glucanase reactivity as demonstrated by exhaustive hydrolysis reactions with purified laminarioligosaccharides. Two additional aromatic residues that line the substrate-binding channel show substrate-dependent conformational flexibility that may promote processive reactivity of the bound oligosaccharide in the bacterial enzymes. Gene synthesis carried out on ∼30% of the GH55 family gave 34 active enzymes (19% functional coverage of the nonredundant members of GH55). These active enzymes reacted with only laminarin from a panel of 10 different soluble and insoluble polysaccharides and displayed a broad range of specific activities and optima for pH and temperature. Application of this experimental method provides a new, systematic way to annotate glycoside hydrolase phylogenetic space for functional properties. PMID:25752603

  18. Active site and laminarin binding in glycoside hydrolase family 55.

    PubMed

    Bianchetti, Christopher M; Takasuka, Taichi E; Deutsch, Sam; Udell, Hannah S; Yik, Eric J; Bergeman, Lai F; Fox, Brian G

    2015-05-01

    The Carbohydrate Active Enzyme (CAZy) database indicates that glycoside hydrolase family 55 (GH55) contains both endo- and exo-β-1,3-glucanases. The founding structure in the GH55 is PcLam55A from the white rot fungus Phanerochaete chrysosporium (Ishida, T., Fushinobu, S., Kawai, R., Kitaoka, M., Igarashi, K., and Samejima, M. (2009) Crystal structure of glycoside hydrolase family 55 β-1,3-glucanase from the basidiomycete Phanerochaete chrysosporium. J. Biol. Chem. 284, 10100-10109). Here, we present high resolution crystal structures of bacterial SacteLam55A from the highly cellulolytic Streptomyces sp. SirexAA-E with bound substrates and product. These structures, along with mutagenesis and kinetic studies, implicate Glu-502 as the catalytic acid (as proposed earlier for Glu-663 in PcLam55A) and a proton relay network of four residues in activating water as the nucleophile. Further, a set of conserved aromatic residues that define the active site apparently enforce an exo-glucanase reactivity as demonstrated by exhaustive hydrolysis reactions with purified laminarioligosaccharides. Two additional aromatic residues that line the substrate-binding channel show substrate-dependent conformational flexibility that may promote processive reactivity of the bound oligosaccharide in the bacterial enzymes. Gene synthesis carried out on ∼30% of the GH55 family gave 34 active enzymes (19% functional coverage of the nonredundant members of GH55). These active enzymes reacted with only laminarin from a panel of 10 different soluble and insoluble polysaccharides and displayed a broad range of specific activities and optima for pH and temperature. Application of this experimental method provides a new, systematic way to annotate glycoside hydrolase phylogenetic space for functional properties. PMID:25752603

  19. Active Site Loop Conformation Regulates Promiscuous Activity in a Lactonase from Geobacillus kaustophilus HTA426

    PubMed Central

    Zhang, Yu; An, Jiao; Yang, Guang-Yu; Bai, Aixi; Zheng, Baisong; Lou, Zhiyong; Wu, Geng; Ye, Wei; Chen, Hai-Feng; Feng, Yan; Manco, Giuseppe

    2015-01-01

    Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL) from Geobacillus kaustophilus HTA426 (GkaP) exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a “hot spot” in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (kcat/Km) toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ∼6.6 Å outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity. PMID:25706379

  20. Synthesis and anti-inflammatory activity of 1-acetyl-5-substituted aryl-3-(beta-aminonaphthyl)-2-pyrazolines and beta-(substituted aminoethyl) amidonaphthalenes.

    PubMed

    Bansal, E; Srivastava, V K; Kumar, A

    2001-01-01

    The title compounds were prepared by reaction of beta-acetylamino-naphthalene with different aromatic aldehydes followed by cyclisation with hydrazine hydrate and with different primary or secondary amines (Mannich's reaction), respectively. The structures of new compounds were confirmed by 1H-NMR and IR spectral data. Anti-inflammatory and ulcerogenic activities in vivo were evaluated and compared with the standard drugs, phenylbutazone and indomethacin. Some compounds of the series exhibited promising anti-inflammatory activity with a lower ulcerogenic liability than the standard drugs. PMID:11231052

  1. Structural Characterization of Mutations at the Oxygen Activation Site in Monomeric Sarcosine Oxidase

    SciTech Connect

    Schuman Jorns, Marilyn; Chen, Zhi-wei; Mathews, F. Scott

    2010-04-30

    Oxygen reduction and sarcosine oxidation in monomeric sarcosine oxidase (MSOX) occur at separate sites above the si- and re-faces, respectively, of the flavin ring. Mutagenesis studies implicate Lys265 as the oxygen activation site. Substitution of Lys265 with a neutral (Met, Gln, or Ala) or basic (Arg) residue results in an {approx}10{sup 4}- or 250-fold decrease, respectively, in the reaction rate. The overall structure of MSOX and residue conformation in the sarcosine binding cavity are unaffected by replacement of Lys265 with Met or Arg. The side chain of Met265 exhibits the same configuration in each molecule of Lys265Met crystals and is nearly congruent with Lys265 in wild-type MSOX. The side chain of Arg265 is, however, dramatically shifted (4-5 {angstrom}) compared with Lys265, points in the opposite direction, and exhibits significant conformational variability between molecules of the same crystal. The major species in solutions of Lys265Arg is likely to contain a 'flipped-out' Arg265 and exhibit negligible oxygen activation, similar to Lys265Met. The 400-fold higher oxygen reactivity observed with Lys265Arg is attributed to a minor (<1%) 'flipped-in' Arg265 conformer whose oxygen reactivity is similar to that of wild-type MSOX. A structural water (WAT1), found above the si-face of the flavin ring in all previously determined MSOX structures, is part of an apparent proton relay system that extends from FAD N(5) to bulk solvent. WAT1 is strikingly absent in Lys265Met and Lys265Arg, a feature that may account for the apparent kinetic stabilization of a reductive half-reaction intermediate that is detectable with the mutants but not wild-type MSOX.

  2. Ligand-dependent dynamics of the active-site lid in bacterial dimethylarginine dimethylaminohydrolase.

    PubMed

    Rasheed, Masooma; Richter, Christine; Chisty, Liisa T; Kirkpatrick, John; Blackledge, Martin; Webb, Martin R; Driscoll, Paul C

    2014-02-18

    The dimethylarginine dimethylaminohydrolase (DDAH) enzyme family has been the subject of substantial investigation as a potential therapeutic target for the regulation of vascular tension. DDAH enzymes catalyze the conversion of asymmetric N(η),N(η)-dimethylarginine (ADMA) to l-citrulline. Here the influence of substrate and product binding on the dynamic flexibility of DDAH from Pseudomonas aeruginosa (PaDDAH) has been assessed. A combination of heteronuclear NMR spectroscopy, static and time-resolved fluorescence measurements, and atomistic molecular dynamics simulations was employed. A monodisperse monomeric variant of the wild-type enzyme binds the reaction product l-citrulline with a low millimolar dissociation constant. A second variant, engineered to be catalytically inactive by substitution of the nucleophilic Cys249 residue with serine, can still convert the substrate ADMA to products very slowly. This PaDDAH variant also binds l-citrulline, but with a low micromolar dissociation constant. NMR and molecular dynamics simulations indicate that the active site "lid", formed by residues Gly17-Asp27, exhibits a high degree of internal motion on the picosecond-to-nanosecond time scale. This suggests that the lid is open in the apo state and allows substrate access to the active site that is otherwise buried. l-Citrulline binding to both protein variants is accompanied by an ordering of the lid. Modification of PaDDAH with a coumarin fluorescence reporter allowed measurement of the kinetic mechanism of the PaDDAH reaction. A combination of NMR and kinetic data shows that the catalytic turnover of the enzyme is not limited by release of the l-citrulline product. The potential to develop the coumarin-PaDDAH adduct as an l-citrulline sensor is discussed. PMID:24484052

  3. QSAR modeling of aromatase inhibitory activity of 1-substituted 1,2,3-triazole analogs of letrozole.

    PubMed

    Nantasenamat, Chanin; Worachartcheewan, Apilak; Prachayasittikul, Supaluk; Isarankura-Na-Ayudhya, Chartchalerm; Prachayasittikul, Virapong

    2013-11-01

    Aromatase is an estrogen biosynthesis enzyme belonging to the cytochrome P450 family that catalyzes the rate-limiting step of converting androgens to estrogens. As it is pertinent toward tumor cell growth promotion, aromatase is a lucrative therapeutic target for breast cancer. In the pursuit of robust aromatase inhibitors, a set of fifty-four 1-substituted mono- and bis-benzonitrile or phenyl analogs of 1,2,3-triazole letrozole were employed in quantitative structure-activity relationship (QSAR) study using multiple linear regression (MLR), artificial neural network (ANN) and support vector machine (SVM). Such QSAR models were developed using a set of descriptors providing coverage of the general characteristics of a molecule encompassing molecular size, flexibility, polarity, solubility, charge and electronic properties. Important physicochemical properties giving rise to good aromatase inhibition were obtained by means of exploring its chemical space as a function of the calculated molecular descriptors. The optimal subset of 3 descriptors (i.e. number of rings, ALogP and HOMO-LUMO) was further used for QSAR model construction. The predicted pIC₅₀ values were in strong correlation with their experimental values displaying correlation coefficient values in the range of 0.72-0.83 for the cross-validated set (QCV) while the external test set (Q(Ext)) afforded values in the range of 0.65-0.66. Insights gained from the present study are anticipated to provide pertinent information contributing to the origins of aromatase inhibitory activity and therefore aid in our on-going quest for aromatase inhibitors with robust properties. PMID:24012714

  4. The effect of A-site and B-site substitution on BaFeO3-δ: An investigation as a cathode material for intermediate-temperature solid oxide fuel cells

    NASA Astrophysics Data System (ADS)

    Wang, Jian; Saccoccio, Mattia; Chen, Dengjie; Gao, Yang; Chen, Chi; Ciucci, Francesco

    2015-11-01

    This work systematically investigates the effects of single A-site dopant (5 mol% La3+, Sm3+ and Gd3+) and single B-site dopant (5 mol% Zr4+ and Ce4+) on the structure and oxygen reduction reaction of BaFeO3-δ (BFO) used as a cathode for solid oxide fuel cells. The materials are prepared by solid-state method and their structural, electronic, electrocatalytic properties are characterized and compared. X-ray diffraction reveals 5 mol% A-site or B-site dopant is sufficient to stabilize the cubic phase of BFO, as predicted by the lattice calculation. X-ray photoelectron spectroscopy and iodometric titration demonstrates that neither of the two doping sites has obvious advantage over the other towards the formation of additional oxygen vacancies. B-site doped BFO shows a lower electrical conductivity than A-site doped ones, however, they have much quicker response to electrical conductivity relaxation, likely originating from the expanded lattice size. With the largest oxygen vacancy concentrations, Ba0.95La0.05FeO3-δ and BaFe0.95Zr0.05O3-δ stand out from the A-site and B-site doped BFO, respectively, and polarization resistances of 0.029 Ω cm2 and 0.020 Ω cm2 are achieved at 700 °C, PO2 = 0.2atm . With a similar amount of oxygen vacancies, B-site doping is more advantageous for enhancing oxygen bulk diffusion kinetics, and thus ORR activity.

  5. In vitro-in vivo activity relationship of substituted benzimidazole cell division inhibitors with activity against Mycobacteria tuberculosis

    PubMed Central

    Knudson, Susan E.; Kumar, Kunal; Awasthi, Divya; Ojima, Iwao; Slayden, Richard A.

    2014-01-01

    Structure based drug design was used to develop a compound library of novel 2,5,6- and 2,5,7-trisubstituted benzimidazoles. Three structural analogs, SB-P1G10, SB-P8B2 and SB-P3G2 were selected from this library based on previous studies for advanced study. In vitro studies revealed that SB-P8B2 and SB-P3G2 had sigmoidal kill-curves while in contrast SB-P1G10 showed a narrow zonal susceptibility. The in vitro studies also demonstrated that exposure to SB-P8B2 or SB-P3G2 was bactericidal, while SB-P1G10 treatment never resulted in complete killing. The dose curves for the three compounds against clinical isolates were comparable to their respective dose curves in the laboratory strain of M. tuberculosis. SB-P8B2 and SB-P3G2 exhibited antibacterial activity against non-replicating bacilli under low oxygen conditions. SB-P3G2 and SB-P1G10 were assessed in acute short-term animal models of tuberculosis, which showed that SB-P3G2 treatment demonstrated activity against M. tuberculosis. Together, these studies reveal an in vitro- in vivo relationship of the 2,5,6-trisubstituted benzimidazoles that serves as a criterion for advancing this class of cell division inhibitors into more resource intensive in vivo efficacy models such as the long-term murine model of tuberculosis and Pre-IND PK/PD studies. Specifically, these studies are the first demonstration of efficacy and an in vitro–in vivo activity relationship for 2,5,6-trisubstituted benzimidazoles. The in vivo activity presented in this manuscript substantiates this class of cell division inhibitors as having potency and efficacy against M. tuberculosis. PMID:24746463

  6. Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site.

    PubMed

    Sunden, Fanny; Peck, Ariana; Salzman, Julia; Ressl, Susanne; Herschlag, Daniel

    2015-01-01

    Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called 'catalytic residues' are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modes of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes. PMID:25902402

  7. Docking studies, synthesis, characterization of some novel oxazine substituted 9-anilinoacridine derivatives and evaluation for their antioxidant and anticancer activities as topoisomerase II inhibitors.

    PubMed

    Kalirajan, R; Kulshrestha, Vivek; Sankar, S; Jubie, S

    2012-10-01

    A series of 9-anilinoacridines substituted with oxazine derivatives were synthesized to evaluate their antioxidant and anticancer activity against Daltons Lymphoma Ascites (DLA) cell growth by in vitro method. It was revealed that these conjugates exhibited significant antioxidant and anticancer activity (inhibition of DLA cell proliferation). Among these agents, compounds 5a, 5h, 5i, 5j were the most cytotoxic with CTC(50) value of 140-250 μg/mL. The docking studies of the synthesized compounds were performed towards the key Topoisomerase II (1QZR) by using Schrodinger Maestro 9.2 version. The oxazine substituted 9-anilinoacridine derivatives 5a, 5h, 5i, 5j have significant anticancer activity as topoisomerase II inhibitors. PMID:22982526

  8. Biological activities of 2alpha-substituted analogues of 1alpha,25-dihydroxyvitamin D3 in transcriptional regulation and human promyelocytic leukemia (HL-60) cell proliferation and differentiation.

    PubMed

    Takahashi, Eiji; Nakagawa, Kimie; Suhara, Yoshitomo; Kittaka, Atsushi; Nihei, Ken-ichi; Konno, Katsuhiro; Takayama, Hiroaki; Ozono, Keiichi; Okano, Toshio

    2006-11-01

    Biological activities of 2alpha-substituted 1alpha,25-dihydroxyvitamin D3 analogues were evaluated in vitro. Their binding affinity was examined with calf thymus cytosolic vitamin D receptor (VDR) and rat plasma vitamin D-binding protein (DBP). In addition, the transcriptional activity of the analogues was measured using a rat 25-hydroxyvitamin D3-24-hydroxylase gene promoter, a human osteocalcin gene promoter, and VDR-GAL4 system. This study investigated the biological activities of 2alpha-substituted analogues in comparison with 2beta-substitued analogues at the molecular level, with regard to the structural differences of alkyl, hydroxyalkyl, hydroxyalkoxy substituents at the 2-position of 1alpha,25-dihydroxyvitamin D3. PMID:17077522

  9. Metavanadate at the active site of the phosphatase VHZ.

    PubMed

    Kuznetsov, Vyacheslav I; Alexandrova, Anastassia N; Hengge, Alvan C

    2012-09-01

    Vanadate is a potent modulator of a number of biological processes and has been shown by crystal structures and NMR spectroscopy to interact with numerous enzymes. Although these effects often occur under conditions where oligomeric forms dominate, the crystal structures and NMR data suggest that the inhibitory form is usually monomeric orthovanadate, a particularly good inhibitor of phosphatases because of its ability to form stable trigonal-bipyramidal complexes. We performed a computational analysis of a 1.14 Å structure of the phosphatase VHZ in complex with an unusual metavanadate species and compared it with two classical trigonal-bipyramidal vanadate-phosphatase complexes. The results support extensive delocalized bonding to the apical ligands in the classical structures. In contrast, in the VHZ metavanadate complex, the central, planar VO(3)(-) moiety has only one apical ligand, the nucleophilic Cys95, and a gap in electron density between V and S. A computational analysis showed that the V-S interaction is primarily ionic. A mechanism is proposed to explain the formation of metavanadate in the active site from a dimeric vanadate species that previous crystallographic evidence has shown to be able to bind to the active sites of phosphatases related to VHZ. Together, the results show that the interaction of vanadate with biological systems is not solely reliant upon the prior formation of a particular inhibitory form in solution. The catalytic properties of an enzyme may act upon the oligomeric forms primarily present in solution to generate species such as the metavanadate ion observed in the VHZ structure. PMID:22876963

  10. Multiple transport-active binding sites are available for a single substrate on human P-glycoprotein (ABCB1).

    PubMed

    Chufan, Eduardo E; Kapoor, Khyati; Sim, Hong-May; Singh, Satyakam; Talele, Tanaji T; Durell, Stewart R; Ambudkar, Suresh V

    2013-01-01

    P-glycoprotein (Pgp, ABCB1) is an ATP-Binding Cassette (ABC) transporter that is associated with the development of multidrug resistance in cancer cells. Pgp transports a variety of chemically dissimilar amphipathic compounds using the energy from ATP hydrolysis. In the present study, to elucidate the binding sites on Pgp for substrates and modulators, we employed site-directed mutagenesis, cell- and membrane-based assays, molecular modeling and docking. We generated single, double and triple mutants with substitutions of the Y307, F343, Q725, F728, F978 and V982 residues at the proposed drug-binding site with cys in a cysless Pgp, and expressed them in insect and mammalian cells using a baculovirus expression system. All the mutant proteins were expressed at the cell surface to the same extent as the cysless wild-type Pgp. With substitution of three residues of the pocket (Y307, Q725 and V982) with cysteine in a cysless Pgp, QZ59S-SSS, cyclosporine A, tariquidar, valinomycin and FSBA lose the ability to inhibit the labeling of Pgp with a transport substrate, [(125)I]-Iodoarylazidoprazosin, indicating these drugs cannot bind at their primary binding sites. However, the drugs can modulate the ATP hydrolysis of the mutant Pgps, demonstrating that they bind at secondary sites. In addition, the transport of six fluorescent substrates in HeLa cells expressing triple mutant (Y307C/Q725C/V982C) Pgp is also not significantly altered, showing that substrates bound at secondary sites are still transported. The homology modeling of human Pgp and substrate and modulator docking studies support the biochemical and transport data. In aggregate, our results demonstrate that a large flexible pocket in the Pgp transmembrane domains is able to bind chemically diverse compounds. When residues of the primary drug-binding site are mutated, substrates and modulators bind to secondary sites on the transporter and more than one transport-active binding site is available for each

  11. Mimicking enzymatic active sites on surfaces for energy conversion chemistry.

    PubMed

    Gutzler, Rico; Stepanow, Sebastian; Grumelli, Doris; Lingenfelder, Magalí; Kern, Klaus

    2015-07-21

    Metal-organic supramolecular chemistry on surfaces has matured to a point where its underlying growth mechanisms are well understood and structures of defined coordination environments of metal atoms can be synthesized in a controlled and reproducible procedure. With surface-confined molecular self-assembly, scientists have a tool box at hand which can be used to prepare structures with desired properties, as for example a defined oxidation number and spin state of the transition metal atoms within the organic matrix. From a structural point of view, these coordination sites in the supramolecular structure resemble the catalytically active sites of metallo-enzymes, both characterized by metal centers coordinated to organic ligands. Several chemical reactions take place at these embedded metal ions in enzymes and the question arises whether these reactions also take place using metal-organic networks as catalysts. Mimicking the active site of metal atoms and organic ligands of enzymes in artificial systems is the key to understanding the selectivity and efficiency of enzymatic reactions. Their catalytic activity depends on various parameters including the charge and spin configuration in the metal ion, but also on the organic environment, which can stabilize intermediate reaction products, inhibits catalytic deactivation, and serves mostly as a transport channel for the reactants and products and therefore ensures the selectivity of the enzyme. Charge and spin on the transition metal in enzymes depend on the one hand on the specific metal element, and on the other hand on its organic coordination environment. These two parameters can carefully be adjusted in surface confined metal-organic networks, which can be synthesized by virtue of combinatorial mixing of building synthons. Different organic ligands with varying functional groups can be combined with several transition metals and spontaneously assemble into ordered networks. The catalytically active metal

  12. Crystal Structure, Exogenous Ligand Binding and Redox Properties of an Engineered Diiron Active Site in a Bacterial Hemerythrin

    PubMed Central

    Okamoto, Yasunori; Onoda, Akira; Sugimoto, Hiroshi; Takano, Yu; Hirota, Shun; Kurtz, Donald M.; Shiro, Yoshitsugu; Hayashi, Takashi

    2013-01-01

    A non-heme diiron active site in a 13-kDa hemerythrin-like domain of the bacterial chemotaxis protein, DcrH-Hr, contains an oxo bridge, two bridging carboxylate groups from Glu and Asp residues, and five terminally ligated His residues. We created a unique diiron coordination sphere containing five His and three Glu/Asp residues by replacing an Ile residue with Glu in DcrH-Hr. Direct coordination of the carboxylate group of E119 to Fe2 of the diiron site in the I119E variant was confirmed by X-ray crystallography. The substituted Glu is adjacent to an exogenous ligand-accessible tunnel. UV-vis absorption spectra indicate that the additional coordination of E119 inhibits the binding of the exogenous ligands, azide and phenol, to the diiron site. The extent of azide binding to the diiron site increases at pH ≤ 6, which is ascribed to protonation of the carboxylate ligand of E119. The diferrous state (deoxy form) of the engineered diiron site with the extra Glu residue is found to react more slowly than wild type with O2 to yield the diferric state (met form). The additional coordination of E119 to the diiron site also slows the rate of reduction from the met form. All these processes were found to be pH-dependent, which can be attributed to protonation state and coordination status of the E119 carboxylate. These results demonstrate that modifications of the endogenous coordination sphere can produce significant changes in the ligand binding and redox properties in a prototypical non-heme diiron-carboxylate protein active site. PMID:24187962

  13. Sensory Substitution

    NASA Astrophysics Data System (ADS)

    Verrillo, Ronald T.

    The idea that the cutaneous surface may be employed as a substitute for the eyes and ears is by no means a modern notion. Although the sense of touch has long been considered as a surrogate for both the visual and auditory modalities, the focus of this chapter will be on the efforts to develop a tactile substitute for hearing, especially that of human speech. The visual system is our primary means of processing information about environmental space such as orientation, distance, direction and size. It is much less effective in making temporal discriminations. The auditory system is unparalleled in processing information that involves rapid sequences of temporal events, such as speech and music. The tactile sense is capable of processing both spatial and temporal information although not as effective in either domain as the eye or the ear.

  14. Increasing the reaction rate of hydroxynitrile lyase from Hevea brasiliensis toward mandelonitrile by copying active site residues from an esterase that accepts aromatic esters.

    PubMed

    von Langermann, Jan; Nedrud, David M; Kazlauskas, Romas J

    2014-09-01

    The natural substrate of hydroxynitrile lyase from rubber tree (HbHNL, Hevea brasiliensis) is acetone cyanohydrin, but synthetic applications usually involve aromatic cyanohydrins such as mandelonitrile. To increase the activity of HbHNL toward this unnatural substrate, we replaced active site residues in HbHNL with the corresponding ones from esterase SABP2 (salicylic acid binding protein 2). Although this enzyme catalyzes a different reaction (hydrolysis of esters), its natural substrate (methyl salicylate) contains an aromatic ring. Three of the eleven single-amino-acid-substitution variants of HbHNL reacted more rapidly with mandelonitrile. The best was HbHNL-L121Y, with a kcat 4.2 times higher and high enantioselectivity. Site-saturation mutagenesis at position 121 identified three other improved variants. We hypothesize that the smaller active site orients the aromatic substrate more productively. PMID:25044660

  15. Hybrid [FeFe]-hydrogenases with modified active sites show remarkable residual enzymatic activity.

    PubMed

    Siebel, Judith F; Adamska-Venkatesh, Agnieszka; Weber, Katharina; Rumpel, Sigrun; Reijerse, Edward; Lubitz, Wolfgang

    2015-02-24

    [FeFe]-hydrogenases are to date the only enzymes for which it has been demonstrated that the native inorganic binuclear cofactor of the active site Fe2(adt)(CO)3(CN)2 (adt = azadithiolate = [S-CH2-NH-CH2-S](2-)) can be synthesized on the laboratory bench and subsequently inserted into the unmaturated enzyme to yield fully functional holo-enzyme (Berggren, G. et al. (2013) Nature 499, 66-70; Esselborn, J. et al. (2013) Nat. Chem. Biol. 9, 607-610). In the current study, we exploit this procedure to introduce non-native cofactors into the enzyme. Mimics of the binuclear subcluster with a modified bridging dithiolate ligand (thiodithiolate, N-methylazadithiolate, dimethyl-azadithiolate) and three variants containing only one CN(-) ligand were inserted into the active site of the enzyme. We investigated the activity of these variants for hydrogen oxidation as well as proton reduction and their structural accommodation within the active site was analyzed using Fourier transform infrared spectroscopy. Interestingly, the monocyanide variant with the azadithiolate bridge showed ∼50% of the native enzyme activity. This would suggest that the CN(-) ligands are not essential for catalytic activity, but rather serve to anchor the binuclear subsite inside the protein pocket through hydrogen bonding. The inserted artificial cofactors with a propanedithiolate and an N-methylazadithiolate bridge as well as their monocyanide variants also showed residual activity. However, these activities were less than 1% of the native enzyme. Our findings indicate that even small changes in the dithiolate bridge of the binuclear subsite lead to a rather strong decrease of the catalytic activity. We conclude that both the Brønsted base function and the conformational flexibility of the native azadithiolate amine moiety are essential for the high catalytic activity of the native enzyme. PMID:25633077

  16. Inhibition of trace element release during Fe(II)-activated recrystallization of Al-, Cr-, and Sn-substituted goethite and hematite

    SciTech Connect

    Frierdich, Andrew J.; Scherer, M.; Bachman, Jonathan E.; Engelhard, Mark H.; Rapponotti, Brett W.; Catalano, Jeffrey G.

    2012-09-18

    Aqueous Fe(II) reacts with Fe(III) oxides by coupled electron transfer and atom exchange (ETAE) resulting in mineral recrystallization, contaminant reduction, and trace element cycling. Previous studies of Fe(II)-Fe(III) ETAE have explored the reactivity of either pure iron oxide phases or those containing small quantities of soluble trace elements. Naturally occurring iron oxides, however, contain substantial quantities of insoluble impurities (e.g., Al) which are known to affect the chemical properties of such minerals. Here we explore the effect of Al(III), Cr(III), and Sn(IV) substitution on trace element release from Ni(II)-substituted goethite and Zn(II)-substituted hematite during reaction with aqueous Fe(II). Fe(II)-activated trace element release is substantially inhibited from both minerals when an insoluble element is co-substituted into the structure, and the total amount of release decreases exponentially with increasing co substituent. The limited changes in surface composition that occur following reaction with Fe(II) indicate that Al, Cr, and Sn do not exsolve from the structure and that Ni and Zn released to solution originate primarily from the bulk rather than the particle exterior (upper ~3 nm). Incorporation of Al into goethite substantially decreases the amount of iron atom exchange with aqueous Fe(II) and, consequently, the amount of Ni release from the structure. This implies that trace element release inhibition caused by substituting insoluble elements results from a decrease in the amount of mineral recrystallization. These results suggest that naturally occurring iron oxides containing insoluble elements are less susceptible to Fe(II)-activated recrystallization and exhibit a greater retention of trace elements and contaminants than pure mineral phases.

  17. Site-specific PEGylation of lidamycin and its antitumor activity.

    PubMed

    Li, Liang; Shang, Boyang; Hu, Lei; Shao, Rongguang; Zhen, Yongsu

    2015-05-01

    In this study, N-terminal site-specific mono-PEGylation of the recombinant lidamycin apoprotein (rLDP) of lidamycin (LDM) was prepared using a polyethyleneglycol (PEG) derivative (M w 20 kDa) through a reactive terminal aldehyde group under weak acidic conditions (pH 5.5). The biochemical properties of mPEG-rLDP-AE, an enediyne-integrated conjugate, were analyzed by SDS-PAGE, RP-HPLC, SEC-HPLC and MALDI-TOF. Meanwhile, in vitro and in vivo antitumor activity of mPEG-rLDP-AE was evaluated by MTT assays and in xenograft model. The results indicated that mPEG-rLDP-AE showed significant antitumor activity both in vitro and in vivo. After PEGylation, mPEG-rLDP still retained the binding capability to the enediyne AE and presented the physicochemical characteristics similar to that of native LDP. It is of interest that the PEGylation did not diminish the antitumor efficacy of LDM, implying the possibility that this derivative may function as a payload to deliver novel tumor-targeted drugs. PMID:26579455

  18. Canine distemper virus neutralization activity is low in human serum and it is sensitive to an amino acid substitution in the hemagglutinin protein

    SciTech Connect

    Zhang, Xinsheng; Wallace, Olivia L.; Domi, Arban; Wright, Kevin J.; Driscoll, Jonathan; Anzala, Omu; Sanders, Eduard J.; Kamali, Anatoli; Allen, Susan; Fast, Pat; Gilmour, Jill; Price, Matt A.; Parks, Christopher L.

    2015-08-15

    Serum was analyzed from 146 healthy adult volunteers in eastern Africa to evaluate measles virus (MV) and canine distemper virus (CDV) neutralizing antibody (nAb) prevalence and potency. MV plaque reduction neutralization test (PRNT) results indicated that all sera were positive for MV nAbs. Furthermore, the 50% neutralizing dose (ND50) for the majority of sera corresponded to antibody titers induced by MV vaccination. CDV nAbs titers were low and generally were detected in sera with high MV nAb titers. A mutant CDV was generated that was less sensitive to neutralization by human serum. The mutant virus genome had 10 nucleotide substitutions, which coded for single amino acid substitutions in the fusion (F) and hemagglutinin (H) glycoproteins and two substitutions in the large polymerase (L) protein. The H substitution occurred in a conserved region involved in receptor interactions among morbilliviruses, implying that this region is a target for cross-reactive neutralizing antibodies. - Highlights: • Screened 146 serum samples for measles virus (MV) and canine distemper virus (CDV) neutralizing antibody (nAb). • MV nAb is prevalent in the sera. • CDV neutralizing activity is generally low or absent and when detected it is present in sera with high MV nAb titers. • A neutralization-resistant CDV mutant was isolated using human serum selection. • A mutation was identified in the receptor-binding region of CDV hemagglutinin protein that confers the neutralization resistance.

  19. Improving Functional Annotation in the DRE-TIM Metallolyase Superfamily through Identification of Active Site Fingerprints.

    PubMed

    Kumar, Garima; Johnson, Jordyn L; Frantom, Patrick A

    2016-03-29

    Within the DRE-TIM metallolyase superfamily, members of the Claisen-like condensation (CC-like) subgroup catalyze C-C bond-forming reactions between various α-ketoacids and acetyl-coenzyme A. These reactions are important in the metabolic pathways of many bacterial pathogens and serve as engineering scaffolds for the production of long-chain alcohol biofuels. To improve functional annotation and identify sequences that might use novel substrates in the CC-like subgroup, a combination of structural modeling and multiple-sequence alignments identified active site residues on the third, fourth, and fifth β-strands of the TIM-barrel catalytic domain that are differentially conserved within the substrate-diverse enzyme families. Using α-isopropylmalate synthase and citramalate synthase from Methanococcus jannaschii (MjIPMS and MjCMS), site-directed mutagenesis was used to test the role of each identified position in substrate selectivity. Kinetic data suggest that residues at the β3-5 and β4-7 positions play a significant role in the selection of α-ketoisovalerate over pyruvate in MjIPMS. However, complementary substitutions in MjCMS fail to alter substrate specificity, suggesting residues in these positions do not contribute to substrate selectivity in this enzyme. Analysis of the kinetic data with respect to a protein similarity network for the CC-like subgroup suggests that evolutionarily distinct forms of IPMS utilize residues at the β3-5 and β4-7 positions to affect substrate selectivity while the different versions of CMS use unique architectures. Importantly, mapping the identities of residues at the β3-5 and β4-7 positions onto the protein similarity network allows for rapid annotation of probable IPMS enzymes as well as several outlier sequences that may represent novel functions in the subgroup. PMID:26935545

  20. Second generation bisheteroarylpiperazine (BHAP) HIV-1 reverse transcriptasae inhibitors: Enhancement of antiviral activity and aqueous solubility via 5- and 6-substitution of the indole ring

    SciTech Connect

    Poel, T.; Thomas, R.C.; Romero, D.L.; Hosley, M.J.; Morge, R.A.; Biles, C.; Reusser, F.; Althaus, I.W.; Schinzer, W.C.; Platzer, D.J.

    1993-12-31

    U-87201E, a potent HIV-1 reverse transcriptase inhibitor (RTI) discovered at Upjohn, is currently in Phase II clinical trials. Additional structure-activity studies have identified second-generation BHAPs with enhanced antiviral activity and improved pharmaceutical properties, notably increased aqueous solubility. Capitalizing on initial SAR studies which demonstrated a tolerance for substitution in the indole ring, a series of BHAPs bearing 5- and 6-substituted indoles was evaluated. Substituents such as ethers, sulfonamides, ureas, and sulfamides containing water-solubilizing groups such as polyethers or basic amines provided highly potent BHAPs with greatly enhanced solubility, such as U-93923. The synthesis, antiviral evaluation and solubility properties of these potent HIV-1 RTIs will be detailed.

  1. Synthesis and biological evaluation of isomeric methoxy substitutions on anti-cancer indolyl-pyridinyl-propenones: Effects on potency and mode of activity.

    PubMed

    Trabbic, Christopher J; George, Sage M; Alexander, Evan M; Du, Shengnan; Offenbacher, Jennifer M; Crissman, Emily J; Overmeyer, Jean H; Maltese, William A; Erhardt, Paul W

    2016-10-21

    Certain indolyl-pyridinyl-propenone analogues kill glioblastoma cells that have become resistant to conventional therapeutic drugs. Some of these analogues induce a novel form of non-apoptotic cell death called methuosis, while others primarily cause microtubule disruption. Ready access to 5-indole substitution has allowed characterization of this position to be important for both types of mechanisms when a simple methoxy group is present. We now report the syntheses and biological effects of isomeric methoxy substitutions on the indole ring. Additionally, analogues containing a trimethoxyphenyl group in place of the pyridinyl moiety were evaluated for anticancer activity. The results demonstrate that the location of the methoxy group can alter both the potency and the mechanism of cell death. Remarkably, changing the methoxy from the 5-position to the 6-position switched the biological activity from induction of methuosis to disruption of microtubules. The latter may represent a prototype for a new class of mitotic inhibitors with potential therapeutic utility. PMID:27343855

  2. Allosteric site-mediated active site inhibition of PBP2a using Quercetin 3-O-rutinoside and its combination.

    PubMed

    Rani, Nidhi; Vijayakumar, Saravanan; P T V, Lakshmi; Arunachalam, Annamalai

    2016-08-01

    Recent crystallographic study revealed the involvement of allosteric site in active site inhibition of penicillin binding protein (PBP2a), where one molecule of Ceftaroline (Cef) binds to the allosteric site of PBP2a and paved way for the other molecule (Cef) to bind at the active site. Though Cef has the potency to inhibit the PBP2a, its adverse side effects are of major concern. Previous studies have reported the antibacterial property of Quercetin derivatives, a group of natural compounds. Hence, the present study aims to evaluate the effect of Quercetin 3-o-rutinoside (Rut) in allosteric site-mediated active site inhibition of PBP2a. The molecular docking studies between allosteric site and ligands (Rut, Que, and Cef) revealed a better binding efficiency (G-score) of Rut (-7.790318) and Cef (-6.194946) with respect to Que (-5.079284). Molecular dynamic (MD) simulation studies showed significant changes at the active site in the presence of ligands (Rut and Cef) at allosteric site. Four different combinations of Rut and Cef were docked and their G-scores ranged between -6.320 and -8.623. MD studies revealed the stability of the key residue (Ser403) with Rut being at both sites, compared to other complexes. Morphological analysis through electron microscopy confirmed that combination of Rut and Cefixime was able to disturb the bacterial cell membrane in a similar fashion to that of Rut and Cefixime alone. The results of this study indicate that the affinity of Rut at both sites were equally good, with further validations Rut could be considered as an alternative for inhibiting MRSA growth. PMID:26360629

  3. An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions.

    PubMed

    Sengupta, Raghuvir N; Van Schie, Sabine N S; Giambaşu, George; Dai, Qing; Yesselman, Joseph D; York, Darrin; Piccirilli, Joseph A; Herschlag, Daniel

    2016-01-01

    Biological catalysis hinges on the precise structural integrity of an active site that binds and transforms its substrates and meeting this requirement presents a unique challenge for RNA enzymes. Functional RNAs, including ribozymes, fold into their active conformations within rugged energy landscapes that often contain misfolded conformers. Here we uncover and characterize one such "off-pathway" species within an active site after overall folding of the ribozyme is complete. The Tetrahymena group I ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine (G) cofactor. We tested whether specific catalytic interactions with G are present in the preceding E•S•G and E•G ground-state complexes. We monitored interactions with G via the effects of 2'- and 3'-deoxy (-H) and -amino (-NH(2)) substitutions on G binding. These and prior results reveal that G is bound in an inactive configuration within E•G, with the nucleophilic 3'-OH making a nonproductive interaction with an active site metal ion termed MA and with the adjacent 2'-OH making no interaction. Upon S binding, a rearrangement occurs that allows both -OH groups to contact a different active site metal ion, termed M(C), to make what are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA's difficulty in specifying a unique conformation and highlighting Nature's potential to use local transitions of RNA in complex function. PMID:26567314

  4. Active site hydrophobicity is critical to the bioluminescence activity of Vibrio harveyi luciferase.

    PubMed

    Li, Chi-Hui; Tu, Shiao-Chun

    2005-10-01

    Vibrio harveyi luciferase is an alphabeta heterodimer containing a single active site, proposed earlier to be at a cleft in the alpha subunit. In this work, six conserved phenylalanine residues at this proposed active site were subjected to site-directed mutations to investigate their possible functional roles and to delineate the makeup of luciferase active site. After initial screening of Phe --> Ala mutants, alphaF46, alphaF49, alphaF114, and alphaF117 were chosen for additional mutations to Asp, Ser, and Tyr. Comparisons of the general kinetic properties of wild-type and mutated luciferases indicated that the hydrophobic nature of alphaF46, alphaF49, alphaF114, and alphaF117 was important to luciferase V(max) and V(max)/K(m), which were reduced by 3-5 orders of magnitude for the Phe --> Asp mutants. Both alphaF46 and alphaF117 also appeared to be involved in the binding of reduced flavin substrate. Additional studies on the stability and yield of the 4a-hydroperoxyflavin intermediate II and measurements of decanal substrate oxidation by alphaF46D, alphaF49D, alphaF114D, and alphaF117D revealed that their marked reductions in the overall quantum yield (phi( degrees )) were a consequence of diminished yields of luciferase intermediates and, with the exception of alphaF114D, emission quantum yield of the excited emitter due to the replacement of the hydrophobic Phe by the anionic Asp. The locations of these four critical Phe residues in relation to other essential and/or hydrophobic residues are depicted in a refined map of the active site. Functional implications of these residues are discussed. PMID:16185065

  5. New Isotopic Constraints on the Sources of Methane at Sites of Active Continental Serpentinization

    NASA Astrophysics Data System (ADS)

    Wang, D. T.; Gruen, D.; Morrill, P. L.; Rietze, A.; Nealson, K. H.; Kubo, M. D.; Cardace, D.; Schrenk, M. O.; Hoehler, T. M.; McCollom, T. M.; Etiope, G.; Hosgormez, H.; Schoell, M.; Ono, S.

    2014-12-01

    At continental sites of serpentinization, high concentrations of reduced gases (e.g., H2, CH4) are frequently found in association with highly-alkaline groundwater. Identification of the process(es) responsible for the generation of methane—as well as the source(s) of C & H—in these environments has been challenging. The difficulty is due to both the wide range of processes (microbial, thermal, abiotic) that could be involved, and the limited number of parameters that are accessible to currently-available analytical technologies (e.g., δ13C, δD). The recent development of a new technique based on tunable infrared laser spectroscopy [1] has enabled the fully-resolved quantification of four isotopologues of methane: 12CH4, 13CH4, 12CH3D, and 13CH3D, a doubly-substituted ("clumped") isotopologue. We used this technique to measure 13CH3D in gases sampled from continental sites of serpentinization, in order to provide independent constraints on C-H bond-forming processes involved in the generation of the methane found in these systems. Our study sites are hosted in ultramafic units that are presently undergoing serpentinization. These include The Cedars peridotite body (Calif., USA) [2], the Coast Range Ophiolite Microbial Observatory (Calif., USA) [3], and the Chimaera seep (Tekirova Ophiolite, Turkey) [4]. Preliminary measurements indicate that Δ13CH3D (the deviation of the abundance of 13CH3D from the stochastic distribution) in methane sampled from these sites spans nearly the entire range of thermodynamically-predicted values, from >+5‰ (13CH3D-based apparent equilibrium temperature < 45 °C) to ~0‰ (Tapparent → ∞). The new 13CH3D data is complemented by conventional geochemical analyses (e.g., dissolved ions/organics, δ13C, δD) on samples collected during the same field campaigns. Our study demonstrates that the measurement of 13CH3D provides a new dimension of isotopic constraints for unraveling the complex processes controlling the distribution

  6. A proposed definition of the 'activity' of surface sites on lactose carriers for dry powder inhalation.

    PubMed

    Grasmeijer, Floris; Frijlink, Henderik W; de Boer, Anne H

    2014-06-01

    A new definition of the activity of surface sites on lactose carriers for dry powder inhalation is proposed which relates to drug detachment during dispersion. The new definition is expected to improve the understanding of 'carrier surface site activity', which stimulates the unambiguous communication about this subject and may aid in the rational design and interpretation of future formulation studies. In contrast to the currently prevailing view on carrier surface site activity, it follows from the newly proposed definition that carrier surface site activity depends on more variables than just the physicochemical properties of the carrier surface. Because the term 'active sites' is ambiguous, it is recommended to use the term 'highly active sites' instead to denote carrier surface sites with a relatively high activity. PMID:24613490

  7. Synthesis and Structure-Activity Relationship Analysis of 5-HT₇ Receptor Antagonists: Piperazin-1-yl Substituted Unfused Heterobiaryls.

    PubMed

    Strekowski, Lucjan; Sączewski, Jarosław; Raux, Elizabeth A; Fernando, Nilmi T; Klenc, Jeff; Paranjpe, Shirish; Raszkiewicz, Aldona; Blake, Ava L; Ehalt, Adam J; Barnes, Samuel; Baranowski, Timothy C; Sullivan, Shannon M; Satała, Grzegorz; Bojarski, Andrzej J

    2016-01-01

    A series of piperazin-1-yl substituted unfused heterobiaryls was synthesized as ligands of the 5-HT₇ receptors. The goal of this project was to elucidate the structural features that affect the 5-HT₇ binding affinity of this class of compounds represented by the model ligand 4-(3-furyl)-2-(4-methylpiperazin-1-yl)pyrimidine (2). The SAR studies included systematical structural changes of the pyrimidine core moiety in 2 to quinazoline, pyridine and benzene, changes of the 3-furyl group to other heteroaryl substituents, the presence of various analogs of the 4-methylpiperazin-1-yl group, as well as additional substitutions at positions 5 and 6 of the pyrimidine. Substitution of position 6 of the pyrimidine in the model ligand with an alkyl group results in a substantial increase of the binding affinity (note a change in position numbers due to the nomenclature rules). It was also demonstrated that 4-(3-furyl) moiety is crucial for the 5-HT₇ binding affinity of the substituted pyrimidines, although, the pyrimidine core can be replaced with a pyridine ring without a dramatic loss of the binding affinity. The selected ethylpyrimidine (12) and butylpyrimidine (13) analogs of high 5-HT₇ binding affinity showed antagonistic properties in cAMP functional test and varied selectivity profile-compound 12 can be regarded as a dual 5-HT₇/5-HT2AR ligand, and 13 as a multi-receptor (5-HT₇, 5-HT2A, 5-HT₆ and D₂) agent. PMID:27043518

  8. Base-mediated decomposition of amide-substituted furfuryl tosylhydrazones: synthesis and cytotoxic activities of enynyl-ketoamides.

    PubMed

    Ji, Fanghua; Peng, Hui; Zhang, Xiaoting; Lu, Wenhua; Liu, Shubin; Jiang, Huanfeng; Liu, Bo; Yin, Biaolin

    2015-02-20

    Base-mediated decomposition of amide-substituted furfuryl tosylhydrazones afforded practical access to novel multifunctionalized enynyl-ketoamides. In addition, furfuryl tosylhydrazones with stable furan rings underwent an interesting tosyl-group migration to form sulfones, which have potential synthetic applications. Some of the obtained enynyl-ketoamides demonstrated good cytotoxicities against human tumor cell lines. PMID:25635471

  9. Disturbance opens recruitment sites for bacterial colonization in activated sludge.

    PubMed

    Vuono, David C; Munakata-Marr, Junko; Spear, John R; Drewes, Jörg E

    2016-01-01

    Little is known about the role of immigration in shaping bacterial communities or the factors that may dictate success or failure of colonization by bacteria from regional species pools. To address these knowledge gaps, the influence of bacterial colonization into an ecosystem (activated sludge bioreactor) was measured through a disturbance gradient (successive decreases in the parameter solids retention time) relative to stable operational conditions. Through a DNA sequencing approach, we show that the most abundant bacteria within the immigrant community have a greater probability of colonizing the receiving ecosystem, but mostly as low abundance community members. Only during the disturbance do some of these bacterial populations significantly increase in abundance beyond background levels and in few cases become dominant community members post-disturbance. Two mechanisms facilitate the enhanced enrichment of immigrant populations during disturbance: (i) the availability of resources left unconsumed by established species and (ii) the increased availability of niche space for colonizers to establish and displace resident populations. Thus, as a disturbance decreases local diversity, recruitment sites become available to promote colonization. This work advances our understanding of microbial resource management and diversity maintenance in complex ecosystems. PMID:25727891

  10. Construction of DNA recognition sites active in Haemophilus transformation.

    PubMed Central

    Danner, D B; Smith, H O; Narang, S A

    1982-01-01

    Competent Haemophilus cells recognize and preferentially take up Haemophilus DNA during genetic transformation. This preferential uptake is correlated with the presence on incoming DNA of an 11-base-pair (bp) sequence, 5'-A-A-G-T-G-C-G-G-T-C-A-3'. To prove that this sequence is the recognition site that identifies Haemophilus DNA to the competent cell, we have now constructed a series of plasmids, each of which contains the 11-bp sequence. Using two different assay systems we have tested the ability of fragments from these plasmids to compete with cloned Haemophilus DNA fragments that naturally contain the 11-bp sequence. We find that the addition of the 11-bp sequence to a DNA fragment is necessary and sufficient for preferential uptake of that fragment. However, plasmid DNAs containing this sequence may vary as much as 48-fold in uptake activity, and this variation correlates with the A+T-richness of the DNA flanking the 11-mer. Images PMID:6285382

  11. Characterization of active site residues of nitroalkane oxidase.

    PubMed

    Valley, Michael P; Fenny, Nana S; Ali, Shah R; Fitzpatrick, Paul F

    2010-06-01

    The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the corresponding aldehydes and ketones plus nitrite. The structure of the enzyme shows that Ser171 forms a hydrogen bond to the flavin N5, suggesting that it plays a role in catalysis. Cys397 and Tyr398 were previously identified by chemical modification as potential active site residues. To more directly probe the roles of these residues, the S171A, S171V, S171T, C397S, and Y398F enzymes have been characterized with nitroethane as substrate. The C397S and Y398 enzymes were less stable than the wild-type enzyme, and the C397S enzyme routinely contained a substoichiometric amount of FAD. Analysis of the steady-state kinetic parameters for the mutant enzymes, including deuterium isotope effects, establishes that all of the mutations result in decreases in the rate constants for removal of the substrate proton by approximately 5-fold and decreases in the rate constant for product release of approximately 2-fold. Only the S171V and S171T mutations alter the rate constant for flavin oxidation. These results establish that these residues are not involved in catalysis, but rather are required for maintaining the protein structure. PMID:20056514

  12. Detection limit for activation measurements in ultralow background sites

    NASA Astrophysics Data System (ADS)

    Trache, Livius; Chesneanu, D.; Margineanu, R.; Pantelica, A.; Ghita, D. G.; Burducea, I.; Straticiuc, M.; Tang, X. D.

    2014-09-01

    We used 12C +13C fusion at the beam energies E = 6, 7 and 8 MeV to determine the sensitivity and the limits of activation method measurements in ultralow background sites. A 13C beam of 0.5 μA from the 3 MV Tandem accelerator of the Horia Hulubei National Institute of Physics and Nuclear Engineering - IFIN HH impinged on thick graphite targets. After about 24 hrs of irradiation targets were measured in two different laboratories: one with a heavy shielded Ge detector in the institute (at the surface) and one located underground in the microBequerel laboratory, in the salt mine of Slanic-Prahova, Romania. The 1369- and 2754 keV peaks from 24Na deactivation were clearly observed in the γ-ray spectra obtained for acquisitions lasting a few hours, or a few days. Determination of the detection limit in evaluating the cross sections for the target irradiated at Ec . m = 3 MeV indicates the fact that it is possible to measure gamma spectrum in underground laboratory down to Ec . m = 2 . 6 MeV. Cleaning the spectra with beta-gamma coincidences and increasing beam intensity 20 times will take as further down. The measurements are motivated by the study of the 12 C +12 C reaction at astrophysical energies.

  13. N6-Methyldeoxyadenosine Marks Active Transcription Start Sites in Chlamydomonas

    PubMed Central

    Chen, Kai; Deng, Xin; Yu, Miao; Han, Dali; Hao, Ziyang; Liu, Jianzhao; Lu, Xingyu; Dore, Louis C; Weng, Xiaocheng; Ji, Quanjiang; Mets, Laurens; He, Chuan

    2015-01-01

    SUMMARY N6-methyldeoxyadenosine (6mA or m6A) is a DNA modification preserved in prokaryotes to eukaryotes. It is widespread in bacteria, and functions in DNA mismatch repair, chromosome segregation, and virulence regulation. In contrast, the distribution and function of 6mA in eukaryotes have been unclear. Here we present a comprehensive analysis of the 6mA landscape in the genome of Chlamydomonas using new sequencing approaches. We identified the 6mA modification in 84% of genes in Chlamydomonas. We found that 6mA mainly locates at ApT dinucleotides around transcription start sites (TSS) with a bimodal distribution, and appears to mark active genes. A periodic pattern of 6mA deposition was also observed at base resolution, which is associated with nucleosome distribution near the TSS, suggesting a possible role in nucleosome positioning. The new genome-wide mapping of 6mA and its unique distribution in the Chlamydomonas genome suggest potential regulatory roles of 6mA in gene expression in eukaryotic organisms. PMID:25936837

  14. Imidase catalyzing desymmetric imide hydrolysis forming optically active 3-substituted glutaric acid monoamides for the synthesis of gamma-aminobutyric acid (GABA) analogs.

    PubMed

    Nojiri, Masutoshi; Hibi, Makoto; Shizawa, Hiroaki; Horinouchi, Nobuyuki; Yasohara, Yoshihiko; Takahashi, Satomi; Ogawa, Jun

    2015-12-01

    The recent use of optically active 3-substituted gamma-aminobutyric acid (GABA) analogs in human therapeutics has identified a need for an efficient, stereoselective method of their synthesis. Here, bacterial strains were screened for enzymes capable of stereospecific hydrolysis of 3-substituted glutarimides to generate (R)-3-substituted glutaric acid monoamides. The bacteria Alcaligenes faecalis NBRC13111 and Burkholderia phytofirmans DSM17436 were discovered to hydrolyze 3-(4-chlorophenyl) glutarimide (CGI) to (R)-3-(4-chlorophenyl) glutaric acid monoamide (CGM) with 98.1% enantiomeric excess (e.e.) and 97.5% e.e., respectively. B. phytofirmans DSM17436 could also hydrolyze 3-isobutyl glutarimide (IBI) to produce (R)-3-isobutyl glutaric acid monoamide (IBM) with 94.9% e.e. BpIH, an imidase, was purified from B. phytofirmans DSM17436 and found to generate (R)-CGM from CGI with specific activity of 0.95 U/mg. The amino acid sequence of BpIH had a 75% sequence identity to that of allantoinase from A. faecalis NBRC13111 (AfIH). The purified recombinant BpIH and AfIH catalyzed (R)-selective hydrolysis of CGI and IBI. In addition, a preliminary investigation of the enzymatic properties of BpIH and AfIH revealed that both enzymes were stable in the range of pH 6-10, with an optimal pH of 9.0, stable at temperatures below 40 °C, and were not metalloproteins. These results indicate that the use of this class of hydrolase to generate optically active 3-substituted glutaric acid monoamide could simplify the production of specific chiral GABA analogs for drug therapeutics. PMID:26205522

  15. Unexpected reactivity of 2-fluorolinalyl diphosphate in the active site of crystalline 2-methylisoborneol synthase

    PubMed Central

    Köksal, Mustafa; Chou, Wayne K. W.; Cane, David E.; Christianson, David W.

    2013-01-01

    The crystal structure of 2-methylisoborneol synthase (MIBS) from Streptomyces coelicolor A3(2) has been determined in its unliganded state and in complex with 2 Mg2+ ions and cis-2-fluorogeranyl diphosphate at 1.85 Å and 2.00 Å resolution, respectively. Under normal circumstances, MIBS catalyzes the cyclization of the naturally-occurring, non-canonical 11-carbon isoprenoid substrate, 2-methylgeranyl diphosphate, which first undergoes an ionization-isomerization-ionization sequence through the tertiary diphosphate intermediate 2-methyllinalyl diphosphate to enable subsequent cyclization chemistry. MIBS does not exhibit catalytic activity with 2-fluorogeranyl diphosphate, and we recently reported the crystal structure of MIBS complexed with this unreactive substrate analogue [Köksal, M., Chou, W. K. W., Cane, D. E., Christianson, D. W. (2012) Biochemistry 51, 3011–3020]. However, cocrystallization of MIBS with the fluorinated analogue of the tertiary allylic diphosphate intermediate, 2-fluorolinalyl diphosphate, reveals unexpected reactivity for the intermediate analogue and yields the crystal structure of the complex with the primary allylic diphosphate, 2-fluoroneryl diphosphate. Comparison with the structure of the unliganded enzyme reveals that the crystalline enzyme active site remains partially open, presumably due to the binding of only 2 Mg2+ ions. Assays in solution indicate that MIBS catalyzes the generation of (1R)-(+)-camphor from the substrate 2-fluorolinalyl diphosphate, suggesting that both 2-fluorolinalyl diphosphate and 2-methyllinalyl diphosphate follow the identical cyclization mechanism leading to 2-substituted isoborneol products; however, the initially generated 2-fluoroisoborneol cyclization product is unstable and undergoes elimination of hydrogen fluoride to yield (1R)-(+)-camphor. PMID:23844678

  16. A Binding Site Model and Structure-Activity Relationships for the Rat A3 Adenosine Receptor

    PubMed Central

    VAN GALEN, PHILIP J. M.; VAN BERGEN, ANDREW H.; GALLO-RODRIGUEZ, CAROLA; MELMAN, NELI; OLAH, MARK E.; IJZERMAN, AD P.; STILES, GARY L.; JACOBSON, KENNETH A.

    2012-01-01

    SUMMARY A novel adenosine receptor, the A3 receptor, has recently been cloned. We have systematically investigated the hitherto largely unexplored structure-activity relationships (SARs) for binding at A3 receptors, using 125I-N6-2-(4-aminophenyl)ethyladenosine as a radioligand and membranes from Chinese hamster ovary cells stably transfected with the rat A3-cDNA. As is the case for A1 and A2a, receptors, substitutions at the N6 and 5′ positions of adenosine, the prototypic agonist ligand, may yield fairly potent compounds. However, the highest affinity and A3 selectivity is found for N6,5′-disubstituted compounds, in contrast to A1 and A2a receptors. Thus, N6-benzyladenosine-5′-N-ethylcarboxamide is highly potent (Ki, 6.8 nM) and moderately selective (13- and 14-fold versus A1 and A2a). The N6 region of the A3 receptor also appears to tolerate hydrophilic substitutions, in sharp contrast to the other subtypes. Potencies of N6,5′-disubstituted compounds in inhibition of adenylate cyclase via A3 receptors parallel their high affinity in the binding assay. None of the typical xanthine or nonxanthine (A1/A2) antagonists tested show any appreciable affinity for rat A3 receptors. 1,3-Dialkylxanthines did not antagonize the A3 agonist-induced inhibition of adenylate cyclase. A His residue in helix 6 that is absent in A3 receptors but present in A1/A2 receptors may be causal in this respect. In a molecular model for the rat A3 receptor, this mutation, together with an increased bulkiness of residues surrounding the ligand, make antagonist binding unfavorable when compared with a previously developed A1 receptor model. Second, this A3 receptor model predicted similarities with A1 and A2 receptors in the binding requirements for the ribose moiety and that xanthine-7-ribosides would bind to rat A3 receptors. This hypothesis was supported experimentally by the moderate affinity (Ki 6 μM) of 7-riboside of 1,3-dibutylxanthine, which appears to be a partial agonist at

  17. Active site of the mRNA-capping enzyme guanylyltransferase from Saccharomyces cerevisiae: similarity to the nucleotidyl attachment motif of DNA and RNA ligases.

    PubMed Central

    Fresco, L D; Buratowski, S

    1994-01-01

    Nascent mRNA chains are capped at the 5' end by the addition of a guanylyl residue to form a G(5')ppp(5')N ... structure. During the capping reaction, the guanylyltransferase (GTP:mRNA guanylyltransferase, EC 2.7.7.50) is reversibly and covalently guanylylated. In this enzyme-GMP (E-GMP) intermediate, GMP is linked to the epsilon-amino group of a lysine residue via a phosphoamide bond. Lys-70 was identified as the GMP attachment site of the Saccharomyces cerevisiae guanylyltransferase (encoded by the CEG1 gene) by guanylylpeptide sequencing. CEG1 genes with substitutions at Lys-70 were unable to support viability in yeast and produced proteins that were not guanylylated in vitro. The CEG1 active site exhibits sequence similarity to the active sites of viral guanylyltransferases and polynucleotide ligases, suggesting similarity in the mechanisms of nucleotidyl transfer catalyzed by these enzymes. Images PMID:8022828

  18. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  19. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  20. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  1. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  2. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  3. Systematic Tuning of the Luminescent Properties of Self-Activated ZnGa 2O 4 Phosphors by Cd Ion Substitution

    <