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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. 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

  3. 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.

  4. A 7-phenyl substituted triazolopyridazine has inverse agonist activity at the benzodiazepine receptor site.

    PubMed Central

    Biziere, K.; Bourguignon, J. J.; Chambon, J. P.; Heaulme, M.; Perio, A.; Tebib, S.; Wermuth, C. G.

    1987-01-01

    To investigate further the structural requirements for benzodiazepine (BZD) receptor ligands, we synthesized SR 95195, [7-phenyl-3-methyl-1,2,4-triazolo-(4,3-b) pyridazine], a positional isomer of the 6-phenyl-triazolo-pyridazines, which were the first non-BZD derivatives to exhibit high affinity for the BZD receptor and BZD-like activity in vivo. In vitro, SR 95195 displaced specifically bound [3H]-flunitrazepam from rat cerebellar and hippocampal membranes with respective IC50 values of 4 and 8 microM. In vivo, SR 95195 lacked BZD-like activity. At high doses SR 95195 induced clonic seizures in mice (threshold convulsant dose: 150 mg kg-1; CD50: 160 mg kg-1 i.p.) which were antagonized by Ro 15-1788. At non-convulsant doses (25 mg kg-1 i.p. and 100 mg kg-1 i.p.) SR 95195 significantly decreased punished responding in an operant conflict procedure in the rat, suggesting SR 95195 has intrinsic anxiogenic activity. SR 95195, in mice, reversed the anticonvulsant and myorelaxant actions of diazepam 3 mg kg-1, orally (respective ED50 values: 45 mg kg-1 i.p. and 44 mg kg-1 i.p.). In an operant-conflict test in rats, SR 95195 at non-anxiogenic doses, antagonized the disinhibitory action of diazepam 4 mg kg-1, i.p. (ED50: 8.6 mg kg-1, i.p.), but not that of pentobarbitone 15 mg kg-1, i.p. It is concluded that SR 95195 has the pharmacological profile of an inverse BZD agonist and that displacing the phenyl from the 6- to the 7-position in the triazolopyridazine series causes a shift from agonist to inverse agonist type activity at the BZD receptor site. PMID:3028557

  5. 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

  6. Fluorophenyl-substituted Fe-only hydrogenases active site ADT models: different electrocatalytic process for proton reduction in HOAc and HBF4/Et2O.

    PubMed

    Wang, Wen-Guang; Wang, Hong-Yan; Si, Gang; Tung, Chen-Ho; Wu, Li-Zhu

    2009-04-21

    A set of fluorophenyl-substituted adt-bridged Fe2S2 active site models of Fe-only hydrogenase, [(micro-SCH2)2NR]Fe2(CO)6 (, R=C6F4CF3-p; , R=C6H4CF3-p) and [(micro-SCH2)2NR]Fe2(CO)5(PPh3) (, R=C6F4CF3-p; , R=C6H4CF3-p), have been synthesized and well characterized. Spectroscopic and electrochemical studies demonstrate that the aryl-substituted complexes are stable toward a strong acid HBF4/Et2O, and electrocatalytic process for the hydrogen production is mostly dependent on the strength of the available proton source. When CH3COOH is used as the proton source, the electrocatalytic process begins with successively two one-electron reduction processes to produce H2 at Fe(0)Fe(0) (E2pc); whereas in the presence of strong acid, HBF4/Et2O, the process is initiated by protonation of a N-bridged atom followed by reduction of the protonated N-bridged atom around -1.29 V, and then release of H2 at Fe(0)Fe(I) (E1pc). Varying the strength of acid leads to the initial electron-transfer step from the reduction of a protonated N-bridged atom to the active site of [Fe(I)Fe(I)].

  7. 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.

  8. Short circuiting a sulfite oxidising enzyme with direct electrochemistry: active site substitutions and their effect on catalysis and electron transfer.

    PubMed

    Rapson, Trevor D; Kappler, Ulrike; Hanson, Graeme R; Bernhardt, Paul V

    2011-01-01

    Sulfite dehydrogenase (SDH) from Starkeya novella is a heterodimeric enzyme comprising a Mo active site and a heme c electron relay, which mediates electron transfer from the Mo cofactor to cytochrome c following sulfite oxidation. Studies on the wild type enzyme (SDH(WT)) and its variants have identified key amino acids at the active site, specifically Arg-55 and His-57. We report the Mo(VI/V), Mo(V/IV) and Fe(III/II) (heme) redox potentials of the variants SDH(R55K), SDH(R55M), SDH(R55Q) and SDH(H57A) in comparison with those of SDH(WT). For SDH(R55M), SDH(R55Q) and SDH(H57A) the heme potentials are lowered from ca. 240mV in SDH(WT) to ca. 200mV, while the heme potential in SDH(R55K) remains unchanged and the Mo redox potentials are not affected significantly in any of these variants. Protein film voltammetry reveals a pH dependence of the electrochemical catalytic half-wave potential (E(cat)) of -59mV/pH in SDH(WT) and SDH(R55K) which tracks the pH dependence of the Mo(VI/V) redox potential. By contrast, the catalytic potentials for SDH(R55M) and SDH(H57A) are pH-independent and follow the potential of the heme cofactor. These results highlight a switch in the pathway of electron exchange as a function of applied potential that is revealed by protein film voltammetry where an actuation of rate limiting intramolecular electron transfer (IET, Mo to heme) at high potential attenuates the catalytic current relative to faster, direct electron transfer (Mo to electrode) at lower potential. The same change in electron transfer pathway is linked to an unusual peak-shaped profile of the ideally sigmoidal steady state voltammogram in SDH(WT) alone, which has been associated with a potential dependent change in the orientation of the enzyme on the electrode surface. All other variants show purely sigmoidal voltammetry due to their inherently slower turnover numbers which are always lower than IET rates.

  9. 47 CFR 54.646 - Site and service substitutions.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 47 Telecommunication 3 2013-10-01 2013-10-01 false Site and service substitutions. 54.646 Section 54.646 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES (CONTINUED) UNIVERSAL SERVICE Universal Service Support for Health Care Providers Healthcare Connect Fund § 54.646 Site and service substitutions. (a)...

  10. [Clastogenic activity of dietary sugar substitutes].

    PubMed

    Durnev, A D; Oreshchenko, A V; Kulakova, A V; Beresten', N F; Seredenin, S B

    1995-01-01

    The present paper describes the possible clastogenic activity of the following synthetic sugar substitutes, such as cyclamate in daily doses of 11 and 110 mg/kg, saccharin, 5 and 50 mg/kg, acesulfam, 15 and 150 mg/kg, sucralose, 15 and 150 mg/kg, aspartame, 40 and 400 mg/kg, orally given to C57Bl/6 mice during 5 days. No clastogenic activity was found in the compounds tested.

  11. Structural and Catalytic Effects of Proline Substitution and Surface Loop Deletion in the Extended Active Site of Human Carbonic Anhydrase II

    PubMed Central

    Boone, Christopher D.; Rasi, Valerio; Tu, Chingkuang; McKenna, Robert

    2015-01-01

    The bioengineering of a thermophilic enzyme starting from a mesophilic scaffold has proven to be a significant challenge as several stabilizing elements have been proposed to be the foundation of thermal stability including disulfide bridges, surface loop reduction, ionic pair networks, proline substitutions, and aromatic clusters. This study emphasizes the impact of increasing the rigidity of human carbonic anhydrase II (HCA II) via incorporation of proline residues at positions 170 and 234, which are located in surface loops that are able to accommodate restrictive main-chain conformations without rearrangement of the surrounding peptide backbone. Additionally, the effect of compactness of HCA II was examined by way of deletion of a surface loop (residues 230 through 240), which had been previously identified as a possible source of thermal stability for the hyperthermophilic CA isolated from the bacterium Sulfurihydrogenibium yellowstonense YO3AOP1. Differential scanning calorimetry analysis of these HCA II variants revealed that these structural modifications had a minimum effect on the thermal stability of the enzyme while kinetic studies showed unexpected effects on the catalytic efficiency and proton transfer rates. X-ray crystallographic analysis of these HCA II variants showed the electrostatic potential and configuration of the highly acidic loop (residues 230 and 240) plays an important role in its high catalytic activity. Based on these observations and the literature, a picture is emerging of the various components within the general structural architecture of HCA II that are key to stability. These elements could provide the blueprints for the rational thermal stability engineering of other enzymes. PMID:25683338

  12. 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

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

    PubMed

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

    2015-08-11

    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.

  14. Increasing the binding affinity of VEGFR-2 inhibitors by extending their hydrophobic interaction with the active site: Design, synthesis and biological evaluation of 1-substituted-4-(4-methoxybenzyl)phthalazine derivatives.

    PubMed

    Eldehna, Wagdy M; Abou-Seri, Sahar M; El Kerdawy, Ahmed M; Ayyad, Rezk R; Hamdy, Abdallah M; Ghabbour, Hazem A; Ali, Mamdouh M; Abou El Ella, Dalal A

    2016-05-01

    A series of anilinophthalazine derivatives 4a-j was initially synthesized and tested for its VEGFR-2 inhibitory activity where it showed promising activity (IC50 = 0.636-5.76 μM). Molecular docking studies guidance was used to improve the binding affinity for series 4a-j towards VEGFR-2 active site. This improvement was achieved by increasing the hydrophobic interaction with the hydrophobic back pocket of the VEGFR-2 active site lined with the hydrophobic side chains of Ile888, Leu889, Ile892, Val898, Val899, Leu1019 and Ile1044. Increasing the hydrophobic interaction was accomplished by extending the anilinophthalazine scaffold with a substituted phenyl moiety through an uriedo linker which should give this extension the flexibility required to accommodate itself deeply into the hydrophobic back pocket. As planned, the designed uriedo-anilinophthalazines 7a-i showed superior binding affinity than their anilinophthalazine parents (IC50 = 0.083-0.473 μM). In particular, compounds 7g-i showed IC50 of 0.086, 0.083 and 0.086 μM, respectively, which are better than that of the reference drug sorafenib (IC50 = 0.09 μM).

  15. Human liver alcohol dehydrogenase: amino acid substitution in the beta 2 beta 2 Oriental isozyme explains functional properties, establishes an active site structure, and parallels mutational exchanges in the yeast enzyme.

    PubMed Central

    Jörnvall, H; Hempel, J; Vallee, B L; Bosron, W F; Li, T K

    1984-01-01

    The homodimeric Oriental beta 2 beta 2 isozyme of human liver alcohol dehydrogenase, corresponding to an allelic variant at the ADH2 gene locus, was studied in order to define the amino acid exchange in relation to the beta 1 beta 1 isozyme, the predominant allelic form among Caucasians. Sequence analysis reveals that the amino acid substitution occurs at position 7 of the largest CNBr fragment, corresponding to position 47 of the whole protein chain. Here, the beta 2 form has a histidine residue, while, in common with other characterized mammalian liver alcohol dehydrogenases, the beta 1 form has an arginine residue. This exchange does not affect the adjacent cysteine-46 residue, which is a protein ligand to the active-site zinc atom, thus clarifying previously inconsistent results. The histidine/arginine-47 mutational replacement corresponds to a position that binds the pyrophosphate group of the coenzyme NAD(H); this explains the functional differences between the beta 1 beta 1 and beta 2 beta 2 isozymes, including both a lower pH optimum and higher turnover number of beta 2 beta 2, which is likely to be the mutant form. The exchange demonstrates the existence of parallel but separate mutations in the evolution of alcohol dehydrogenases because these mammalian enzymes differ at exactly the same position by the same type of substitution as is found between a mutant and the wild-type constitutive forms of the corresponding yeast enzyme. PMID:6374651

  16. 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.

  17. Synthesis and antiviral activity of substituted quercetins.

    PubMed

    Thapa, Mahendra; Kim, Yunjeong; Desper, John; Chang, Kyeong-Ok; Hua, Duy H

    2012-01-01

    Influenza viruses are important pathogens that cause respiratory infections in humans and animals. In addition to vaccination, antiviral drugs against influenza virus play a significant role in controlling viral infections by reducing disease progression and virus transmission. Plant derived polyphenols are associated with antioxidant activity, anti-carcinogenic, and cardio- and neuro-protective actions. Some polyphenols, such as resveratrol and epigallocatechin gallate (EGCG), showed significant anti-influenza activity in vitro and/or in vivo. Recently we showed that quercetin and isoquercetin (quercetin-3-β-d-glucoside), a glucoside form of quercetin, significantly reduced the replication of influenza viruses in vitro and in vivo (isoquercetin). The antiviral effects of isoquercetin were greater than that of quercetin with lower IC(50) values and higher in vitro therapeutic index. Thus, we investigated the synthesis and antiviral activities of various quercetin derivatives with substitution of C3, C3', and C5 hydroxyl functions with various phenolic ester, alkoxy, and aminoalkoxy moieties. Among newly synthesized compounds, quercetin-3-gallate which is structurally related to EGCG showed comparable antiviral activity against influenza virus (porcine H1N1 strain) to that of EGCG with improved in vitro therapeutic index.

  18. Active site-directed inhibitors of cytochrome P-450scc. Structural and mechanistic implications of a side chain-substituted series of amino-steroids.

    PubMed

    Sheets, J J; Vickery, L E

    1983-10-10

    A series of analogues of cholesterol, each having a shortened side chain and a primary amine group, were prepared and tested for their effects on bovine adrenocortical cholesterol side chain cleavage cytochrome P-450 (P-450scc). A previous study had shown that one derivative, 22-amino-23,24-bisnor-5-cholen-3 beta-ol, is a potent competitive inhibitor of the enzyme and forms a complex in which the steroid ring binds to the cholesterol site and the side chain amine forms a bond with the heme iron (Sheets, J. J., and Vickery, L. E. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 5773-5777). In the studies reported here, the 23-amine derivative, 23-amino-24-nor-5-cholen-3 beta-ol, was found to be an equally potent inhibitor and to be competitive with respect to cholesterol (Ki = 38 nM). Binding of the 23-amine to P-450scc also caused formation of a low spin complex with an absorption maximum at 422 nm, indicative of a nitrogen-donor ligand. Other derivatives in which the side chain amine was linked closer to the steroid, 17 beta-amino-5-androsten-3 beta-ol and (20 R + S)-20-amino-5-pregnen-3 beta-ol, were found to be only very weak inhibitors (I50 greater than 100 microM) and did not produce the 422 nm spectral form when bound. Derivatives in which the amine was attached a greater distance from the steroid ring, 24-amino-5-cholen-3 beta-ol and 25-amino-26,27-bisnor-5-cholesten-3 beta-ol, caused a progressive decrease in inhibitory potency and a failure to produce the 422 nm form on binding. The dependence of the type of interaction of these amino-steroids with P-450scc upon the amine position establishes that the steroid binding site and the heme catalytic site of the enzyme are fixed within a specific distance of one another. The heme appears to be located sufficiently close to the position that the side chain of cholesterol would occupy to allow for direct attack of an iron-bound oxidant to occur during hydroxylation and side chain cleavage.

  19. Orally active 7-substituted (4-benzylphthalazin-1-yl)-2-methylpiperazin-1-yl]nicotinonitriles as active-site inhibitors of sphingosine 1-phosphate lyase for the treatment of multiple sclerosis.

    PubMed

    Weiler, Sven; Braendlin, Nadine; Beerli, Christian; Bergsdorf, Christian; Schubart, Anna; Srinivas, Honnappa; Oberhauser, Berndt; Billich, Andreas

    2014-06-26

    Sphingosine 1-phosphate (S1P) lyase has recently been implicated as a therapeutic target for the treatment of multiple sclerosis (MS), based on studies in a genetic mouse model. Potent active site directed inhibitors of the enzyme are not known so far. Here we describe the discovery of (4-benzylphthalazin-1-yl)-2-methylpiperazin-1-yl]nicotinonitrile 5 in a high-throughput screen using a biochemical assay, and its further optimization. This class of compounds was found to inhibit catalytic activity of S1PL by binding to the active site of the enzyme, as seen in the cocrystal structure of derivative 31 with the homodimeric human S1P lyase. 31 induces profound reduction of peripheral T cell numbers after oral dosage and confers pronounced protection in a rat model of multiple sclerosis. In conclusion, this novel class of direct S1P lyase inhibitors provides excellent tools to further explore the therapeutic potential of T cell-targeted therapies in multiple sclerosis and other autoimmune and inflammatory diseases.

  20. Substitution of arginine for histidine-47 in the coenzyme binding site of yeast alcohol dehydrogenase I

    SciTech Connect

    Gould, R.M.; Plapp, B.V. )

    1990-06-12

    Molecular modeling of alcohol dehydrogenases suggests that His-47 in the yeast enzyme (His-44 in the protein sequence, corresponding to Arg-47 in the horse liver enzyme) binds the pyrophosphate of the NAD coenzyme. His-47 in the Saccharomyces cerevisiae isoenzyme I was substituted with an arginine by a directed mutation. Steady-state kinetic results at pH 7.3 and 30{degree}C of the mutant and wild-type enzymes were consistent with an ordered Bi-Bi mechanism. The substitution decreased dissociation constants by 4-fold for NAD{sup +} and 2-fold for NADH while turnover numbers were decreased by 4-fold for ethanol oxidation and 6-fold for acetaldehyde reduction. The magnitudes of these effects are smaller than those found for the same mutation in the human liver {beta} enzyme, suggesting that other amino acid residues in the active site modulate the effects of the substitution. The pH dependencies of dissociation constants and other kinetic constants were similar in the two yeast enzymes. Thus, it appears that His-47 is not solely responsible for a pK value near 7 that controls activity and coenzyme binding rates in the wild-type enzyme. The small substrate deuterium isotope effect above pH 7 and the single exponential phase of NADH production during the transient oxidation of ethanol by the Arg-47 enzyme suggest that the mutation makes an isomerization of the enzyme-NAD{sup +} complex limiting for turnover with ethanol.

  1. Prediction of contact residue pairs based on co-substitution between sites in protein structures.

    PubMed

    Miyazawa, Sanzo

    2013-01-01

    Residue-residue interactions that fold a protein into a unique three-dimensional structure and make it play a specific function impose structural and functional constraints in varying degrees on each residue site. Selective constraints on residue sites are recorded in amino acid orders in homologous sequences and also in the evolutionary trace of amino acid substitutions. A challenge is to extract direct dependences between residue sites by removing phylogenetic correlations and indirect dependences through other residues within a protein or even through other molecules. Rapid growth of protein families with unknown folds requires an accurate de novo prediction method for protein structure. Recent attempts of disentangling direct from indirect dependences of amino acid types between residue positions in multiple sequence alignments have revealed that inferred residue-residue proximities can be sufficient information to predict a protein fold without the use of known three-dimensional structures. Here, we propose an alternative method of inferring coevolving site pairs from concurrent and compensatory substitutions between sites in each branch of a phylogenetic tree. Substitution probability and physico-chemical changes (volume, charge, hydrogen-bonding capability, and others) accompanied by substitutions at each site in each branch of a phylogenetic tree are estimated with the likelihood of each substitution, and their direct correlations between sites are used to detect concurrent and compensatory substitutions. In order to extract direct dependences between sites, partial correlation coefficients of the characteristic changes along branches between sites, in which linear multiple dependences on feature vectors at other sites are removed, are calculated and used to rank coevolving site pairs. Accuracy of contact prediction based on the present coevolution score is comparable to that achieved by a maximum entropy model of protein sequences for 15 protein families

  2. Pr Substitution at Y and Ba sites in YBCO (123) System

    NASA Astrophysics Data System (ADS)

    Sharma, P. K.; Kumar, Sudhish; Dolia, S. N.; Singhal, R. K.

    2011-07-01

    We present structural, transport, and iodometric study of YBa2Cu3O7-δ system with Pr3+ substituting both the Y3+ and the Ba2+sites. The rate of the Tc depression in the case when with Pr3+ substitutes the Ba2+ is much higher than the case when Pr3+ is substituted at the Y3+ site. This is explained due to a composite effect of the depletion of itinerant holes due to the progressive depletion of the oxygen content, and the loss of orthorhombicity. Further studies on electronic structure and itinerant holes and their relation with superconductivity are underway.

  3. Do Social Activities Substitute for Food in Youth?

    PubMed Central

    Nitecki, Lauren A.; Epstein, Leonard H.

    2010-01-01

    Background Behavioral economics offers a framework to understand choice among alternatives. There is no research on the interrelationship between food and social activity in overweight and non-overweight children. Purpose The purpose of this study is to test the substitutability of food and social interactions using behavioral economic methods in overweight and non-overweight youth. Methods Fifty-four (24 males and 30 females) overweight and non-overweight youth aged 9 to 11 years old were tested using a behavioral choice paradigm which involved participants responding to earn points exchangeable for food and/or social activity. Results Youth substituted food for social activities when the cost of social time with an unfamiliar peer increased (p<0.05) and substituted food for social activities with an unfamiliar peer when the cost of food increased (p<0.05). However, when interacting with a friend was the alternative, participants did not substitute food for social interactions. Conclusions Social interactions can serve as a substitute for food in both lean and overweight youth. PMID:20052567

  4. Synthesis and immunostimulatory activity of substituted TLR7 agonists.

    PubMed

    Akinbobuyi, Babatope; Wang, Lei; Upchurch, Katherine C; Byrd, Matthew R; Chang, Charles A; Quintana, Jeremy M; Petersen, Rachel E; Seifert, Zacharie J; Boquin, José R; Oh, SangKon; Kane, Robert R

    2016-09-01

    Fifteen new substituted adenines were synthesized as potential TLR7 agonists. These compounds, along with 9 previously reported compounds, were analyzed for TLR7 activity and for the selective stimulation of B cell proliferation. Several functionalized derivatives exhibit significant activity, suggesting their potential for use as vaccine adjuvants. PMID:27476423

  5. Synthesis and immunostimulatory activity of substituted TLR7 agonists.

    PubMed

    Akinbobuyi, Babatope; Wang, Lei; Upchurch, Katherine C; Byrd, Matthew R; Chang, Charles A; Quintana, Jeremy M; Petersen, Rachel E; Seifert, Zacharie J; Boquin, José R; Oh, SangKon; Kane, Robert R

    2016-09-01

    Fifteen new substituted adenines were synthesized as potential TLR7 agonists. These compounds, along with 9 previously reported compounds, were analyzed for TLR7 activity and for the selective stimulation of B cell proliferation. Several functionalized derivatives exhibit significant activity, suggesting their potential for use as vaccine adjuvants.

  6. 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.

  7. 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

  8. 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

  9. Syntheses and biological activities of pyranyl-substituted cinnamates.

    PubMed

    Zhu, J; Majikina, M; Tawata, S

    2001-01-01

    Twenty-two kinds of pyranyl-substituted cinnamates were synthesized by the reaction of 4-hydroxy-6-(2-phenylethyl)-2H-pyran-2-one or 4-hydroxy-6-methyl-2H-pyran-2-one (HMP) with a variety of substituted cinnamic acids, and their antifungal and plant growth inhibitory activities were investigated. Among the compounds prepared, 6-methyl-2-oxo-2H-pyran-4-yl 3-(4-isopropylphenyl)propenoate (H5) showed the strongest antifungal activity against Rhizoctonia solani and Sclerotium dellfinii, and 6-methyl-2-oxo-2H-pyran-4-yl 3-(2-methylphenyl)propenoate (H2) had the highest plant growth inhibitory activity toward Brassica rapa.

  10. 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.

  11. Site selectivity in praseodymium- and bismuth-substituted gadolinium gallium garnet epilayers

    SciTech Connect

    Klages, C.P.

    1984-05-01

    Praseodymium- and bismuth-substituted gadolinium gallium garnet (GGG) layers have been prepared by liquid-phase epitaxy on (111)- and (110)-oriented GGG substrates. By an investigation of linear dichroism of Pr/sup 3 +/ -transitions, the supercooling dependence of dodecahedral site selectivity in Pr-substituted GGG can be shown to differ considerably for (110)- and (111)-epitaxial layers. An increase insite selectivity with supercooling is found in (111) layers while it is constant in (110) layers. The long-wavelength /sup 1/S /sub 0/ ..-->.. /sup 3/ P/sub 1/-transition of the B/sub 1/ /sup 3 +/ ion has been utilized to detect site selectivity in (110)oriented Bi-substituted GGG layers.

  12. 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)

  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. Anticancer activity of methyl-substituted oxaliplatin analogs.

    PubMed

    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

    2012-05-01

    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.

  15. 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.

  16. 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.

  17. Substrate specificity and stereoselectivity of horse liver alcohol dehydrogenase. Kinetic evaluation of binding and activation parameters controlling the catalytic cycles of unbranched, acyclic secondary alcohols and ketones as substrates of the native and active-site-specific Co(II)-substituted enzyme.

    PubMed

    Adolph, H W; Maurer, P; Schneider-Bernlöhr, H; Sartorius, C; Zeppezauer, M

    1991-11-01

    1. The steady-state parameters kcat and Km and the rate constants of hydride transfer for the substrates isopropanol/acetone; (S)-2-butanol, (R)-2-butanol/2-butanone; (S)-2-pentanol, (R)-2-pentanol/2-pentanone; 3-pentanol/3-pentanone; (S)-2-octanol and (R)-2-octanol have been determined for the native Zn(II)-containing horse-liver alcohol dehydrogenase (LADH) and the specific active-site-substituted Co(II)LADH. 2. A combined evaluation of steady-state kinetic data and rate constants obtained from stopped-flow measurements, allowed the determination of all rate constants of the following ordered bi-bi mechanism: E in equilibrium E.NAD in equilibrium E.NAD.R1R2 CHOH in equilibrium E.NADH.R1R2CO in equilibrium E.NADH in equilibrium E. 3. On the basis of the different substrate specificities of LADH and yeast alcohol dehydrogenase (YADH), a procedure has been developed to evaluate the enantiomeric product composition of ketone reductions. 2-Butanone and 2-pentanone reductions revealed (S)-2-butanol (86%) and (S)-2-pentanol (95%) as the major products. 4. The observed enantioselectivity implies the existence of two productive ternary complexes; E.NADH.(pro-S) 2-butanone and E.NADH.(pro-R) 2-butanone. All rate constants describing the kinetic pathways of the system (S)-2-butanol, (R)-2-butanol/2-butanone have been determined. These data have been used to estimate the expected enantiomer product composition of 2-butanone reductions using apparent kcat/Km values for the two different ternary-complex configurations of 2-butanone. Additionally, these data have been used for computer simulations of the corresponding reaction cycles. Calculated, simulated and experimental data were found to be in good agreement. Thus, the system (S)-2-butanol, (R)-2-butanol/2-butanone is the first example of a LADH-catalyzed reaction for which the stereochemical course could be described in terms of rate constants of the underlying mechanism. 5. The effects of Co(II) substitution on the

  18. 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

  19. Synthesis and psychotropic activity of functionally substituted diaziridines and bisdiaziridines

    SciTech Connect

    Kostyanovskii, R.G.; Shustov, G.V.; Nabiev, O.G.; Denisenko, S.N.; Sukhanova, S.A.; Lavretskaya, E.F.

    1987-04-01

    The authors examine the psychotropic activity of diaziridines which differ considerable in their structures and the C- and N-substituents. Diaziridines are monoamine oxidase inhibitors in the brain and, thus, are potential antidepressants. The acute toxicities and some pharmacological effects of diaziridines are shown. Mice were used in the experiments. The bisdiaziridines obtained differ in their /sup 1/H and /sup 13/C NMR spectra. The effect is presented of functionally substituted diaziridines on the effects of reserpine, 5-hydroxytryptophan, tryptamine, corazole, and apomorphine hypothermia.

  20. Too packed to change: side-chain packing and site-specific substitution rates in protein evolution.

    PubMed

    Marcos, María Laura; Echave, Julian

    2015-01-01

    In protein evolution, due to functional and biophysical constraints, the rates of amino acid substitution differ from site to site. Among the best predictors of site-specific rates are solvent accessibility and packing density. The packing density measure that best correlates with rates is the weighted contact number (WCN), the sum of inverse square distances between a site's C α and the C α of the other sites. According to a mechanistic stress model proposed recently, rates are determined by packing because mutating packed sites stresses and destabilizes the protein's active conformation. While WCN is a measure of C α packing, mutations replace side chains. Here, we consider whether a site's evolutionary divergence is constrained by main-chain packing or side-chain packing. To address this issue, we extended the stress theory to model side chains explicitly. The theory predicts that rates should depend solely on side-chain contact density. We tested this prediction on a data set of structurally and functionally diverse monomeric enzymes. We compared side-chain contact density with main-chain contact density measures and with relative solvent accessibility (RSA). We found that side-chain contact density is the best predictor of rate variation among sites (it explains 39.2% of the variation). Moreover, the independent contribution of main-chain contact density measures and RSA are negligible. Thus, as predicted by the stress theory, site-specific evolutionary rates are determined by side-chain packing.

  1. Synthesis and herbicidal activity of substituted pyrazole isothiocyanates.

    PubMed

    Wu, Hua; Feng, Jun-Tao; Lin, Kai-Chun; Zhang, Xing

    2012-01-01

    Isothiocyanates and substituted pyrazoles were combined to form a series of novel isothiocyanates with highly effective herbicidal activity. The target compounds were analyzed by elemental analysis, 1H-NMR, EI-MS and IR spectroscopy. The synthesized compounds, particularly compounds 3-1 and 3-7, exhibited good herbicidal activities against four weeds. The EC(50) values of compound 3-1 against Echinochloa crusgalli L., Cyperus iria L., Dactylis glomerata L., and Trifolium repens L. were 64.32, 65.83, 62.42, and 67.72 µg/mL, respectively. The EC(50) values of compound 3-7 against E. crusgalli L., C. iria L., D. glomerata L., T. repens L. were 65.33, 64.90, 59.41 and 67.41 µg/mL, respectively. Compounds 3-1 and 3-7 may be further optimized as lead compounds for new herbicides. PMID:23075815

  2. Lattice site specific diffusion properties for substitutional and interstitial impurity atoms in ZnO crystals

    NASA Astrophysics Data System (ADS)

    Yaqoob, Faisal; Huang, Mengbing

    2016-09-01

    Fundamental understanding of impurity diffusion in crystals remains a challenge due to lack of experimental capabilities for measuring the diffusion properties of atoms according to their substitutional and interstitial lattice locations. With examples of indium and silver in ZnO crystals, we demonstrate an ion beam based method to experimentally determine the energetics and entropy changes in diffusion of substitutional and interstitial impurity atoms. While the interstitial Ag diffuses much faster than the substitutional Ag, as normally expected, the interstitial In migrates slower than the substitutional In, which is attributed to a large negative entropy change (˜-10 kB), possibly caused by the large atomic size of In. The activation energy and the diffusivity pre-exponential factor for the interstitial Ag are significantly enhanced, being more than a factor of two and ˜13 orders of magnitude, respectively, relative to the case for the interstitial In. This implies two different diffusion mechanisms between these two types of interstitial atoms in ZnO crystals: the direct interstitial diffusion mechanism for the interstitial In and the kick-out diffusion mechanism for the interstitial Ag. In addition, the activation energies and the diffusivity prefactors follow the Meyer-Neldel relationship with an excitation energy of ˜92 meV.

  3. 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

  4. Optically Active Hybrid Materials Constructed from Helically Substituted Polyacetylenes.

    PubMed

    Zhang, Huanyu; Zhao, Biao; Deng, Jianping

    2016-04-01

    Functional materials derived from synthetic helical polymers are attracting increasing interest. Helically substituted polyacetylenes (HSPAs) are especially interesting as typical artificial helical polymers. In recent years, we designed and prepared a series of functional materials based on HSPAs and inorganic materials. The target is to establish some novel hybrid materials that combine the superior properties of both. The examined inorganic materials include silica, graphene, and magnetic Fe3 O4 nanoparticles. Such new functional materials hold great promise and are expected to find practical applications, for instance, as chiral absorbents, chiral sensors, chiral selectors for inducing enantioselective crystallization, chiral catalysts towards asymmetric catalysis, and chiral carriers for enantioselective release. The Personal Account summarizes our major achievements in preparing optically active hybrid materials. We hope it will speed up progress in chiral-related research areas.

  5. 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

  6. 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.

  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. 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

  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. 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.

  11. 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

  12. Stable local moments of vacancies, substitutional and hollow site impurities in graphene.

    PubMed

    Mashkoori, M; Jafari, S A

    2015-04-22

    The two-sublattice nature of graphene lattice in conjunction with three-fold rotational symmetry, allows for the p-wave hybridization of the impurity state with the Bloch states of carbon atoms. Such an opportunity is not available in normal metals where the wave function is scalar. The p-wave hybridization function V(→k) appears when dealing with vacancies, substitutional adatoms and the hollow site impurities while the s-wave mixing on graphene lattice pertains only to the top site impurities. In this work, we compare the local moment formation in these two cases and find that the local moments formed by p-wave mixing compared to the s-wave one are robust against the changes in the parameters of the model. Furthermore, we investigate the stability of the local moments in the above cases. We find that the quantum fluctuations can destroy the local moments in the case of s-wave hybridization, while the local moments formed by p-wave hybridization survive the quantum fluctuations. Based on these findings, we propose vacancies, substitutional adatoms, and hollow site adatoms as possible candidates to produce stable local moments in graphene.

  13. Defining critical residues for substrate binding to 1-deoxy-D-xylulose 5-phosphate synthase--active site substitutions stabilize the predecarboxylation intermediate C2α-lactylthiamin diphosphate.

    PubMed

    Brammer Basta, Leighanne A; Patel, Hetalben; Kakalis, Lazaros; Jordan, Frank; Freel Meyers, Caren L

    2014-06-01

    1-Deoxy-D-xylulose 5-phosphate (DXP) synthase catalyzes the formation of DXP from pyruvate and D-glyceraldehyde 3-phosphate (GraP) in a thiamin diphosphate-dependent manner, and is the first step in the essential pathway to isoprenoids in human pathogens. Understanding the mechanism of this unique enzyme is critical for developing new anti-infective agents that selectively target isoprenoid biosynthesis. The present study used mutagenesis and a combination of protein fluorescence, CD and kinetics experiments to investigate the roles of Arg420, Arg478 and Tyr392 in substrate binding and catalysis. The results support a random sequential, preferred order mechanism, and predict that Arg420 and Arg478 are involved in binding of the acceptor substrate, GraP. D-Glyceraldehyde, an alternative acceptor substrate lacking the phosphoryl group predicted to interact with Arg420 and Arg478, also accelerates decarboxylation of the predecarboxylation intermediate C2α-lactylthiamin diphosphate (LThDP) on DXP synthase, indicating that this binding interaction is not absolutely required, and that the hydroxyaldehyde sufficiently triggers decarboxylation. Unexpectedly, Tyr392 contributes to GraP affinity, and is not required for LThDP formation or its GraP-promoted decarboxylation. Time-resolved CD spectroscopy and NMR experiments indicate that LThDP is significantly stabilized on R420A and Y392F variants as compared with wild-type DXP synthase in the absence of acceptor substrate, but these substitutions do not appear to affect the rate of GraP-promoted LThDP decarboxylation in the presence of high levels of GraP, and LThDP formation remains the rate-limiting step. These results suggest a role of these residues in promoting GraP binding, which in turn facilitates decarboxylation, and also highlight interesting differences between DXP synthase and other thiamin diphosphate-dependent enzymes.

  14. On the Question of Site-Selective Ligand Exchange in Carboxylate-Substituted Metal Oxo Clusters

    PubMed Central

    Kreutzer, Johannes; Czakler, Matthias; Puchberger, Michael; Pittenauer, Ernst; Schubert, Ulrich

    2015-01-01

    Reaction of [Ti4Zr4O6(OBu)4(OMc)16] (OMc = methacrylate) with acetylacetone (acacH) resulted in dissection of the cluster and formation of [Ti(OBu)2(acac)2] and the smaller cluster [Ti2Zr4O4(OMc)16]. In contrast, the same reaction with [Zr6O4(OH)4(OOCR)12]2·6RCOOH (R = Et, CH2CH=CH2) led to site-selective substitution of two carboxylate ligands and formation of isostructural [Zr6O4(OH)4(OOCR)12–x(acac)x]2·6RCOOH (x ≤ 1). PMID:26300687

  15. 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.

  16. Local salt substitutes "Obu-otoyo" activate acetylcholinesterase and butyrylcholinesterase and induce lipid peroxidation in rat brain.

    PubMed

    Akinyemi, Ayodele J; Oboh, Ganiyu; Ademiluyi, Adedayo O

    2015-09-01

    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

  17. 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

  18. 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

  19. The interaction of substituted benzamides with brain benzodiazepine binding sites in vitro.

    PubMed Central

    Horton, R. W.; Lowther, S.; Chivers, J.; Jenner, P.; Marsden, C. D.; Testa, B.

    1988-01-01

    1. The interaction of substituted benzamides with brain benzodiazepine (BDZ) binding sites was examined by their ability to displace [3H]-flunitrazepam ([3H]-FNM) from specific binding sites in bovine cortical membranes in vitro. 2. Clebopride, Delagrange 2674, Delagrange 2335 and BRL 20627 displayed concentration-dependent displacement of [3H]-FNM with IC50 values of 73 nM, 132 nM, 7.7 microM and 5.9 microM, respectively. Other substituted benzamides including metoclopramide, sulpiride, tiapride, sultopride and cisapride were inactive at 10(-5) M. 3. Inhibition by clebopride and Delagrange 2674 of [3H]-FNM binding was apparently competitive and readily reversible. 4. In the presence of gamma-aminobutyric acid (GABA), the ability of diazepam and Delagrange 2674 to displace [3H]-Ro 15-1788 binding was increased 3.6 and 1.6 fold respectively, compared to the absence of GABA, while ethyl beta-carboline-3-carboxylate (beta CCE) and clebopride were less potent in the presence of GABA. 5. Diazepam was 30 fold less potent at displacing [3H]-Ro 15-1788 in membranes that had been photoaffinity labelled with FNM than in control membranes, whereas the potency of beta CCE did not differ. Clebopride and Delagrange 2674 showed a less than two fold loss of potency in photoaffinity labelled membranes. 6. The pattern of binding of clebopride and Delagrange 2674 in these in vitro tests is similar to that found previously with partial agonists or antagonists at BDZ binding sites. 7. Clebopride and Delagrange 2674 inhibited [3H]-FNM binding with similar potency in rat cerebellar and hippocampal membranes, suggesting they have no selectivity for BDZ1 and BDZ2 binding sites.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2850059

  20. 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

  1. 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.

  2. Antifungal activity of 4-substituted crotonic acid esters.

    PubMed

    Gershon, H; Shanks, L; Gawiak, D E

    1976-08-01

    Twenty-three 4-substituted crotonic acid esters were tested for antifungal activity against Candida albicans, Aspergillus niger, Mucor mucedo, and Trichophyton mentagrophytes. For the analogues of the methyl ester containing substituents in the 4 position, the following order of fungitoxicity was observed: I greater than Br greater than Cl greater than CH3S greater than CH3O greater than F=H. Of the homologues of the esters of the 4-iodo and 4-bromo compounds which included methyl, ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl, ethyl 4-iodocrotonate was most toxic to the four fungi at pH 7.0 in the presence of 10% beef serum (C. albicans, 18mug/ml, A. niger, 40 mug/ml, M. mucedo, 5 mug/ml, T. mentagrophytes, 4 mug/ml). It is believed that the mechanism of fungitoxicity is due, in part, to a nucleophilic reaction involving SH-containing compounds. This is based on the correlation of fungitoxicity with the order of leaving groups in the nucleophilic reaction and the protection against the toxicity of the test compounds to the fungi by cysteine and glutathione.

  3. 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.

  4. Synthesis and antimicrobial activity of new substituted anilinobenzimidazoles.

    PubMed

    Nofal, Z M; Fahmy, H H; Mohamed, H S

    2002-06-01

    A series of benzimidazole derivatives carrying different heterocycles such as 1,2,3-thiadiazole, 1,3,4-thiadiazole, thiazolidine, 2,3-dihydro-thiazole, 1,3,4-oxadiazole, semicarbazone and substituted thiosemi-carbazones were synthesized. Also a series of 1-methylbenzimidazole carrying hydroxy ethyl-amide, substituted sulfonyl hydrazide and benzoyl hydrazide from aminobenzoyl group at position 2 of 1-methylbenzimidazole were synthesized. The antimicrobial evaluation of some of the new compounds was carried out. PMID:12135093

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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...

  11. 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

  12. Inhibition of tyrosinase by flavonoids, stilbenes and related 4-substituted resorcinols: structure-activity investigations.

    PubMed

    Shimizu, K; Kondo, R; Sakai, K

    2000-02-01

    Several flavonoids, stilbenes and related 4-substituted resorcinols, obtained from Artocarpus incisus and other plants or synthesized, were tested for their inhibitory activity against tyrosinase. The structure-activity relationships suggested that specific natural or synthesized compounds having the 4-substituted resorcinol skeleton have potent tyrosinase inhibitory ability. Kinetic studies have indicated that specific compounds having the 4-substituted resorcinol skeleton exhibit competitive inhibition of the oxidation of DL-beta-(3,4-dihydroxyphenyl)alanine (DL-DOPA) by mushroom tyrosinase. These findings could lead to the design and discovery of new tyrosinase inhibitors.

  13. 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.

  14. Frequent substitution polymorphisms in African green monkey CCR5 cluster at critical sites for infections by simian immunodeficiency virus SIVagm, implying ancient virus-host coevolution.

    PubMed

    Kuhmann, S E; Madani, N; Diop, O M; Platt, E J; Morvan, J; Müller-Trutwin, M C; Barré-Sinoussi, F; Kabat, D

    2001-09-01

    In contrast to humans, several primate species are believed to have harbored simian immunodeficiency viruses (SIVs) since ancient times. In particular, the geographically dispersed species of African green monkeys (AGMs) are all infected with highly diversified SIVagm viruses at high prevalences (greater than 50% of sexually mature individuals) without evident diseases, implying that the progenitor monkeys were infected prior to their dispersal. If this is correct, AGMs would be expected to have accumulated frequent resistance-conferring polymorphisms in host genes that are important for SIV replication. Accordingly, we analyzed the coding sequences of the CCR5 coreceptors from 26 AGMs (52 alleles) in distinct populations of the four species. These samples contained 29 nonsynonymous coding changes and only 15 synonymous nucleotide substitutions, implying intense functional selection. Moreover, 24 of the resulting amino acid substitutions were tightly clustered in the CCR5 amino terminus (D13N in the vervets and Y14N in the tantalus species) or in the first extracellular loop (Q93R and Q93K in all species). The Y14N substitution was extremely frequent in the 12 wild-born African tantalus, with 7 monkeys being homozygous for this substitution and 4 being heterozygous. Although two of these heterozygotes and the only wild-type homozygote were naturally infected with SIVagm, none of the Y14N homozygotes were naturally infected. A focal infectivity assay for SIVagm indicated that all five tested SIVagms efficiently use CCR5 as a coreceptor and that they also use CXCR6 (STRL33/Bonzo) and GPR15 (BOB) with lower efficiencies but not CXCR4. Interestingly, the D13N, Y14N, Q93R, and Q93K substitutions in AGM CCR5 all strongly inhibited infections by the SIVagm isolates in vitro. The Y14N substitution eliminates a tyrosine sulfation site that is important for infections and results in partial N-linked glycosylation (i.e., 60% efficiency) at this position. Nevertheless, the CCR

  15. Effect of amino acid substitutions in a potential metal-binding site of AnfA on expression from the anfH promoter in Azotobacter vinelandii.

    PubMed

    Premakumar, R; Loveless, T M; Bishop, P E

    1994-10-01

    AnfA, an activator required for transcription of the structural genes encoding nitrogenase 3 (anfHDGK) in Azotobacter vinelandii, has a potential metal-binding site [(S19)H(C21)FTGE(C26)R] in its N terminus. Growth studies and expression of an anfH-lacZ fusion in mutants containing amino acid substitutions in this site indicate that Ser-19 is not required for AnfA activity whereas Cys-21 and Cys-26 are required. Residual expression of the anfH-lacZ fusion in AnfA- mutants was found to be due to activation by VnfA, the activator required for expression of genes encoding nitrogenase 2.

  16. 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.

  17. 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

  18. Dilution of two-dimensional antiferromagnetism by Mn site substitution in La1Sr2Mn2-xAlxO7

    NASA Astrophysics Data System (ADS)

    Nair, Sunil; Banerjee, A.

    2004-09-01

    We report the effect of Al substitution on the Mn site of the bilayered half doped manganite La1Sr2Mn2O7 . This substitution dilutes the magnetically active Mn-O-Mn network without introducing an appreciable distortion in the lattice, and ionic considerations lead to a predominant reduction of Mn4+ with increasing Al. The rate of fall of the long-range antiferromagnetic transition temperature as a function of substitution is seen to match well with established quasi-two-dimensional (quasi-2D) Heisenberg systems indicating that the nature of magnetic interactions in this quasi-2D system is of the short-range Heisenberg type. The magnetic contribution of the specific heat estimated using Fisher’s relation could be fitted with a function incorporating the presence of a gapped Fermi surface appropriate for this type of system. The resistivity is seen to increase as a function of substitution due to the weakening of the double exchange within the ferromagnetic layers of this A -type of antiferromagnets and justifiably, in the paramagnetic region the data could be fitted to Mott’s equation for the variable range hopping of polarons in two dimensions.

  19. Radioprotective Activity and Synthesis of Siladithioacetals and Germadithioacetals Derived from N-substituted Naphthylethylimidazoline

    PubMed Central

    Célariès, Benoît; Court, Louis; Lion, Claude; Laval, Jean-Denis

    2001-01-01

    A number of organosilicon and organogermanium derivatives and some related compounds including the N-substituted. 2-[1-naphthylmethyl]-2-imidazoline and 2-[1-(1-naphthyl)ethyl]-2-imidazoline have been prepared and the toxicity of some compounds have been determined in mice. In this paper we report the synthesis and the evaluation of the pharmacological activity of new organosilicon and organogermanium, compounds such as sila- and germadithioacetals derived from N-substituted naphthylimidazolne. PMID:18475997

  20. Antihyperlipidemic morpholine derivatives with antioxidant activity: An investigation of the aromatic substitution.

    PubMed

    Ladopoulou, Eleni M; Matralis, Alexios N; Nikitakis, Anastasios; Kourounakis, Angeliki P

    2015-11-01

    Drugs affecting more than one target could result in a more efficient treatment of multifactorial diseases as well as fewer safety concerns, compared to a one-drug one-target approach. Within our continued efforts towards the design of multifunctional molecules against atherosclerosis, we hereby report the synthesis of 17 new morpholine derivatives which structurally vary in terms of the aromatic substitution on the morpholine ring. These derivatives simultaneously suppress cholesterol biosynthesis through SQS inhibition (IC50 values of the most active compounds are between 0.7 and 5.5 μM) while exhibiting a significant protection of hepatic microsomal membranes against lipid peroxidation (with IC50 values for the most active compounds being between 73 and 200 μM). Further evaluation of these compounds was accomplished in vivo in an animal model of acute experimental hyperlipidemia, where it was observed that compounds reduced the examined lipidemic parameters (TC, TG and LDL) by 15-80%. In order to examine the mode of binding of these molecules in the active catalytic site of SQS, we also performed docking simulation studies. Our results indicate that some of the new compounds can be considered interesting structures in the search for new multifunctional agents of potential application in atherosclerosis.

  1. Restoring enzyme activity in nonfunctional low erucic acid Brassica napus fatty acid elongase 1 by a single amino acid substitution.

    PubMed

    Katavic, Vesna; Mietkiewska, Elzbieta; Barton, Dennis L; Giblin, E Michael; Reed, Darwin W; Taylor, David C

    2002-11-01

    Genomic fatty acid elongation 1 (FAE1) clones from high erucic acid (HEA) Brassica napus, Brassica rapa and Brassica oleracea, and low erucic acid (LEA) B. napus cv. Westar, were amplified by PCR and expressed in yeast cells under the control of the strong galactose-inducible promoter. As expected, yeast cells expressing the FAE1 genes from HEA Brassica spp. synthesized very long chain monounsaturated fatty acids that are not normally found in yeast, while fatty acid profiles of yeast cells expressing the FAE1 gene from LEA B. napus were identical to control yeast samples. In agreement with published findings regarding different HEA and LEA B. napus cultivars, comparison of FAE1 protein sequences from HEA and LEA Brassicaceae revealed one crucial amino acid difference: the serine residue at position 282 of the HEA FAE1 sequences is substituted by phenylalanine in LEA B. napus cv. Westar. Using site directed mutagenesis, the phenylalanine 282 residue was substituted with a serine residue in the FAE1 polypeptide from B. napus cv. Westar, the mutated gene was expressed in yeast and GC analysis revealed the presence of very long chain monounsaturated fatty acids (VLCMFAs), indicating that the elongase activity was restored in the LEA FAE1 enzyme by the single amino acid substitution. Thus, for the first time, the low erucic acid trait in canola B. napus can be attributed to a single amino acid substitution which prevents the biosynthesis of the eicosenoic and erucic acids.

  2. 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

  3. 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

  4. 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

  5. Synthesis and antifungal activities of 3-alkyl substituted thieno[2,3-d]pyrimidinones

    NASA Astrophysics Data System (ADS)

    Wang, H. M.; Deng, S. H.; Zheng, A. H.; Zhang, Q. Y.; Chen, X. B.; Zeng, X. H.; Hu, Y. G.

    2016-08-01

    The 3-aryl substituted thieno[2,3-d]pyrimidinones 3 by sequential reaction of iminophosphorane 1, aromatic isocyanates and various nucleophiles (HY), found some compounds showed good antitumor and antibacterial activities. Meanwhile, aliphatic isocyanates were applied in the reaction to prepare 3-alkyl substituted thieno[2,3- d]pyrimidinones, but there are no reports of their antifungal activities. As a continuation of our research for new biologically active heterocycles, we herein wish to report a facile synthesis and antifungal activities of 3-alkyl substituted thieno[2,3-d]pyrimidinones 6 via easily accessible iminophosphorane 1. The growth inhibitory effect of one concentration (50mg/L) of compounds 6 against five fungus(Fusarium oxysporium, Rhizoctonia solani, Colletotrichum gossypii, Gibberella zeae and Dothiorella gregaria) in vitro was tested by the method of toxic medium. Compound 6d showed the best inhibition rate against Gibberella zeae with 85.68%.

  6. 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

  7. 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.

  8. Codon-substitution models to detect adaptive evolution that account for heterogeneous selective pressures among site classes.

    PubMed

    Yang, Ziheng; Swanson, Willie J

    2002-01-01

    The nonsynonymous to synonymous substitution rate ratio (omega = d(N)/d(S)) provides a sensitive measure of selective pressure at the protein level, with omega values <1, =1, and >1 indicating purifying selection, neutral evolution, and diversifying selection, respectively. Maximum likelihood models of codon substitution developed recently account for variable selective pressures among amino acid sites by employing a statistical distribution for the omega ratio among sites. Those models, called random-sites models, are suitable when we do not know a priori which sites are under what kind of selective pressure. Sometimes prior information (such as the tertiary structure of the protein) might be available to partition sites in the protein into different classes, which are expected to be under different selective pressures. It is then sensible to use such information in the model. In this paper, we implement maximum likelihood models for prepartitioned data sets, which account for the heterogeneity among site partitions by using different omega parameters for the partitions. The models, referred to as fixed-sites models, are also useful for combined analysis of multiple genes from the same set of species. We apply the models to data sets of the major histocompatibility complex (MHC) class I alleles from human populations and of the abalone sperm lysin genes. Structural information is used to partition sites in MHC into two classes: those in the antigen recognition site (ARS) and those outside. Positive selection is detected in the ARS by the fixed-sites models. Similarly, sites in lysin are classified into the buried and solvent-exposed classes according to the tertiary structure, and positive selection was detected at the solvent-exposed sites. The random-sites models identified a number of sites under positive selection in each data set, confirming and elaborating the results of the fixed-sites models. The analysis demonstrates the utility of the fixed-sites models

  9. An in vitro study into the effect of zinc substituted hydroxyapatite on osteoclast number and activity.

    PubMed

    Shepherd, David V; Kauppinen, Kyösti; Brooks, Roger A; Best, Serena M

    2014-11-01

    Zinc ions have been shown to inhibit osteoclast development and proliferation both in vitro and in vivo. The same inhibiting effect has been observed in vitro when zinc was substituted into tri-calcium phosphate (TCP). Because of the solubility of TCP it is not an ideal candidate for a material to inhibit osteoclast activity in the long term. Hydroxyapatite (HA) is less soluble and so potentially offers a more long-term, sustainable effect. Previous work has shown that zinc can successfully be substituted into HA and still retain phase purity after heat treatment. The study reported here presents the effects of zinc substituted HA on the development and activity of osteoclast-like cells. It was found that increasing zinc substitution levels led to a decrease in the number of these cells present after 21 days. When resorption activity was investigated it was found that an increase in the amount of zinc present in the discs led to a significant decrease in the amount of resorption taking place on the discs. These results provide evidence for the potential of zinc substituted HA as a material to reduce resorptive activity to provide long-term bonding of implant to bone.

  10. Site-selective substitutional doping with atomic precision on stepped Al (111) surface by single-atom manipulation.

    PubMed

    Chen, Chang; Zhang, Jinhu; Dong, Guofeng; Shao, Hezhu; Ning, Bo-Yuan; Zhao, Li; Ning, Xi-Jing; Zhuang, Jun

    2014-01-01

    In fabrication of nano- and quantum devices, it is sometimes critical to position individual dopants at certain sites precisely to obtain the specific or enhanced functionalities. With first-principles simulations, we propose a method for substitutional doping of individual atom at a certain position on a stepped metal surface by single-atom manipulation. A selected atom at the step of Al (111) surface could be extracted vertically with an Al trimer-apex tip, and then the dopant atom will be positioned to this site. The details of the entire process including potential energy curves are given, which suggests the reliability of the proposed single-atom doping method.

  11. 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

  12. Catalysis: Elusive active site in focus

    NASA Astrophysics Data System (ADS)

    Labinger, Jay A.

    2016-08-01

    The identification of the active site of an iron-containing catalyst raises hopes of designing practically useful catalysts for the room-temperature conversion of methane to methanol, a potential fuel for vehicles. See Letter p.317

  13. New Substituted Isocoumarins and Dihydroisocoumarins and their Cytotoxic Activities

    PubMed Central

    Hampl, Veronika; Wetzel, Isolde; Bracher, Franz; Krauss, Jürgen

    2011-01-01

    New isocoumarins were prepared in an efficient way from 2-iodobenzoic acid derivatives and hept-1-yne in a Sonogashira reaction, followed by spontaneous cyclization. Catalytic hydrogenation gave the corresponding dihydroisocoumarins. A 4-chloroisocoumarin was prepared on an alternative pathway. Some of the new compounds showed moderate cytotoxic activities against a human leukemia cell line (HL 60). PMID:21617770

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

    NASA Astrophysics Data System (ADS)

    Kohri, Hitoshi; Yagasaki, Takayoshi

    2016-10-01

    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.

  15. 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

  16. 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).

  17. Structure-activity relationships of substituted oxyoxalamides as inhibitors of the human soluble epoxide hydrolase.

    PubMed

    Kim, In-Hae; Lee, In-Hee; Nishiwaki, Hisashi; Hammock, Bruce D; Nishi, Kosuke

    2014-02-01

    We explored both structure-activity relationships among substituted oxyoxalamides used as the primary pharmacophore of inhibitors of the human sEH and as a secondary pharmacophore to improve water solubility of inhibitors. When the oxyoxalamide function was modified with a variety of alkyls or substituted alkyls, compound 6 with a 2-adamantyl group and a benzyl group was found to be a potent sEH inhibitor, suggesting that the substituted oxyoxalamide function is a promising primary pharmacophore for the human sEH, and compound 6 can be a novel lead structure for the development of further improved oxyoxalamide or other related derivatives. In addition, introduction of substituted oxyoxalamide to inhibitors with an amide or urea primary pharmacophore produced significant improvements in inhibition potency and water solubility. In particular, the N,N,O-trimethyloxyoxalamide group in amide or urea inhibitors (26 and 31) was most effective among those tested for both inhibition and solubility. The results indicate that substituted oxyoxalamide function incorporated into amide or urea inhibitors is a useful secondary pharmacophore, and the resulting structures will be an important basis for the development of bioavailable sEH inhibitors.

  18. 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

  19. 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

  20. 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

  1. 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

  2. Exploration of structure-function relationships in Escherichia coli cystathionine γ-synthase and cystathionine β-lyase via chimeric constructs and site-specific substitutions.

    PubMed

    Manders, Adrienne L; Jaworski, Allison F; Ahmed, Mohammed; Aitken, Susan M

    2013-06-01

    Cystathionine γ-synthase (CGS) and cystathionine β-lyase (CBL) share a common structure and several active-site residues, but catalyze distinct side-chain rearrangements in the two-step transsulfuration pathway that converts cysteine to homocysteine, the precursor of methionine. A series of 12 chimeric variants of Escherichia coli CGS (eCGS) and CBL (eCBL) was constructed to probe the roles of two structurally distinct, ~25-residue segments situated in proximity to the amino and carboxy termini and located at the entrance of the active-site. In vivo complementation of methionine-auxotrophic E. coli strains, lacking the genes encoding eCGS and eCBL, demonstrated that exchange of the targeted regions impairs the activity of the resulting enzymes, but does not produce a corresponding interchange of reaction specificity. In keeping with the in vivo results, the catalytic efficiency of the native reactions is reduced by at least 95-fold, and α,β versus α,γ-elimination specificity is not modified. The midpoint of thermal denaturation monitored by circular dichroism, ranges between 59 and 80°C, compared to 66°C for the two wild-type enzymes, indicating that the chimeric enzymes adopt a stable folded structure and that the observed reductions in catalytic efficiency are due to reorganization of the active site. Alanine-substitution variants of residues S32 and S33, as well as K42 of eCBL, situated in proximity to and within, respectively, the targeted amino-terminal region were also investigated to explore their role as determinants of reaction specificity via positioning of key active-site residues. The catalytic efficiency of the S32A, S33A and the K42A site-directed variants of eCBL is reduced by less than 10-fold, demonstrating that, while these residues may participate in positioning S339, which tethers the catalytic base, their role is minor.

  3. 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

  4. 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)].

  5. Histidine at the active site of Neurospora tyrosinase.

    PubMed

    Pfiffner, E; Lerch, K

    1981-10-13

    The involvement of histidyl residues as potential ligands to the binuclear active-site copper of Neurospora tyrosinase was explored by dye-sensitized photooxidation. The enzymatic activity of the holoenzyme was shown to be unaffected by exposure to light in the presence of methylene blue; however, irradiation of the apoenzyme under the same conditions led to a progressive loss of its ability to be reactivated with Cu2+. This photoinactivation was paralleled by a decrease in the histidine content whereas the number of histidyl residues in the holoenzyme remained constant. Copper measurements of photooxidized, reconstituted apoenzyme demonstrated the loss of binding of one copper atom per mole of enzyme as a consequence of photosensitized oxidation of three out of nine histidine residues. Their sequence positions were determined by a comparison of the relative yields of the histidine containing peptides of photooxidized holo- and apotyrosinases. The data obtained show the preferential modification of histidyl residues 188, 193, and 289 and suggest that they constitute metal ligands to one of the two active-site copper atoms. Substitution of copper by cobalt was found to afford complete protection of the histidyl residues from being modified by dye-sensitized photooxidation. PMID:6458322

  6. Effect of Sr-substitution on the restitution of superconductivity in Pr-substituted at rare earth and Ba-site in EuBa 2Cu 3O z

    NASA Astrophysics Data System (ADS)

    Thampi, R. S.; Rayaprol, S.; Mavani, Krushna; Kuberkar, D. G.; Gonal, M. R.; Prasad, R.; Kulkarni, R. G.

    2001-06-01

    We report the effect of Sr-substitution in restoring the superconductivity of Pr-doped (Eu 1- xPr x)Ba 2Cu 3O z [A] and Eu(Ba 2- xPr x)Cu 3O z [B] samples. The structural and superconducting properties of these A and B samples have been investigated using X-ray diffraction, a.c. susceptibility, electrical resistivity, d.c. magnetization and iodometric measurements. It is observed that the superconductivity gets suppressed at the rate of 1.6 K per at.% of Pr substitution at Eu-site due to the localization of mobile holes, while the sample with 15% Pr at Ba-site [B(0.3)] becomes non-superconducting mainly due to the hole filling and localization of holes. However, the increasing substitution of Sr at Ba-site in both A(0.4) and B(0.3) samples upto 25% resulted in the restoration of superconductivity (T c∼33 K) due to the delocalization of holes. Interestingly, the increasing substitution of Sr at Eu-site in the non-superconducting B(0.4) sample upto 30%, increases Tc from 0 to 52 K mainly due to the hole doping mechanism which is much faster and larger restoration of superconductivity than the Ba-site doping of Sr in the same sample.

  7. 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

  8. 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

  9. Co-substitution at the Mn-site in YMnO{sub 3}: Structural stability and physical properties

    SciTech Connect

    Malo, S.; Maignan, A.

    2012-04-15

    Highlights: Black-Right-Pointing-Pointer Solid state synthesis of several series of hexagonal co-substituted YMn{sub 1-x}(M Prime ,M Double-Prime ){sub x}O{sub 3}. Black-Right-Pointing-Pointer Evidencing the critical parameters which govern the stabilization of the hexagonal phase. Black-Right-Pointing-Pointer Inducing a decrease of the antiferromagnetic ordering temperature by co-substitutions. -- Abstract: The YMnO{sub 3} oxide, crystallizing in the P6{sub 3}cm space group is one of the most studied multiferroic material. While these properties can be modified by substitutions at the Mn-site, the peculiar fivefold coordination appears to limit the M solubility range in YMn{sub 1-x}M{sub x}O{sub 3}. This motivated the preparation by solid state reaction of several series of co-substituted YMn{sub 1-x}(M Prime ,M Double-Prime ){sub x}O{sub 3}, which were studied using both powder X-ray diffraction and transmission electron microscopy. The cation pairs have been chosen for their ability to adopt a fivefold coordination similar to that of Mn{sup 3+} in YMnO{sub 3}, to keep a constant average trivalent oxidation state and to maintain an average cationic radius similar to that of Mn{sup 3+}. For YMn{sub 1-x}(Cu{sub 1/2}Ti{sub 1/2}){sub x}O{sub 3}, a complete solid solution is observed without cation ordering. For all other studied cation pairs, the solubility range is always reaching a minimum value of 25% for the Zn containing pairs (Zn{sub 2/3}V{sub 1/3} and Zn{sub 3/4}Mo{sub 1/4}), a maximum of 50% being reached for the Cu containing pairs (Cu{sub 2/3}V{sub 1/3}, Cu{sub 2/3}Nb{sub 1/3} and Cu{sub 3/4}W{sub 1/4}). Interestingly, the non-centrosymmetric P6{sub 3}cm space group is retained within the solubility range without generating cation ordering phenomena. These co-substitutions at the Mn{sup 3+} are found to induce a decrease of the antiferromagnetic ordering temperature, the T{sub N} variations being similar to the temperature decrease of the dielectric anomaly

  10. 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.

  11. Synthesis and anticancer activity of some novel 2-substituted benzimidazole derivatives.

    PubMed

    Refaat, Hanan M

    2010-07-01

    In an effort to establish new candidates with improved anticancer activity, we report here the synthesis of various series of 2-substituted benzimidazoles: 2-[(4-oxothiazolidin-2-ylidene) methyl and (4-amino-2-thioxothiazol-5-yl) benzimidazoles (2 and 3, respectively); 2-[(4-fluorobenzylidene and cycloalkylidene) cyanomethyl] benzimidazoles (4 and 5, respectively), together with the synthesis of certain of 2-[(4- or 5-oxothiazolidin-2-ylidene, 4-substituted thiazolyl-2-ylidene and [1,3]thiazin-2-ylidene)cyanomethyl]benzimidazoles (6, 8, 7 and 9, respectively). Several of the synthesized products were subjected to in vitro anticancer screening that revealed that all the tested compounds exhibited antitumor activity against human hepatocellular carcinoma (HEPG2), human breast adenocarcinoma (MCF7) and human colon carcinoma (HCT 116) cell lines, with IC50's<10 microg/ml. PMID:20399544

  12. 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.

  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. Synthesis and hypoglycemic activity of 9-O-(lipophilic group substituted) berberine derivatives.

    PubMed

    Zhang, Shanshan; Wang, Xiaohong; Yin, Weicheng; Liu, Zhenbao; Zhou, Mi; Xiao, Daipeng; Liu, Yanfei; Peng, Dongming

    2016-10-01

    A series of 9-O-(lipophilic group substituted) berberine derivatives were synthesized and evaluated for their cytotoxicity and hypoglycemic activity against HepG2 cells. All the results indicated that most of the synthesized compounds exhibited lower cytotoxicity and a certain degree of hypoglycemic activity. Especially the compounds 5g and 5h displayed dramatically increased hypoglycemic activity compared with berberine, and the cytotoxicity maintained or even lower than berberine, indicating that they are potential candidates for new anti-type 2 diabetes mellitus drugs. PMID:27561717

  15. 19-Substituted Benzoquinone Ansamycin Heat Shock Protein-90 Inhibitors: Biological Activity and Decreased Off-Target Toxicity

    PubMed Central

    Chang, Chuan-Hsin; Drechsel, Derek A.; Kitson, Russell R. A.; Siegel, David; You, Qiang; Backos, Donald S.; Ju, Cynthia; Moody, Christopher J.

    2014-01-01

    The benzoquinone ansamycins (BQAs) are a valuable class of antitumor agents that serve as inhibitors of heat shock protein (Hsp)-90. However, clinical use of BQAs has resulted in off-target toxicities, including concerns of hepatotoxicity. Mechanisms underlying the toxicity of quinones include their ability to redox cycle and/or arylate cellular nucleophiles. We have therefore designed 19-substituted BQAs to prevent glutathione conjugation and nonspecific interactions with protein thiols to minimize off-target effects and reduce hepatotoxicity. 19-Phenyl– and 19-methyl–substituted versions of geldanamycin and its derivatives, 17-allylamino-17-demethoxygeldanamycin and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), did not react with glutathione, whereas marked reactivity was observed using parent BQAs. Importantly, although 17-DMAG induced cell death in primary and cultured mouse hepatocytes, 19-phenyl and 19-methyl DMAG showed reduced toxicity, validating the overall approach. Furthermore, our data suggest that arylation reactions, rather than redox cycling, are a major mechanism contributing to BQA hepatotoxicity. 19-Phenyl BQAs inhibited purified Hsp90 in a NAD(P)H:quinone oxidoreductase 1 (NQO1)–dependent manner, demonstrating increased efficacy of the hydroquinone ansamycin relative to its parent quinone. Molecular modeling supported increased stability of the hydroquinone form of 19-phenyl-DMAG in the active site of human Hsp90. In human breast cancer cells, 19-phenyl BQAs induced growth inhibition also dependent upon metabolism via NQO1 with decreased expression of client proteins and compensatory induction of Hsp70. These data demonstrate that 19-substituted BQAs are unreactive with thiols, display reduced hepatotoxicity, and retain Hsp90 and growth-inhibitory activity in human breast cancer cells, although with diminished potency relative to parent BQAs. PMID:24682466

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. Substantial increment in critical parameters of MgB2 superconductor by boron site nano-carbon substitution

    NASA Astrophysics Data System (ADS)

    Mudgel, Monika; Awana, V. P. S.; Bhalla, G. L.; Kishan, H.

    2010-12-01

    This paper deals with the determination of critical properties of MgB2 along with the impact of carbon substitution on critical parameters. The change in lattice parameters and decrease of transition temperature, Tc confirms the successful substitution by carbon at boron site. The magneto transport measurements up to 140 kOe are carried out to determine upper critical field (Hc2). The upper critical field values, Hc2 are obtained from ρ-T(H) data based upon the criterion of 90% of normal resistivity. The Ginzburg Landau theory (GL equation) is applied to the ρ-T(H) data which accounts for the temperature dependence behavior of Hc2 in the low temperature high field region along with the determination of Hc(0) value. The Hc(0) value of about 300 kOe is obtained for the carbon doped sample while the same is just near to 160 kOe for the pure MgB2 sample.

  1. Site selective substitution Pt for Ti in KTiOPO4:Ga crystals revealed by electron paramagnetic resonance

    NASA Astrophysics Data System (ADS)

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

    2014-07-01

    Electron Paramagnetic Resonance at low temperatures has been used to characterize potassium titanyl phosphate (KTiOPO4) 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 KTiOPO4 creating Pt3+ centers. Two different Pt3+ 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 39K and two 31P nuclei in their surroundings. In both Pt(A) and Pt(D) centers, Pt3+ ions substitute for Ti4+ ions, but with a preference to one of two electrically distinct crystallographic positions. The site selective substitution can be controlled by the Ga-doping.

  2. Active Sites Environmental Monitoring Program: Action levels

    SciTech Connect

    Ashwood, J.S.; Ashwood, T.L.

    1991-10-01

    The Active Sites Environmental Monitoring Program (ASEMP) was established at Oak Ridge National Laboratory to provide for early leak detection and to monitor performance of the active low-level waste disposal facilities in Solid Waste Storage Area (SWSA) 6 and the transuranic waste storage areas in SWSA 5 North. Early leak detection is accomplished by sampling runoff, groundwater, and perched water in burial trenches. Sample results are compared to action levels that represent background contamination by naturally occurring and fallout-derived radionuclides. 15 refs., 3 figs., 12 tabs.

  3. Synthesis and κ-Opioid Receptor Activity of Furan-Substituted Salvinorin A Analogues

    PubMed Central

    2015-01-01

    The neoclerodane diterpene salvinorin A, found in the leaves of Salvia divinorum, is a potent κ-opioid receptor agonist, making it an attractive scaffold for development into a treatment for substance abuse. Although several successful semisynthetic studies have been performed to elucidate structure–activity relationships, the lack of analogues with substitutions to the furan ring of salvinorin A has prevented a thorough understanding of its role in binding to the κ-opioid receptor. Herein we report the synthesis of several salvinorin A derivatives with modified furan rings. Evaluation of these compounds in a functional assay indicated that sterically less demanding substitutions are preferred, suggesting the furan ring is bound in a congested portion of the binding pocket. The most potent of the analogues successfully reduced drug-seeking behavior in an animal model of drug-relapse without producing the sedation observed with other κ-opioid agonists. PMID:25426797

  4. 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

  5. A tactile vision substitution system for the study of active sensing.

    PubMed

    Hsu, Brian; Hsieh, Cheng-Han; Yu, Sung-Nien; Ahissar, Ehud; Arieli, Amos; Zilbershtain-Kra, Yael

    2013-01-01

    This paper presents a tactile vision substitution system (TVSS) for the study of active sensing. Two algorithms, namely image processing and trajectory tracking, were developed to enhance the capability of conventional TVSS. Image processing techniques were applied to reduce the artifacts and extract important features from the active camera and effectively converted the information into tactile stimuli with much lower resolution. A fixed camera was used to record the movement of the active camera. A trajectory tracking algorithm was developed to analyze the active sensing strategy of the TVSS users to explore the environment. The image processing subsystem showed advantageous improvement in extracting object's features for superior recognition. The trajectory tracking subsystem, on the other hand, enabled accurately locating the portion of the scene pointed by the active camera and providing profound information for the study of active sensing strategy applied by TVSS users.

  6. 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.

  7. 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)].

  8. 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

  9. 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

  10. Characterization of active sites in zeolite catalysts

    SciTech Connect

    Eckert, J.; Bug, A.; Nicol, J.M.

    1997-11-01

    This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). Atomic-level details of the interaction of adsorbed molecules with active sites in catalysts are urgently needed to facilitate development of more effective and/or environmentally benign catalysts. To this end the authors have carried out neutron scattering studies combined with theoretical calculations of the dynamics of small molecules inside the cavities of zeolite catalysts. The authors have developed the use of H{sub 2} as a probe of adsorption sites by observing the hindered rotations of the adsorbed H{sub 2} molecule, and they were able to show that an area near the four-rings is the most likely adsorption site for H{sub 2} in zeolite A while adsorption of H{sub 2} near cations located on six-ring sites decreases in strength as Ni {approximately} Co > Ca > Zn {approximately} Na. Vibrational and rotational motions of ethylene and cyclopropane adsorption complexes were used as a measure for zeolite-adsorbate interactions. Preliminary studies of the binding of water, ammonia, and methylamines were carried out in a number of related guest-host materials.

  11. 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

  12. 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

  13. 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

  14. 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...

  15. Synthesis and fungicidal activities of novel indene-substituted oxime ether strobilurins.

    PubMed

    Tu, Song; Xu, Long-He; Ye, Li-Yi; Wang, Xi; Sha, Yong; Xiao, Zong-Yuan

    2008-07-01

    Nineteen novel indene-substituted oxime ether strobilurins, which used an indene group to stabilize the ( E)-styryl group in SYP-Z071 (an unsaturated oxime strobilurin fungicide under development by the Shenyang Research Institute of Chemical Industry), were designed and synthesized. The biological assay results showed that all compounds possessed good or excellent fungicidal activities. It was found that most of the compounds showed higher fungicidal activities against Pyricularia oryzae, Phytophthora infestans, Erysiphe graminis, and Colletotrichum lagenarium than SYP-Z071 at the tested concentration. The biological assay results also indicated that most of the compounds exhibited higher in vivo fungicidal activities against cucumber Pseudoperonospora cubensis and C. lagenarium than the commercial fungicides trifloxystrobin and kresoxim-methyl at a concentration of 6.25 mg/L. Furthermore, it was found that alpha-(methoxyimino)- N-methylphenylacetamide oxime ethers 6m- s exhibited a broad spectrum and remarkably higher activities against all tested fungi. Especially, the 6-methylindene-substituted compound 6p was identified as the most promising candidate for further study. PMID:18547049

  16. 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

  17. Extraction site preservation using an in-situ hardening alloplastic bone graft substitute.

    PubMed

    Leventis, Minas D; Fairbairn, Peter; Horowitz, Robert A

    2014-01-01

    This case report highlights the use of an in-situ hardening alloplastic bone grafting material composed of beta-tricalcium phosphate (β-TCP) granules coated with poly(lactic-co-glycolic acid) (PLGA) to preserve the dimensions and architecture of the alveolar ridge after atraumatic extraction. This material provided a stable scaffold that, although left uncovered, deterred the ingrowth of unwanted soft tissue, allowing newly formed keratinized soft tissue to proliferate over the healing grafted socket and gradually cover the site. At re-entry after 4 months adequate newly formed bone was observed, allowing for the correct positional placement of an implant. The results of this case suggest that an in-situ hardening alloplastic grafting material can be successfully utilized with minimally invasive procedures to preserve the bone and the soft-tissue profile of the alveolar ridge for future implant rehabilitation.

  18. 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

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

    PubMed

    Kelly, Paul P; Eichler, Anja; Herter, Susanne; Kranz, David C; Turner, Nicholas J; Flitsch, Sabine L

    2015-01-01

    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.

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

    PubMed

    Kelly, Paul P; Eichler, Anja; Herter, Susanne; Kranz, David C; Turner, Nicholas J; Flitsch, Sabine L

    2015-01-01

    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

  1. An intermediate in the phage lambda site-specific recombination reaction is revealed by phosphorothioate substitution in DNA.

    PubMed Central

    Kitts, P A; Nash, H A

    1988-01-01

    It has been proposed that phage lambda site-specific recombination proceeds via two independent strand exchanges: the first exchange forming a Holliday-structure which is then converted into complete recombinant products by the second strand exchange. If this hypothesis is correct, one should be able to trap the putative Holliday intermediate by preventing the second strand exchange. In this paper, we show that substitution of phosphorothioate for phosphate in one strand of a recombination site is an effective way to block recombination while permitting the accumulation of a novel structure. This effect is seen only when phosphorothioate is positioned at a point of potential cleavage by Int recombinase, demonstrating that the inhibition of strand exchange is highly specific. Analysis of the novel structure that accumulates in these reactions proves that it contains a Holliday joint. Holliday-structures can also be detected in unblocked recombinations but are present at very low levels. The characteristics of Holliday-structure formation that we describe substantiate the proposed recombination pathway. Images PMID:2970060

  2. 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

  3. 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.

  4. Molecular docking and QSAR analyses for understanding the antimalarial activity of some 7-substituted-4-aminoquinoline derivatives.

    PubMed

    Shibi, I G; Aswathy, L; Jisha, R S; Masand, V H; Divyachandran, A; Gajbhiye, J M

    2015-09-18

    The quinoline moiety is one of the widely studied scaffolds for generating derivatives with various pharmacophoric groups due to its potential antimalarial activities. In the present study, a series of 7-substituted-4-aminoquinoline derivatives were selected to understand their antimalarial properties computationally by molecular modeling techniques including 2D QSAR, comparative molecular field analysis (CoMFA), comparative molecular similarity indices analysis (CoMSIA) and molecular docking. The 2D-QSAR model built with four descriptors selected by genetic algorithm technique and CoMFA model showed satisfactory statistical results (Q(2)=0.540, R(2)ncv=0.881, F value=157.09). A reliable CoMSIA model out of the fourteen different combinations has a Q(2) value of 0.638. The molecular docking studies of the compounds for 1CET as the protein target revealed that ten compounds showed maximum interactions with the binding site of the protein. The present study highlights the unique binding signatures of the ligands within the active site groove of the target and it explains the subtle differences in their EC50 values and their mechanism of inhibition.

  5. 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.

  6. 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

  7. [Design, synthesis and antiproliferative activities of artemisinin derivatives substituted by N-heterocycles].

    PubMed

    Zuo, Zhi-zhong; Zhong, Hang; Cai, Ting; Bao, Yu; Liu, Zhi-qiang; Liu, Dan; Zhao, Lin-xiang

    2015-07-01

    Increasing attention has been focused on the antitumor activity of artemisinin derivatives in recent years, for artemisinin had been reported to have cytotoxic effects against HL-60, P388 and MCF-7 tumor cells. We report here the synthesis and evaluation for antitumor activity of a series of artemisinin-ether derivatives bearing tetrahydropyrrole, morpholine, piperidine, substituted piperidines and azoles with various linkers. Sixteen 10-O-substituted dihydroartemisinin derivatives were designed and synthesized, all of which have never been reported in literatures and whose antiproliferative effects on human breast cancer MCF-7, MCF-7/Adr and HL-60 cells were determined by MTT assay or direct cell counting. Each of these artemisinin derivatives possessed better effects than dihydroartemisinin evidently against HL-60 and MCF-7 cells growth, while less potent than doxorubicin. All target compounds exhibited significantly improved potency compared to DHA and doxorubicin on the doxorubicin-resistant MCF-7/Adr cells, so did they in their sensitive counterparts MCF-7 cells. Among them, compounds GF02, GH04 and ZH04 showed strong activity against these three cell lines growth. Further research is undergoing. PMID:26552149

  8. 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

  9. 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.

  10. Lethal Factor Active-Site Mutations Affect Catalytic Activity In Vitro

    PubMed Central

    Hammond, S. E.; Hanna, P. C.

    1998-01-01

    The lethal factor (LF) protein of Bacillus anthracis lethal toxin contains the thermolysin-like active-site and zinc-binding consensus motif HEXXH (K. R. Klimpel, N. Arora, and S. H. Leppla, Mol. Microbiol. 13:1093–1100, 1994). LF is hypothesized to act as a Zn2+ metalloprotease in the cytoplasm of macrophages, but no proteolytic activities have been previously shown on any target substrate. Here, synthetic peptides are hydrolyzed by LF in vitro. Mass spectroscopy and peptide sequencing of isolated cleavage products separated by reverse-phase high-pressure liquid chromatography indicate that LF seems to prefer proline-containing substrates. Substitution mutations within the consensus active-site residues completely abolish all in vitro catalytic functions, as does addition of 1,10-phenanthroline, EDTA, and certain amino acid hydroxamates, including the novel zinc metalloprotease inhibitor ZINCOV. In contrast, the protease inhibitors bestatin and lysine CMK, previously shown to block LF activity on macrophages, did not block LF activity in vitro. These data provide the first direct evidence that LF may act as an endopeptidase. PMID:9573135

  11. 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

  12. 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.

  13. 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.

  14. Synthesis and CYP24A1 inhibitory activity of (E)-2-(2-substituted benzylidene)- and 2-(2-substituted benzyl)-6-methoxy-tetralones.

    PubMed

    Aboraia, Ahmed S; Makowski, Bart; Bahja, Alba; Prosser, David; Brancale, Andrea; Jones, Glenville; Simons, Claire

    2010-10-01

    A series of (E)-2-(2-substituted benzylidene)- and 2-(2-substituted benzyl)-6-methoxy-tetralones were prepared, using an efficient synthetic scheme, and evaluated for their inhibitory activity against cytochrome P450C24A1 (CYP24A1) hydroxylase. In general the reduced benzyl tetralones were more active than the parent benzylidene tetralones. The 2-ethyl and 2-trifluoromethyl benzyl tetralone derivatives (4c and 4b) showed optimal activity in this series with IC(50) values of 1.92 microM and 2.08 microM, respectively compared with the standard ketoconazole IC(50) 0.52 microM. The 2-bromobenzyl tetralone (4d) showed a preference for CYP27A1 (IC(50) 59 nM) over CYP24A1 (IC50 16.3 microM) and may be a useful lead in CYP27A1 inhibition studies. The 2-ethylphenyl benzyl derivative (9c), which showed weak activity against the wild type CYP24A1 (IC(50) 25.57 microM), exhibited enhanced inhibitory activity towards L148F and M416T mutants, this difference in activity for the L148F mutant has been explained using molecular modelling.

  15. 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

  16. New insights into the pharmacological chaperone activity of c2-substituted glucoimidazoles for the treatment of Gaucher disease.

    PubMed

    Li, Zhonghua; Li, Tiehai; Dai, Shaoxing; Xie, Xiaoli; Ma, Xiaofeng; Zhao, Wei; Zhang, Weimin; Li, Jing; Wang, Peng George

    2013-07-01

    Mutations in acid β-glucocerebrosidase (GCase) lead to the accumulation of the sphingolipid glucosylceramide, thereby resulting in Gaucher disease (GD). Active-site-specific competitive GCase inhibitors are effective pharmacological chaperones (PCs) that act as folding agents for mutant GCase folding in the endoplasmic reticulum. In this study, we prepared a series of glucoimidazole C2-substituent derivatives, and evaluated their inhibition and PC properties with GCase. A cell-based assay with patient-derived lymphoblasts (N370S or L444P mutations) demonstrated that administration of these compounds can significantly increase GCase activity. Interestingly, the 3,3-dimethyl-N-phenyl-4-amide-1-butyl-substituted moderate inhibitor 11 had the greatest effect on activity: 2.1-fold increase in N370S lymphoblasts at 2.5 μM and 1.2-fold increase in L444P at 0.5 μM following a three-day incubation. Computer docking studies and a protease protection assay were used to elucidate the ligand-enzyme interactions responsible for the chaperone activity of 11. Western blot and immuno-fluorescence assays verified restoration of GCase trafficking to the lysosome. Together, these results indicate that 11 is a promising PC for GD treatment and provide direct evidence of the mechanism of GCase chaperoning. PMID:23775891

  17. 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

  18. 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

  19. 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

  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. 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.

  2. 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.

  3. 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

  4. 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

  5. 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

  6. Synthesis and anti-inflammatory activity of 2-substituted-((N, N-disubstituted)-1, 3-benzoxazole)-5-carboxamides.

    PubMed

    Reena, M; Kiran, G; Rajyalakshmi, G; Venkateshwa, Rao J; Sarangapani, M

    2010-06-01

    A series of 2-substituted-((N, N-disubstituted)-1, 3-benzoxazole)-5-carboxamides derivatives were synthesized by the reaction of 2-substituted-5-carbomethoxy benzoxazole with different secondary amines. The newly synthesized compounds were characterized on the basis of spectral (FT-IR, 1H NMR, MS) & elemental analysis. All these compounds were screened for anti-inflammatory activity using carrageenan induced rat paw edema method. All of these compounds exhibited significant activity. Among the tested compounds Ve, Vg, Vf and Va were considered to have potent anti-inflammatory activity and was comparable with standard.

  7. 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.

  8. Tuning the biological activity profile of antibacterial polymers via subunit substitution pattern.

    PubMed

    Liu, Runhui; Chen, Xinyu; Chakraborty, Saswata; Lemke, Justin J; Hayouka, Zvi; Chow, Clara; Welch, Rodney A; Weisblum, Bernard; Masters, Kristyn S; Gellman, Samuel H

    2014-03-19

    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 sp(3) 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

  9. Trypanocidal Activity of Thioamide-Substituted Imidazoquinolinone: Electrochemical Properties and Biological Effects

    PubMed Central

    Frank, Fernanda M.; Ciccarelli, Alejandra B.; Bollini, Mariela; Bruno, Ana M.; Batlle, Alcira; Lombardo, Maria E.

    2013-01-01

    Three thioamide-substituted imidazoquinolinone, which possess a heterocyclic center similar to tryptanthrin and are named C1, C2, and C3, were studied regarding (a) their in vitro anti-Trypanosoma cruzi activity, (b) their cytotoxicity and electrochemical behaviour, and (c) their effect on cell viability, redox state, and mitochondrial function. The assayed compounds showed a significant activity against the proliferative forms, but only C1 showed activity on the trypomastigote form (for C1, IC50  epi = 1.49 μM; IC50  amas = 1.74 μM; and IC50  try = 34.89 μM). The presence of an antioxidant compound such as ascorbic acid or dithiotreitol induced a threefold increase in the antiparasitic activity, whereas glutathione had a dual effect depending on its concentration. Our results indicate that these compounds, which exhibited low toxicity to the host cells, can be reduced inside the parasite by means of the pool of low molecular weight thiols, causing oxidative stress and parasite death by apoptosis. The antiparasitic activity of the compounds studied could be explained by a loss of the capacity of the antioxidant defense system of the parasite to keep its intracellular redox state. C1 could be considered a good candidate for in vivo evaluation. PMID:23935690

  10. 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.

  11. 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.

  12. 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)

  13. 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.

  14. Control of active sites in flocculation: Concept of equivalent active sites''

    SciTech Connect

    Behl, S.; Moudgil, B.M. . Dept. of Materials Science and Engineering)

    1993-12-01

    Flocculation and dispersion of solids are strong functions of the amount and conformation of the adsorbed polymer. Regions of dispersion and flocculation of solids with particular polymer molecules may be deduced from saturation adsorption data. The concept of equivalent active sites'' is proposed to explain flocculation and dispersion behavior irrespective of the amount or conformation of the adsorbed polymer. The concept has been further extended to study the selective flocculation process.

  15. 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.

  16. Probing the active site loop motif of murine ferrochelatase by random mutagenesis.

    PubMed

    Shi, Zhen; Ferreira, Gloria C

    2004-05-01

    Ferrochelatase catalyzes the terminal step of the heme biosynthetic pathway by inserting ferrous iron into protoporphyrin IX. A conserved loop motif was shown to form part of the active site and contact the bound porphyrin by molecular dynamics calculations and structural analysis. We applied a random mutagenesis approach and steady-state kinetic analysis to assess the role of the loop motif in murine ferrochelatase function, particularly with respect to porphyrin interaction. Functional substitutions in the 10 consecutive loop positions Gln(248)-Leu(257) were identified by genetic complementation in Escherichia coli strain Deltavis. Lys(250), Val(251), Pro(253), Val(254), and Pro(255) tolerated a variety of replacements including single substitutions and contained low informational content. Gln(248), Ser(249), Gly(252), Trp(256), and Leu(257) possessed high informational content, since permissible replacements were limited and only observed in multiply substituted mutants. Selected active loop variants exhibited k(cat) values comparable with or higher than that of wild-type murine ferrochelatase. The K(m) values for porphyrin increased, except for the single mutant V251L. Other than a moderate increase observed in the triple mutant S249A/K250Q/V251C, the K(m) values for Fe(2+) were lowered. The k(cat)/K(m) for porphyrin remained largely unchanged, with the exception of a 10-fold reduction in the triple mutant K250M/V251L/W256Y. The k(cat)/K(m) for Fe(2+) was improved. Molecular modeling of these active loop variants indicated that loop mutations resulted in alterations of the active site architecture. However, despite the plasticity of the loop primary structure, the relative spatial positioning of the loop in the active site appeared to be maintained in functional variants, supporting a role for the loop in ferrochelatase function. PMID:14981080

  17. Study of effect of copper-substitution at Ga site in some Ga-based Heusler alloys from first-principles calculations

    NASA Astrophysics Data System (ADS)

    Roy, Tufan; Chakrabarti, Aparna

    2015-06-01

    In this work we have predicted the mechanical and magnetic properties of cubic austenite phase of the Cu substituted alloys of Ni2MnGa and Mn2NiGa at the Ga site i.e. Ni2MnCu and Mn2NiCu, respectively, as well as of Pt2MnCu and Pd2MnCu. We have observed that the conventional Heusler structure is lower in energy compared to the inverse Heusler structure for the Ni2MnGa, Pt2MnGa and Pd2MnGa and the Cu-substituted materials, whereas Mn2NiGa and its Cu-substituted alloy at Ga site assume the inverse structure. From the calculated elastic constants, we find that Pt2MnCu is expected to be the most ductile material among all the substituted materials studied here. We have calculated and compared the Curie temperatures derived from the Heisenberg exchange coupling parameters.

  18. 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

  19. 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

  20. Influence of terminal substitution on structural, DNA, protein binding, anticancer and antibacterial activities of palladium(II) complexes containing 3-methoxy salicylaldehyde-4(N) substituted thiosemicarbazones.

    PubMed

    Kalaivani, P; Prabhakaran, R; Ramachandran, E; Dallemer, F; Paramaguru, G; Renganathan, R; Poornima, P; Vijaya Padma, V; Natarajan, K

    2012-02-28

    The variable chelating behavior of 3-methoxysalicylaldehyde-4(N)-substituted thiosemicarbazones was observed in equimolar reactions with [PdCl(2)(PPh(3))(2)]. The new complexes were characterized by various analytical, spectroscopic techniques (mass, (1)H-NMR, absorption, IR). All the new complexes were structurally characterized by single crystal X-ray diffraction. Crystallographic results showed that the ligands H(2)L(1) and H(2)L(4) are coordinated as binegative tridentate ONS donor ligands in the complexes 1 and 4 by forming six and five member rings. However, the ligands H(2)L(2) and H(2)L(3) bound to palladium in 2 and 3 as uninegative bidentate NS donors by forming a five member chelate ring. From this study, it was found that the substitution on terminal 4(N)-nitrogen may have an influence on the chelating ability of thiosemicarbazone. The presence of hydrogen bonding in 2 and 3 might be responsible for preventing the coordination of phenolic oxygen to the metal ion. The interaction of the complexes with calf-thymus DNA (CT-DNA) has been explored by absorption and emission titration methods. Based on the observations, an electrostatic binding mode of DNA has been proposed. The protein binding studies were monitored by quenching of tryptophan and tyrosine residues in the presence of complexes using Lysozyme as model protein. Antibacterial activity studies of the complexes have been screened against pathogenic bacteria such as Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia and Pseudomonas aeruginosa. MIC50 values of the complexes showed that they exhibited significant activity against the pathogens and among them, 3 exhibited higher activity. Further, anticancer activity of the complexes on the lung cancer cell line A549 has also been studied. PMID:22222360

  1. Synthesis and activity of substituted anthraquinones against a human filarial parasite, Brugia malayi.

    PubMed

    Dhananjeyan, Mugunthu R; Milev, Youli P; Kron, Michael A; Nair, Muraleedharan G

    2005-04-21

    Lymphatic filariasis (elephantiasis) is a global public health problem caused by the parasitic nematodes Wuchereria bancrofti and Brugia malayi. We have previously reported anthraquinones from daylily roots with potent activity against pathogenic trematode Schistosoma mansoni. Here we report the synthesis of novel anthraquinones A-S and their antifilrarial activity. Anthraquinones A-S were synthesized by a single-step Friedel-Crafts acylation reaction between phthalic anhydrides and substituted benzenes. The antifilarial properties of these synthetic anthraquinones were tested against microfilaria as well as adult male and female worms of B. malayi. The most active anthraquinone was K, which showed 100% mortality within 1, 5, and 3 days, respectively, against microfilaria and adult male and female worms at 5 ppm concentration. Albendazole, an oral drug currently used to treat parasitic infections, was used as a positive control. Methylated products of anthraquinones did not affect the microfilaria. Histological examination of treated adult female parasites showed most of the anthraquinones caused marked effects on intrauterine embryos. PMID:15828820

  2. Effect of ring substitution on the metabolic fate and anti-inflammatory activity of some prodolic acid analogs.

    PubMed

    Robinson, W T; Martel, R R; Ferdinandi, E; Kraml, M

    1977-12-01

    Prodolic acid, 1-n-propyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-acetic acid, exhibits potent anti-inflammatory activity in adjuvant arthritic rats. The potency of prodolic acid is enhanced by indole ring substitution. This increase correlated well with higher and sustained drug concentrations in the serum of normal animals. Pharmacokinetic studies demonstrated that ring substitution prolonged the serum half-life without affecting the absorption or volume of distribution. Because, in the rat, indole ring hydroxylation is a major pathway for the disposition of prodolic acid, we ascribe the increased pharmacological activity of ring substituted derivatives to the interference of substituents with the hydroxylation reaction. PMID:925966

  3. A strong magneto-optical activity in rare-earth La{sup 3+} substituted M-type strontium ferrites

    SciTech Connect

    Hu Feng; Liu Xiansong; Zhu Deru; Fernandez-Garcia, Lucia; Suarez, Marta; Luis Menendez, Jose

    2011-06-01

    M-type strontium ferrites with substitution of Sr{sup 2+} by rare-earth La{sup 3+} were prepared by conventional ceramic technology. The structure, magnetic properties, and magneto-optical Kerr activity of Sr{sub 1-x}La{sub x}Fe{sub 12}O{sub 19} (x = 0, 0.05, 0.10, 0.15, 0.20) were investigated by x-ray diffraction (XRD), vibrating sample magnetometer (VSM), and magneto-optical ellipsometry, respectively. X-ray diffraction showed that the samples sintered at 1290 deg. C for 3 h were single M-type hexagonal ferrites. The magnetic properties were remarkably changed due to the valence change of Fe ions induced by the substitution of La ions. Most significantly, an important magneto-optical activity was induced in the La{sup 3+} substituted M-type strontium ferrites around 3 eV.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. Nucleotide substitutions within U5 are critical for efficient reverse transcription of human immunodeficiency virus type 1 with a primer binding site complementary to tRNA(His).

    PubMed Central

    Li, Y; Zhang, Z; Wakefield, J K; Kang, S M; Morrow, C D

    1997-01-01

    Sequence analysis of integrated proviruses of human immunodeficiency virus type 1 (HIV-1) which utilize tRNA(His) to initiate reverse transcription [virus derived from pHXB2(His-AC-TGT)] revealed five additional nucleotide substitutions in the U5 and primer binding site (PBS) regions (ATGAC for CCTGT at nucleotides 152, 160, 174, 181, and 200, respectively) (Z. Zhang et al., Virology 226:306-317, 1996). We constructed a mutant proviral genome [pHXB2(His-AC-GAC)] which contained the ATGAC substitutions to test if they represented a necessary adaptation by the virus for use of tRNA(His) to initiate reverse transcription. Viruses from pHXB2(His-AC-TGT) and pHXB2(His-AC-GAC) were infectious. Sequence analysis of the U5 and PBS regions of integrated provirus from a cell culture infected with virus derived from pHXB2(His-AC-TGT) revealed a G-to-A change in CCTGT at nucleotide 181 after limited in vitro culture, suggesting that this nucleotide change represented an adaptation by the virus to efficiently utilize tRNA(His) to initiate reverse transcription. To further address this possibility, we used a specific mutation in reverse transcriptase (RT), a methionine-to-valine change in the highly conserved YMDD amino acid motif of HIV-1 RT (M184V), which has been shown in previous studies to influence the fidelity and activity of the enzyme. The M184V RT mutation was cloned into pHXB2(His-AC-GAC) and pHXB2(His-AC-TGT). Virus derived from pHXB2(His-AC-GAC) with M184V RT had slightly delayed replication compared to the virus from pHXB2(His-AC-GAC) with wild-type RT; in contrast, virus from pHXB2(His-AC-TGT) with M184V RT was severely compromised in replication. Using an endogenous reverse transcription-PCR assay to analyze the reverse transcription of viruses obtained after transfection, we found that viruses derived from pHXB2(His-AC-GAC) with the wildtype RT were slightly faster in the initiation of reverse transcription than viruses with M184V RT. The initiation of reverse

  9. Simulation analysis of formycin 5'-monophosphate analog substrates in the ricin A-chain active site.

    PubMed

    Olson, M A; Scovill, J P; Hack, D C

    1995-06-01

    Ricin is an RNA N-glycosidase that hydrolyzes a single adenine base from a conserved loop of 28S ribosomal RNA, thus inactivating protein synthesis. Molecular-dynamics simulation methods are used to analyze the structural interactions and thermodynamics that govern the binding of formycin 5'-monophosphate (FMP) and several of its analogs to the active site of ricin A-chain. Simulations are carried out initiated from the X-ray crystal structure of the ricin-FMP complex with the ligand modeled as a dianion, monoanion and zwitterion. Relative changes in binding free energies are estimated for FMP analogs constructed from amino substitutions at the 2- and 2'-positions, and from hydroxyl substitution at the 2'-position.

  10. 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

  11. A-Site (MCe) Substitution Effects on the Structures and Properties of CaBi4Ti4O15 Ceramics

    NASA Astrophysics Data System (ADS)

    Yan, Haixue; Li, Chengen; Zhou, Jiaguang; Zhu, Weimin; He, Lianxin; Song, Yuxin

    2000-11-01

    We investigated the effect of A-site compound substitution on the structures and properties of Ca0.8(MCe)0.1Bi4Ti4O15 (M denotes Li, Na and K) ceramics. The samples were prepared by the conventional ceramic technique. Sintering characteristics of Ca0.8(MCe)0.1Bi4Ti4O15 and CaBi4Ti4O15 ceramics were discussed. X-ray powder diffraction patterns of the three modified CBT-based compounds show a single phase of bismuth oxide layer type structure with m=4. The hysteresis loops of polarization versus electric field of the four compounds were also measured. A-site compound substitution improves the piezoelectric properties and the high-temperature resistivity of these materials. A-site (LiCe) and (KCe) substitution not only improves the Curie temperature but also decreases the temperature coefficient of dielectric constant (TK\\varepsilon). Among the three modified ceramics, only the Curie temperature of Ca0.8(NaCe)0.1Bi4Ti4O15 is lower than that of CaBi4Ti4O15; however, its TK\\varepsilon is the lowest. As a result, all the three modified CBT-based ceramics were found to be excellent high-temperature piezoelectric materials.

  12. 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?

  13. 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.

  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

    2016-08-01

    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

  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. 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.

  18. 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.

  19. 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.

  20. Bovine rhodopsin: amino acid substitutions Asp-83----Asn and Glu-134----Gln prevent activation of cyclic GMP phosphodiesterase.

    PubMed

    Gurevich, V V; Zozulya, S A; Zerf, E P; Pokrovskaya, I D; Obukhova, T A; Garnovskaya, M N; Dumler, I L; Rychkova, M P

    1990-01-01

    Two bovine rhodopsin mutants with substitutions of negatively charged residues within transmembrane domains II and III by uncharged ones (Asp-83----Asn and Glu-134----Gln) were constructed. Both mutants stimulated transducin GTPase with slightly lowered efficiency, but were completely unable to activate cyclic GMP phosphodiesterase. PMID:1966786

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. Activation of Inhibitors by Sortase Triggers Irreversible Modification of the Active Site*S

    PubMed Central

    Maresso, Anthony W.; Wu, Ruiying; Kern, Justin W.; Zhang, Rongguang; Janik, Dorota; Missiakas, Dominique M.; Duban, Mark-Eugene; Joachimiak, Andrzej; Schneewind, Olaf

    2011-01-01

    Sortases anchor surface proteins to the cell wall of Gram-positive pathogens through recognition of specific motif sequences. Loss of sortase leads to large reductions in virulence, which identifies sortase as a target for the development of antibacterials. By screening 135,625 small molecules for inhibition, we report here that aryl (β-amino)ethyl ketones inhibit sortase enzymes from staphylococci and bacilli. Inhibition of sortases occurs through an irreversible, covalent modification of their active site cysteine. Sortases specifically activate this class of molecules via β-elimination, generating a reactive olefin intermediate that covalently modifies the cysteine thiol. Analysis of the three-dimensional structure of Bacillus anthracis sortase B with and without inhibitor provides insights into the mechanism of inhibition and reveals binding pockets that can be exploited for drug discovery. PMID:17545669

  6. 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

  7. 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

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

    PubMed Central

    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.

    2016-01-01

    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

  9. Synthesis and biological activity of new (E)-alpha-(Methoxyimino)benzeneacetate derivatives containing a substituted pyrazole ring.

    PubMed

    Li, Miao; Liu, Chang-Ling; Yang, Ji-Chun; Zhang, Jin-Bo; Li, Zhi-Nian; Zhang, Hong; Li, Zheng-Ming

    2010-03-10

    Strobilurins are one of the most important classes of agricultural fungicides. To discover new strobilurin analogues with high activity, a series of new strobilurin derivatives containing a substituted pyrazole in the side chain were synthesized and their biological activities were tested. The compounds were identified by (1)H nuclear magnetic resonance, infrared, and elemental analysis. The test results indicated that the compounds exhibited strong fungicidal activities against Pyricularia oryzae , Phytophthora infestans , Pseudoperonospora cubensis , and Erysiphe graminis . The relationship between structure and biological activity is discussed in terms of the effects of the substituents on the pyrazole ring. The present work demonstrates that strobilurin analogues with a 3-(substituted phenyl)-1H-pyrazol-5-oxy side chain can be used as possible lead compounds for the development of potential agrochemicals. PMID:19961182

  10. 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

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

    PubMed Central

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

    2016-01-01

    CphA is a Zn2+-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

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  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. Mutations of fumarase that distinguish between the active site and a nearby dicarboxylic acid binding site.

    PubMed Central

    Weaver, T.; Lees, M.; Banaszak, L.

    1997-01-01

    Two mutant forms of fumarase C from E. coli have been made using PCR and recombinant DNA. The recombinant form of the protein included a histidine arm on the C-terminal facilitating purification. Based on earlier studies, two different carboxylic acid binding sites, labeled A- and B-, were observed in crystal structures of the wild type and inhibited forms of the enzyme. A histidine at each of the sites was mutated to an asparagine. H188N at the A-site resulted in a large decrease in specific activity, while the H129N mutation at the B-site had essentially no effect. From the results, we conclude that the A-site is indeed the active site, and a dual role for H188 as a potential catalytic base is proposed. Crystal structures of the two mutant proteins produced some unexpected results. Both mutations reduced the affinity for the carboxylic acids at their respective sites. The H129N mutant should be particularly useful in future kinetic studies because it sterically blocks the B-site with the carboxyamide of asparagine assuming the position of the ligand's carboxylate. In the H188N mutation at the active site, the new asparagine side chain still interacts with an active site water that appears to have moved slightly as a result of the mutation. PMID:9098893

  1. 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

  2. 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

  3. Isosteric substitutions of urea to thiourea and selenourea in aliphatic oligourea foldamers: site-specific perturbation of the helix geometry.

    PubMed

    Nelli, Yella Reddy; Antunes, Stéphanie; Salaün, Arnaud; Thinon, Emmanuelle; Massip, Stéphane; Kauffmann, Brice; Douat, Céline; Guichard, Gilles

    2015-02-01

    Nearly isosteric oxo to thioxo substitution was employed to interrogate the structure of foldamers with a urea backbone and explore the relationship between helical folding and hydrogen-bonding interactions. A series of oligomers with urea bonds substituted by thiourea bonds at discrete or all positions in the sequence have been prepared and their folding propensity was studied by using a combination of spectroscopic methods and X-ray diffraction. The outcome of oxo to thioxo replacements on the helical folding was found to depend on whether central or terminal ureas were modified. The canonical helix geometry was not affected upon insertion of thioureas close to the negative end of the helix dipole, whereas thioureas close to the positive pole were found to increase the terminal flexibility and cause helix fraying. Perturbation was amplified when a selenourea was incorporated instead, leading to a structure that is only partly folded.

  4. 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.

  5. In Vivo Activity of the Pyrrolopyrazolyl-Substituted Oxazolidinone RWJ-416457▿

    PubMed Central

    Hilliard, Jamese J.; Fernandez, Jeffrey; Melton, John; Macielag, Mark J.; Goldschmidt, Raul; Bush, Karen; Abbanat, Darren

    2009-01-01

    RWJ-416457 is an investigational pyrrolopyrazolyl-substituted oxazolidinone with activity against antibiotic-susceptible and -resistant gram-positive pathogens. Efficacies of RWJ-416457, linezolid, and vancomycin against methicillin-susceptible Staphylococcus aureus (MSSA) and community-associated methicillin-resistant S. aureus (CA-MRSA) in murine skin and systemic infections were compared, as were efficacies against Streptococcus pneumoniae in a lower respiratory infection. In staphylococcal systemic infections, RWJ-416457 was equipotent with to twofold more potent than linezolid, with 50% effective dose values ranging from 1.5 to 5 mg/kg of body weight/day. RWJ-416457 was two- to fourfold less potent than vancomycin against MSSA but up to fourfold more potent than vancomycin against CA-MRSA. In MSSA and CA-MRSA skin infections, RWJ-416457 demonstrated an efficacy similar to that of linezolid, reducing CFU/g skin approximately 1.0 log10 at all doses tested; vancomycin yielded greater reductions than the oxazolidinones, with decreases in CFU/g skin of 3 log10 (MSSA) and 2 log10 (CA-MRSA). In the pneumococcal model, RWJ-416457 was two- to fourfold more potent than linezolid. The free-drug area under the concentration-time curves at 24 h (fAUC24) were similar for RWJ-416457 and linezolid. The half-life of RWJ-416457 was up to threefold longer than that of linezolid for all routes of administration. The fAUC24/MIC ratio, the pharmacodynamic parameter considered predictive of oxazolidinone efficacy, was approximately twofold greater for RWJ-416457 than for linezolid. Since the fAUC values were similar for both compounds, the higher fAUC/MIC ratios of RWJ-416457 appear to result from its greater in vitro potency. These results demonstrate that RWJ-416457 is a promising new oxazolidinone with efficacy in S. aureus or S. pneumoniae mouse infection models. PMID:19273686

  6. Amide-controlled, one-pot synthesis of tri-substituted purines generates structural diversity and analogues with trypanocidal activity.

    PubMed

    Pineda de las Infantas y Villatoro, Maria J; Unciti-Broceta, Juan D; Contreras-Montoya, Rafael; Garcia-Salcedo, Jose A; Gallo Mezo, Miguel A; Unciti-Broceta, Asier; Diaz-Mochon, Juan J

    2015-01-01

    A novel one-pot synthesis of tri-substituted purines and the discovery of purine analogues with trypanocidal activity are reported. The reaction is initiated by a metal-free oxidative coupling of primary alkoxides and diaminopyrimidines with Schiff base formation and subsequent annulation in the presence of large N,N-dimethylamides (e.g. N,N-dimethylpropanamide or larger). This synthetic route is in competition with a reaction previously-reported by our group, allowing the generation of a combinatorial library of tri-substituted purines by the simple modification of the amide and the alkoxide employed. Among the variety of structures generated, two purine analogues displayed trypanocidal activity against the protozoan parasite Trypanosoma brucei with IC50 < 5 μM, being each of those compounds obtained through each of the synthetic pathways.

  7. Synthesis, spectral characterization, and biological activity of some new substituted 10H-phenothiazines, its ribofuranosides, and sulfones.

    PubMed

    Gautam, Naveen; Ajmera, Neha; Gupta, Shikha; Gautam, D C

    2010-03-01

    This article describes the synthesis of new substituted 10 H-phenothiazines by Smiles rearrangement. These compounds are then used as a base to form ribofuranosides by treating them with a sugar (1-O-acetyl-2,3,5-tri-O-benzoyl-beta-ribofuranose). On oxidation with hydrogen peroxide in glacial acetic acid, these phenothiazines yield their sulfones. These compounds are screened for antioxidant and antimicrobial activity and their structure has been established by elemental analysis and spectroscopic data. PMID:20408049

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. Substitution of a single amino acid (aspartic acid for histidine) converts the functional activity of human complement C4B to C4A

    SciTech Connect

    Carroll, M.C.; Fathallah, D.M.; Bergamaschini, L.; Alicot, E.M. ); Isenman, D.E. )

    1990-09-01

    The C4B isotype of the fourth component of human complement (C4) displays 3- to 4-fold greater hemolytic activity than does its other isotype C4A. This correlates with differences in their covalent binding efficiencies to erythrocytes coated with antibody and complement C1. C4A binds to a greater extent when C1 is on IgG immune aggregates. The differences in covalent binding properties correlate only with amino acid changes between residues 1101 and 1106 (pro-C4 numbering)-namely, Pro-1101, Cys-1102, Leu-1105, and Asp-1106 in C4A and Leu-1101, Ser-1102, Ile-1105, and His-1106 in C4B, which are located in the C4d region of the {alpha} chain. To more precisely identify the residues that are important for the functional differences, C4A-C4B hybrid proteins were constructed by using recombinant DNA techniques. Comparison of these by hemolytic assay and binding to IgG aggregates showed that the single substitution of aspartic acid for histidine at position 1106 largely accounted for the change in functional activity and nature of the chemical bond formed. Surprisingly, substitution of a neutral residue, alanine, for histidine at position 1106 resulted in an increase in binding to immune aggregates without subsequent reduction in the hemolytic activity. This result strongly suggests that position 1106 is not catalytic as previously proposed but interacts sterically/electrostatically with potential acceptor sites and serves to select binding sites on potential acceptor molecules.

  15. 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.

  16. Influence of Cu-site substitution on La2Ca1Ba2Cu5Oz superconducting system

    NASA Astrophysics Data System (ADS)

    Bhalodia, J. A.; Mankadia, S. R.; Dalsaniya, S. M.; Gonal, M. R.

    2012-07-01

    We have prepared a series of La2CaBa2Cu5-xCoxOz; x = 0.0, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0 (La-2125) compounds by the standard solid state reaction method and characterized for their structural, superconducting, magnetic properties and oxygen content through X-ray diffraction, scanning electron microscopy, d. c. resistivity, magnetic susceptibility and iodometric titration respectively. All the compounds crystallize with the tetragonal LaBa2Cu3Oz type structure, space group P4/mmm. Here the effect of higher Co substitution for Cu in the La2CaBa2Cu5-xCoxOz system has been studied. It is observed that only 2 at. % Co substitution for Cu destroys the superconductivity of the sample. For heavily doped samples (with x >= 0.1) are found non-superconducting presumably because of magnetic pair- breaking effect. These samples do not show superconductivity but are of interest for understanding the interplay between superconductivity and magnetism. Possible reasons for destruction of superconductivity are discussed in this communication.

  17. A novel approach to predict active sites of enzyme molecules.

    PubMed

    Chou, Kuo-Chen; Cai, Yu-dong

    2004-04-01

    Enzymes are critical in many cellular signaling cascades. With many enzyme structures being solved, there is an increasing need to develop an automated method for identifying their active sites. However, given the atomic coordinates of an enzyme molecule, how can we predict its active site? This is a vitally important problem because the core of an enzyme molecule is its active site from the viewpoints of both pure scientific research and industrial application. In this article, a topological entity was introduced to characterize the enzymatic active site. Based on such a concept, the covariant discriminant algorithm was formulated for identifying the active site. As a paradigm, the serine hydrolase family was demonstrated. The overall success rate by jackknife test for a data set of 88 enzyme molecules was 99.92%, and that for a data set of 50 independent enzyme molecules was 99.91%. Meanwhile, it was shown through an example that the prediction algorithm can also be used to find any typographic error of a PDB file in annotating the constituent amino acids of catalytic triad and to suggest a possible correction. The very high success rates are due to the introduction of a covariance matrix in the prediction algorithm that makes allowance for taking into account the coupling effects among the key constituent atoms of active site. It is anticipated that the novel approach is quite promising and may become a useful high throughput tool in enzymology, proteomics, and structural bioinformatics. PMID:14997541

  18. Site specific ligand substitution in cubane-type Mo3FeS(4)(4+) clusters: kinetics and mechanism of reaction and isolation of mixed ligand Cl/SPh complexes.

    PubMed

    Algarra, Andrés G; Basallote, Manuel G; Fernandez-Trujillo, M J; Llusar, Rosa; Pino-Chamorro, Jose A; Sorribes, Ivan; Vicent, Cristian

    2010-04-21

    The synthesis, crystal structure and solution characterization of the cubane-type [Mo(3)(FeCl)S(4)(dmpe)(3)Cl(3)] (1) (dmpe = 1,2-bis(dimethylphophane-ethane)) cluster are reported and the ligand substitution processes of chloride by thiophenolate investigated. The kinetics and the intimate mechanism of these substitutions reveal that compound 1 undergoes a number of Fe and Mo site specific ligand substitution reactions in acetonitrile solutions. In particular, PhS(-) coordination at the tetrahedral Fe site proceeds in a single resolved kinetic step whereas such substitutions at the Mo sites proceed more slowly. The effect of the presence of acids in the reaction media is also investigated and reveals that an acid excess hinders substitution reactions both at the Fe and Mo sites; however, an acid-promoted solvolysis of the Fe-Cl bonds is observed. Electrospray ionization (ESI) and tandem (ESI-MS/MS) mass spectrometry allow the identification of all the reaction intermediates proposed on the basis of stopped-flow measurements. The distinctive site specific reactivity made it possible to isolate two new clusters of the Mo(3)FeS(4)(4+) family featuring mixed chlorine/thiophenolate ligands, namely Mo(3)S(4)(FeSPh)(dmpe)(3)Cl(3) (2) and [Mo(3)S(4)(FeSPh)(dmpe)(3)(SPh)(3)] (3). A detailed computational study has also been carried out to understand the details of the mechanism of substitution at the M-Cl (M = Mo and Fe) bonds as well as the solvolysis at the Fe-Cl sites, with particular emphasis on the role of acids on the substitution process. The results of the calculations are in agreement with the experimental observations, thus justifying the non-existence of an accelerating effect of acids on the thiophenolate substitution reaction, which differs from previous proposals for the Fe(4)S(4) and MoFe(3)S(4) clusters and some related compounds.

  19. Synthesis, characterization and anticonvulsant activity evaluation of some 1,4-dihydropyridines and 3,5-(substituted)oxycarbonyl-1,4-dihydro-2,6-dimethyl-N-[2-(4-sulfamoylphenylamino)-acetyl]-4-(substituted)pyridines.

    PubMed

    Subudhi, Bharat Bhusan; Panda, Prasanna K; Swain, Sarada P; Sarangi, Priyambada

    2009-01-01

    A series of 3,5-(substituted)oxycarbonyl-1,4-dihydro-2,6-dimethyl-4-(substituted)pyridines (1a-j) were synthesized by Hantzsch method for pyridine synthesis. Treatment with chloroacetyl chloride produced N-(2-chloroacetyl)-3,5-(substituted)oxycarbonyl-1,4-dihydro-2,6-dimethyl-4-(substituted)pyridines (2a-e), which on further treatment with sulfanilamide resulted in 3,5-(substituted)oxycarbonyl-1,4-dihydro-2,6-dimethyl-N-[2-(4-sulfamoylphenylamino)-acetyl]-4-(substituted)pyridines (3a-e). The structures has been established on the basis of spectral (IR, 1H-NMR, mass) and elemental analysis. Compounds 1a-j and 3a-e (5 mg/kg and 10 mg/kg) were evaluated for their anticonvulsant effect against pentylenetetrazole-induced convulsions with diazepam (4 mg/kg) as the reference. Compounds 3a-e exhibited significant (p<0.01) anticonvulsant activity compared to the control. PMID:19719048

  20. 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

  1. Growth exponents in surface models with non-active sites

    NASA Astrophysics Data System (ADS)

    Santos, M.; Figueiredo, W.; Aarão Reis, F. D. A.

    2006-11-01

    In this work, we studied the role played by the inactive sites present on the substrate of a growing surface. In our model, one particle sticks at the surface if the site where it falls is an active site. However, we allow the deposited particle to diffuse along the surface in accordance with some mechanism previously defined. Using Monte Carlo simulations, and some analytical results, we have investigated the model in (1+1) and (2+1) dimensions considering different relaxation mechanisms. We show that the consideration of non-active sites is a crucial point in the model. In fact, we have seen that the saturation regime is not observed for any value of the density of inactive sites. Besides, the growth exponent β turns to be one, at long times, whatever the mechanism of diffusion we consider in one and two dimensions.

  2. 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

  3. Anti-Inflammatory Activity Comparison among Scropoliosides-Catalpol Derivatives with 6-O-Substituted Cinnamyl Moieties.

    PubMed

    Zhu, Tiantian; Zhang, Liuqiang; Ling, Shuang; Qian, Fei; Li, Yiming; Xu, Jin-Wen

    2015-11-03

    We have previously shown that scropolioside B has higher anti-inflammatory activity than catalpol does after the inhibition of nuclear factor (NF)-κB activity and IL-1β expression, maturation, and secretion. Various scropoliosides were extracted, isolated, and purified from Scrophularia dentata Royle ex Benth. We then compared their anti-inflammatory activities against LPS-induced NF-κB activity, cytokines mRNA expression, IL-1β secretion, and cyclooxygenase-2 activity. The inhibitory effects of the scropoliosides varied depending on whether the 6-O-substituted cinnamyl moiety was linked to C'' 2-OH, C''3-OH, or C''4-OH, and on the number of moieties linked, which is closely related to the enhancement of antiinflammatory activity. Among these compounds, scropolioside B had the strongest antiinflammatory effects.

  4. 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.

  5. 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

  6. Tilting and Tumbling in Transmembrane Anion Carriers: Activity Tuning through n‐Alkyl Substitution

    PubMed Central

    Edwards, Sophie J.; Marques, Igor; Dias, Christopher M.; Tromans, Robert A.; Lees, Nicholas R.

    2016-01-01

    Abstract Anion transport by synthetic carriers (anionophores) holds promise for medical applications, especially the treatment of cystic fibrosis. Among the factors which determine carrier activity, the size and disposition of alkyl groups is proving remarkably important. Herein we describe a series of dithioureidodecalin anionophores, in which alkyl substituents on one face are varied from C0 to C10 in two‐carbon steps. Activities increase then decrease as the chain length grows, peaking quite sharply at C6. Molecular dynamics simulations showed the transporter chloride complexes releasing chloride as they approach the membrane‐aqueous interface. The free transporter then stays at the interface, adopting an orientation that depends on the alkyl substituent. If chloride release is prevented, the complex is positioned similarly. Longer chains tilt the binding site away from the interface, potentially freeing the transporter or complex to move through the membrane. However, chains which are too long can also slow transport by inhibiting movement, and especially reorientation, within the phospholipid bilayer. PMID:26748870

  7. Bacteriorhodopsin mutants containing single tyrosine to phenylalanine substitutions are all active in proton translocation.

    PubMed Central

    Mogi, T; Stern, L J; Hackett, N R; Khorana, H G

    1987-01-01

    To study the possible role of the tyrosine residues in proton translocation by bacteriorhodopsin, we have replaced these residues individually by phenylalanine. The required codon changes were introduced in the bacterioopsin gene by replacement of appropriate restriction fragments by synthetic counterparts containing the desired nucleotide changes. The denatured opsin polypeptides obtained by expression of the mutant genes in Escherichia coli were purified and treated with a mixture of detergents, phospholipids, and retinal in a previously established renaturation procedure. All of the mutant proteins folded to regenerate bacteriorhodopsin-like chromophores. Three mutants with tyrosine to phenylalanine substitutions at positions 57, 83, and 185 regenerated the chromophore more slowly than the wild-type protein, and two of these mutants, Phe-57 and -83, showed slightly blue-shifted chromophores. When reconstituted into liposomes all of the mutant proteins with single Tyr----Phe substitutions pumped protons at rates and levels comparable to those of the wild-type bacteriorhodopsin. We conclude that single substitutions of tyrosine by phenylalanine do not affect folding, retinal binding, or light-dependent proton pumping in bacteriorhodopsin. PMID:3039495

  8. The role of amino acid residues in the active site of L-methionine γ-lyase from Pseudomonas putida.

    PubMed

    Fukumoto, Mitsuki; Kudou, Daizou; Murano, Shouko; Shiba, Tomoo; Sato, Dan; Tamura, Takashi; Harada, Shigeharu; Inagaki, Kenji

    2012-01-01

    Cys116, Lys240*, and Asp241* (asterisks indicate residues from the second subunit of the active dimer) at the active site of L-methionine γ-lyase of Pseudomonas putida (MGL_Pp) are highly conserved among heterologous MGLs. In a previous study, we found that substitution of Cys116 for His led to a drastic increase in activity toward L-cysteine and a decrease in that toward L-methionine. In this study, we examined some properties of the C116H mutant by kinetic analysis and 3D structural analysis. We assumed that substitution of Cys116 for His broke the original hydrogen-bond network and that this induced a significant effect of Tyr114 as a general acid catalyst, possibly due to the narrow space in the active site. The C116H mutant acquired a novel β-elimination activity and lead a drastic conformation change in the histidine residue at position 116 by binding the substrate, suggesting that this His residue affects the reaction specificity of C116H. Furthermore, we suggest that Lys240* is important for substrate recognition and structural stability and that Asp241* is also involved in substrate specificity in the elimination reaction. Based on this, we suggest that the hydrogen-bond network among Cys116, Lys240*, and Asp241* contributes to substrate specificity that is, to L-methionine recognition at the active site in MGL_Pp.

  9. Ring-substituted histaprodifen analogues as partial agonists for histamine H(1) receptors: synthesis and structure-activity relationships.

    PubMed

    Elz, S; Kramer, K; Leschke, C; Schunack, W

    2000-01-01

    Thirteen racemic benzene ring-substituted analogues of histaprodifen (8a; 2-[2-(3,3-diphenylpropyl)-1H-imidazol-4-yl]ethanamine), a novel lead for potent and selective histamine H(1)-receptor agonists, have been prepared from substituted 4,4-diphenylbutyronitriles 5 via cyclization of the corresponding methyl butyrimidates 6 with 2-oxo-4-phthalimido-1-butyl acetate in liquid ammonia, followed by deprotection. Nitriles 5 were accessible by alkylation of either substituted diphenylmethanes with 3-bromopropionitrile or diethyl malonate with substituted 1-chloro-diphenylmethanes and subsequent standard reactions. The title compounds 8 displayed partial agonism on contractile H(1) receptors of the guinea-pig ileum (E(max) = 2-98% relative to histamine) and, compared with the endogenous agonist, were endowed with agonist potencies of 4-92%. The meta fluorinated (8c) and meta chlorinated (8f) analogues showed the highest relative potency in this series (95% confidence limits 85-99% and 78-102%), but did not exceed the value of the lead 8a (99-124%). Compound 8c (2-[2-[3-(3-fluorophenyl)-3-phenylpropyl]-1H-imidazol-4-yl]ethanamine ) was a partial agonist at contractile H(1) receptors of the guinea-pig aorta (relative potency 154% vs. 100% for histamine) and at relaxation-mediating endothelial H(1) receptors of the rat aorta (relative potency 556% vs. 100% for histamine) and matched with the functional behaviour of 8a. Agonism observed for each compound was sensitive to blockade by the selective H(1)-receptor antagonist mepyramine (pA(2) approximately 9 (guinea-pig) and pA(2) approximately 8 (rat aorta)). All histaprodifen analogues 8 stimulated neither histaminergic H(2)/H(3) nor cholinergic M(3) receptors. They displayed only low to moderate affinity for these sites (H(2): pD'(2) < 5; H(3)/M(3): pA(2) < 6). With regard to the substitution pattern on the benzene ring, there was no correlation between the histaprodifen series and the corresponding derivatives of another

  10. 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

  11. 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)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

  12. 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

  13. Computational characterization of ketone-ketal transformations at the active site of matrix metalloproteinases.

    PubMed

    Khrenova, Maria G; Nemukhin, Alexander V; Savitsky, Alexander P

    2014-04-24

    We modeled the first steps of hydrolysis reactions of a natural oligopeptide substrate of matrix metalloproteinase MMP-2 as well as of a substrate analogue. In the latter, the scissile amide group is substituted by a ketomethylene group which can be transformed to the ketal group upon binding of this compound to the enzyme active site. According to our quantum mechanical-molecular mechanical (QM/MM) calculations, the reaction of the ketone-ketal transformation proceeds with a low energy barrier (3.4 kcal/mol) and a high equilibrium constant (10(4)). The reaction product with the ketal group formed directly at the active site of the enzyme works as an inhibitor that chelates the zinc ion. On the other hand, the oligopeptide mimetic retains molecular groups responsible for binding of this compound to the enzyme active site. This example illustrates a strategy to design MMP inhibitors in situ by using data on binding specificity of substrates to a particular type of MMP and details of the reaction mechanism. PMID:24684684

  14. 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

  15. Structure activity relationship of pyridoxazinone substituted RHS analogs of oxabicyclooctane-linked 1,5-naphthyridinyl novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents (Part-6).

    PubMed

    Singh, Sheo B; Kaelin, David E; Wu, Jin; Miesel, Lynn; Tan, Christopher M; Meinke, Peter T; Olsen, David B; Lagrutta, Armando; Wei, Changqing; Liao, Yonggang; Peng, Xuanjia; Wang, Xiu; Fukuda, Hideyuki; Kishii, Ryuta; Takei, Masaya; Yajima, Masanobu; Shibue, Taku; Shibata, Takeshi; Ohata, Kohei; Nishimura, Akinori; Fukuda, Yasumichi

    2015-09-01

    Oxabicyclooctane linked 1,5-naphthyridinyl-pyridoxazinones are novel broad-spectrum bacterial topoisomerase inhibitors (NBTIs) targeting bacterial DNA gyrase and topoisomerase IV at a site different than quinolones. Due to lack of cross-resistance to known antibiotics they present excellent opportunity to combat drug-resistant bacteria. A structure activity relationship of the pyridoxazinone moiety is described in this Letter. Chemical synthesis and activities of NBTIs with substitutions at C-3, C-4 and C-7 of the pyridoxazinone moiety with halogens, alkyl groups and methoxy group has been described. In addition, substitutions of the linker NH proton and its transformation into amide analogs of AM-8085 and AM-8191 have been reported. Fluoro, chloro, and methyl groups at C-3 of the pyridoxazinone moiety retained the potency and spectrum. In addition, a C-3 fluoro analog showed 4-fold better oral efficacy (ED50 3.9 mg/kg) as compared to the parent AM-8085 in a murine bacteremia model of infection of Staphylococcus aureus. Even modest polarity (e.g., methoxy) is not tolerated at C-3 of the pyridoxazinone unit. The basicity and NH group of the linker is important for the activity when CH2 is at the linker position-8. However, amides (with linker position-8 ketone) with a position-7 NH or N-methyl group retained potency and spectrum suggesting that neither basicity nor hydrogen-donor properties of the linker amide NH is essential for the activity. This would suggest likely an altered binding mode of the linker position-7,8 amide containing compounds. The amides showed highly improved hERG (functional IC50 >30 μM) profile.

  16. 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

  17. Synthesis and structure-activity relationship studies of novel 3,9-substituted α-carboline derivatives with high cytotoxic activity against colorectal cancer cells.

    PubMed

    Lin, Yi-Chien; Chen, Yi-Fong; Tseng, Li-Shin; Lee, Yueh-Hsuan; Morris-Natschke, Susan L; Kuo, Sheng-Chu; Yang, Ning-Sun; Lee, Kuo-Hsiung; Huang, Li-Jiau

    2016-03-01

    In our continued focus on 1-benzyl-3-(5-hydroxymethyl-2-furyl)indazole (YC-1) analogs, we synthesized a novel series of 3,9-substituted α-carboline derivatives and evaluated the new compounds for antiproliferactive effects. Structure activity relationships revealed that a COOCH3 or CH2OH group at position-3 and substituted benzyl group at position-9 of the α-carboline nucleus were crucial for maximal activity. The most active compound, 11, showed high levels of cytotoxicity against HL-60, COLO 205, Hep 3B, and H460 cells with IC50 values of 0.3, 0.49, 0.7, and 0.8 μM, respectively. The effect of compound 11 on the cell cycle distribution demonstrated G2/M arrest in COLO 205 cells. Furthermore, mechanistic studies indicated that compound 11 induced apoptosis by activating death receptor and mitochondria dependent apoptotic signaling pathways in COLO 205 cells. The new 3,9-substituted α-carboline derivatives exhibited excellent anti-proliferative activities, and compound 11 can be used as a promising pro-apoptotic agent for future development of new antitumor agents.

  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.

  19. Systematic synthesis of galloyl-substituted procyanidin B1 and B2, and their ability of DPPH radical scavenging activity and inhibitory activity of DNA polymerases.

    PubMed

    Saito, Akiko; Mizushina, Yoshiyuki; Ikawa, Hiroshi; Yoshida, Hiromi; Doi, Yuki; Tanaka, Akira; Nakajima, Noriyuki

    2005-04-15

    Six galloyl-substituted procyanidin B1 and B2, 3-O-gallate, 3''-O-gallate, and 3,3''-di-O-gallate, were systematically synthesized with the condensation method using TMSOTf as a catalyst. Their ability of DPPH radical scavenging activity and DNA polymerase inhibitory activity were also investigated. The results indicated that the galloyl group of these compounds is very important for both activities. 3,3''-Di-O-gallate dimers acted as strong inhibitor against DNA polymerase alpha and beta, whereas the desgalloyl and monogalloyl compounds did not exhibit any appreciable inhibitory activity against the DNA polymerase beta.

  20. The structure and activation of substrate water molecules in Sr(2+)-substituted photosystem II.

    PubMed

    Chatterjee, Ruchira; Milikisiyants, Sergey; Coates, Christopher S; Koua, Faisal H M; Shen, Jian-Ren; Lakshmi, K V

    2014-10-14

    The mechanism of solar water oxidation by photosystem II (PSII) is of fundamental interest and it is the object of extensive studies both in the past and present. The solar water oxidation reaction of PSII occurs in the oxygen-evolving complex (OEC). The OEC consists of a tetranuclear manganese calcium-oxo (Mn4Ca-oxo) cluster that is surrounded by amino acid residues and inorganic cofactors. The role of the Ca(2+) ion in the water oxidation reaction is one of the most interesting questions that is yet to be answered. In this study, we probe the structural and functional differences induced by metal ion substitution in the Mn4Ca-oxo cluster by substituting the Ca(2+) ion in the OEC by a Sr(2+) ion. We apply two-dimensional (2D) hyperfine sublevel correlation (HYSCORE) spectroscopy to detect weak magnetic interactions between the paramagnetic Mn4Sr-oxo cluster and the surrounding protons in the S2 state of the OEC of Sr(2+)-substituted PSII. We identify three groups of protons that are magnetically interacting with the Mn4Sr-oxo cluster. Using the recently reported 1.9 Å resolution X-ray structure of the OEC in the S1 state [Umena et al.] and the high-resolution 2D HYSCORE spectroscopy studies of the S2 state of the OEC of Ca(2+)-containing PSII [Milikisiyants et al., Energy Environ. Sci., 2012, 5, 7747], we discuss the assignments of the three groups of protons that are magnetically coupled to the Mn4Sr-oxo cluster. Since hyperfine interactions are highly sensitive to small perturbations in the electronic and geometric structure of paramagnetic centers, a comparison of the 2D HYSCORE spectra of Sr(2+)-substituted and Ca(2+)-containing PSII allows us to draw important conclusions with respect to the structure of the substrate water molecules in the OEC and the role of the Ca(2+) ion in the water oxidation reaction. In addition, for the first time, we determine the experimental value of the spin projection factor for the Mn(III) ion of the Mn4Ca-oxo cluster as ρ1

  1. 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

  2. Site-directed substitution of Ser1406 of hamster CAD with glutamic acid alters allosteric regulation of carbamyl phosphate synthetase II.

    PubMed

    Banerjei, L C; Davidson, J N

    1997-01-01

    Ser1406 of the allosteric region of the hamster CAD enzyme, carbamyl phosphate synthetase II (CPSase), is known to be phosphorylated in vitro by cAMP-dependent protein kinase (PKA). Metabolic labeling experiments described here demonstrate that CAD is phosphorylated in somatic cells in culture. Phosphorylation is stimulated by treating cells with 8-bromo-cAMP, a PKA activator. The stimulation is essentially prevented by pretreatment with H-89, a PKA specific inhibitor. Substitution of Ser1406 with alanine results in an enzyme with kinetics and allosteric regulation indistinguishable from unsubstituted CAD. However, substitution to glutamic acid increases CPSase activity by reducing the apparent Km (ATP). The UTP concentration required to give 50% inhibition is increased rendering this altered enzyme significantly less sensitive to feedback inhibition, but allosteric activation by PRPP is unaffected. While these data do not prove that Ser1406 is phosphorylated in vivo, they do indicate that a specific alteration at this residue can affect allosteric regulation. PMID:9218000

  3. 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

  4. Annealing of Cd-implanted GaAs: Defect removal, lattice site occupation, and electrical activation

    NASA Astrophysics Data System (ADS)

    Moriya, N.; Brener, I.; Kalish, R.; Pfeiffer, W.; Deicher, M.; Keller, R.; Magerle, R.; Recknagel, E.; Skudlik, H.; Wichert, Th.; Wolf, H.

    1993-05-01

    A systematic investigation of the behavior of Cd-implanted GaAs after rapid thermal annealing is presented. The use of various experimental techniques gives a detailed picture regarding the annealing process in the low-dose regime (1012 and 1013 cm-2) on a microscopic as well as on a macroscopic scale. Perturbed angular correlation experiments, using the radioactive probe 111mCd, yield information on the immediate environment of the Cd implant on an atomic scale. Rutherford backscattering channeling and photoluminescence spectroscopy give complementary information concerning the overall damage level in the implanted layer, Hall measurements are used to determine the degree of electrical activation of the implanted Cd acceptors. The outdiffusion of the implanted radioactive Cd atoms is also investigated. The removal of defects in the next-nearest neighborhood of the Cd atoms takes place after annealing at 700 K and is accompanied by a general recovering of the crystal lattice. Between 600 and 900 K more distant defects are removed. The observed outdiffusion of about one-third of the dopant atoms after annealing above 600 K is discussed in context with their partial incorporation in extended defects. Although already at 700 K, 80% of the implanted Cd atoms are on substitutional lattice sites with no defects in their immediate environment, an annealing temperature in excess of 1000 K is necessary to obtain electrical activation of the implants. It is concluded that compensating defects, present in ion-implanted GaAs, are the reason for the significantly higher temperature required for electrical activation as compared to the incorporation of the dopants on defect-free, substitutional lattice sites.

  5. 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

  6. 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.

  7. 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.

  8. Design and Synthesis of C-2 Substituted Thiazolo and Dihydrothiazolo Ring-Fused 2-Pyridones; Pilicides with Increased Antivirulence Activity

    PubMed Central

    Chorell, Erik; Pinkner, Jerome S.; Phan, Gilles; Edvinsson, Sofie; Buelens, Floris; Remaut, Han; Waksman, Gabriel; Hultgren, Scott J.; Almqvist, Fredrik

    2010-01-01

    Pilicides block pili formation by binding to pilus chaperones and blocking their function in the chaperone/usher pathway in E. coli. Various C-2 substituents were introduced on the pilicide scaffold by design and synthetic method developments. Experimental evaluation showed that proper substitution of this position affected the biological activity of the compound. Aryl substituents resulted in pilicides with significantly increased potencies as measured in pili-dependent biofilm and hemagglutination assays. The structural basis of the PapD chaperone-pilicide interactions was determined by X ray crystallography. PMID:20586493

  9. 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

  10. 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.

  11. 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

  12. Chlorodifluoromethyl-substituted monosaccharide derivatives--radical activation of the carbon-chlorine-bond.

    PubMed

    Wegert, Anita; Hein, Martin; Reinke, Helmut; Hoffmann, Norbert; Miethchen, Ralf

    2006-11-27

    The dithionite-mediated addition of BrCF(2)Cl to 3,4-di-O-pivaloyl-D-xylal (1) generated preferably 1-CF(2)Cl-substituted products, that is, (2-bromo-2-deoxy-3,4-di-O-pivaloyl-beta-D-xylopyranosyl)-chlorodifluoromethane and (2-deoxy-3,4-di-O-pivaloyl-beta-D-threo-pentopyranosyl)-chlorodifluoromethane. Selected chlorodifluoromethyl-substituted monosaccharide derivatives were hydrodechlorinated or alkylated at the CF(2)Cl-group using tin reagents under radical reaction conditions. Thus, hydrodechlorinations of (2,3,4-tri-O-acetyl-6-deoxy-alpha-L-galactopyranosyl)-chlorodifluoromethane and of methyl 3,4-di-O-acetyl-2-C-chlorodifluoromethyl-2,6-dideoxy-alpha/beta-L-glucopyranoside are reported using tri-n-butyltin hydride initiated by AIBN. UV-initiated allylations are reported for reactions of (2-deoxy-3,4-di-O-pivaloyl-beta-D-threo-pentopyranosyl)-chlorodifluoromethane, (2,3,4-tri-O-acetyl-6-deoxy-alpha-L-galactopyranosyl)-chlorodifluoromethane, 1,3,4,6-tetra-O-acetyl-2-C-chlorodifluoromethyl-2-deoxy-alpha-D-glucopyranose, 1,3,4,6-tetra-O-acetyl-2-C-chlorodifluoromethyl-2-deoxy-alpha-D-mannopyranose and methyl 3,4-di-O-acetyl-2-C-chlorodifluoromethyl-2-deoxy-alpha/beta-D-rabinopyranoside with allyltri-n-butyltin.

  13. 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

  14. 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.

  15. Studies on the active site of pig plasma amine oxidase.

    PubMed Central

    Collison, D; Knowles, P F; Mabbs, F E; Rius, F X; Singh, I; Dooley, D M; Cote, C E; McGuirl, M

    1989-01-01

    Amine oxidase from pig plasma (PPAO) has two bound Cu2+ ions and at least one pyrroloquinoline quinone (PQQ) moiety as cofactors. It is shown that recovery of activity by copper-depleted PPAO is linear with respect to added Cu2+ ions. Recovery of e.s.r. and optical spectral characteristics of active-site copper parallel the recovery of catalytic activity. These results are consistent with both Cu2+ ions contributing to catalysis. Further e.s.r. studies indicate that the two copper sites in PPAO, unlike those in amine oxidases from other sources, are chemically distinct. These comparative studies establish that non-identity of the Cu2+ ions in PPAO is not a requirement for amine oxidase activity. It is shown through the use of a new assay procedure that there are two molecules of PQQ bound per molecule of protein in PPAO; only the more reactive of these PQQ moieties is required for activity. PMID:2559715

  16. 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

  17. Opioids and efflux transporters. Part 4: influence of N-substitution on P-glycoprotein substrate activity of noroxymorphone analogues.

    PubMed

    Metcalf, Matthew D; Rosicky, Andrew D; Hassan, Hazem E; Eddington, Natalie D; Coop, Andrew; Cunningham, Christopher W; Mercer, Susan L

    2014-08-01

    The efflux transporter protein P-glycoprotein (P-gp) is capable of affecting the central distribution of diverse neurotherapeutics, including opioid analgesics, through their active removal from the brain. P-gp located at the blood brain barrier has been implicated in the development of tolerance to opioids and demonstrated to be up-regulated in rats tolerant to morphine and oxycodone. We have previously examined the influence of hydrogen-bonding oxo-substitutents on the P-gp-mediated efflux of 4,5-epoxymorphinan analgesics, as well as that of N-substituted analogues of meperidine. Structure-activity relationships (SAR) governing N-substituent effects on opioid efficacy is well-established, however the influence of such structural modifications on P-gp-mediated efflux is unknown. Here, we present SAR describing P-gp recognition of a short series of N-modified 4,5-epoxymorphinans. Oxymorphone, naloxone, naltrexone, and nalmexone all failed to demonstrate P-gp substrate activity, indicating these opioid scaffolds contain structural features that preclude recognition by the transporter. These results are examined using mathematical molecular modeling and discussed in comparison to other opioid scaffolds bearing similar N-substituents. PMID:24915880

  18. 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

  19. Mutagenesis of Zinc Ligand Residue Cys221 Reveals Plasticity in the IMP-1 Metallo-β-Lactamase Active Site

    PubMed Central

    Horton, Lori B.; Shanker, Sreejesh; Mikulski, Rose; Brown, Nicholas G.; Phillips, Kevin J.; Lykissa, Ernest; Venkataram Prasad, B. V.

    2012-01-01

    Metallo-β-lactamases catalyze the hydrolysis of a broad range of β-lactam antibiotics and are a concern for the spread of drug resistance. To analyze the determinants of enzyme structure and function, the sequence requirements for the subclass B1 IMP-1 β-lactamase zinc binding residue Cys221 were tested by saturation mutagenesis and evaluated for protein expression, as well as hydrolysis of β-lactam substrates. The results indicated that most substitutions at position 221 destabilized the enzyme. Only the enzymes containing C221D and C221G substitutions were expressed well in Escherichia coli and exhibited catalytic activity toward β-lactam antibiotics. Despite the lack of a metal-chelating group at position 221, the C221G enzyme exhibited high levels of catalytic activity in the presence of exogenous zinc. Molecular modeling suggests the glycine substitution is unique among substitutions in that the complete removal of the cysteine side chain allows space for a water molecule to replace the thiol and coordinate zinc at the Zn2 zinc binding site to restore function. Multiple methods were used to estimate the C221G Zn2 binding constant to be 17 to 43 μM. Studies of enzyme function in vivo in E. coli grown on minimal medium showed that both IMP-1 and the C221G mutant exhibited compromised activity when zinc availability was low. Finally, substitutions at residue 121, which is the IMP-1 equivalent of the subclass B3 zinc-chelating position, failed to rescue C221G function, suggesting the coordination schemes of subclasses B1 and B3 are not interchangeable. PMID:22908171

  20. Stabilized G protein binding site in the structure of constitutively active metarhodopsin-II.

    PubMed

    Deupi, Xavier; Edwards, Patricia; Singhal, Ankita; Nickle, Benjamin; Oprian, Daniel; Schertler, Gebhard; Standfuss, Jörg

    2012-01-01

    G protein-coupled receptors (GPCR) are seven transmembrane helix proteins that couple binding of extracellular ligands to conformational changes and activation of intracellular G proteins, GPCR kinases, and arrestins. Constitutively active mutants are ubiquitously found among GPCRs and increase the inherent basal activity of the receptor, which often correlates with a pathological outcome. Here, we have used the M257Y(6.40) constitutively active mutant of the photoreceptor rhodopsin in combination with the specific binding of a C-terminal fragment from the G protein alpha subunit (GαCT) to trap a light activated state for crystallization. The structure of the M257Y/GαCT complex contains the agonist all-trans-retinal covalently bound to the native binding pocket and resembles the G protein binding metarhodopsin-II conformation obtained by the natural activation mechanism; i.e., illumination of the prebound chromophore 11-cis-retinal. The structure further suggests a molecular basis for the constitutive activity of 6.40 substitutions and the strong effect of the introduced tyrosine based on specific interactions with Y223(5.58) in helix 5, Y306(7.53) of the NPxxY motif and R135(3.50) of the E(D)RY motif, highly conserved residues of the G protein binding site.

  1. Participation and strength of interaction of lysine 95(beta) in the polymerization of hemoglobin S as determined by its site-directed substitution by isoleucine.

    PubMed

    Himanen, J P; Schneider, K; Chait, B; Manning, J M

    1995-06-01

    The role of Lys-95(beta), which is on the exterior of the hemoglobin (HbS) tetramer, in the aggregation process has been addressed because there is a lack of agreement on its importance. The early studies on the aggregation of HbS in the presence of other mutant hemoglobins are consistent with the subsequent electron microscopic studies in demonstrating the participation of Lys-95(beta) in gelation; the results of the crystal structure do not agree with these conclusions. Therefore, with the objective of clarifying its role we have carried out site-directed substitution of Lys-95(beta) to an isoleucine residue. The mutation was introduced by polymerase chain reaction recombination methodology, and the absence of other mutations in the beta-globin gene was established by sequencing the gene in its entirety. The recombinant mutant hemoglobin was expressed in yeast and characterized by peptide mapping and sequencing, which demonstrated that the only different tryptic peptide had the Ile substitution at position 95(beta). The recombinant hemoglobin had the correct amino acid composition and molecular weight by mass spectrometric analysis. It was also pure as judged by isoelectric focusing. It was fully functional because it had an average Hill coefficient of 3.1 and responded normally to the allosteric regulators, chloride, 2,3-diphosphoglycerate, and inositol hexaphosphate. Of particular interest was the finding that this hemoglobin mutant aggregated at a concentration of about 40 g/dl, nearly twice that at which HbS itself aggregated (24 g/dl). Therefore, Lys-95(beta) has a very important role in the aggregation process and is a good candidate site for the design of a therapeutic agent for sickle cell anemia. PMID:7775447

  2. Synthesis and evaluation of M. tuberculosis salicylate synthase (MbtI) inhibitors designed to probe plasticity in the active site.

    PubMed

    Manos-Turvey, Alexandra; Cergol, Katie M; Salam, Noeris K; Bulloch, Esther M M; Chi, Gamma; Pang, Angel; Britton, Warwick J; West, Nicholas P; Baker, Edward N; Lott, J Shaun; Payne, Richard J

    2012-12-14

    Mycobacterium tuberculosis salicylate synthase (MbtI) catalyses the first committed step in the biosynthesis of mycobactin T, an iron-chelating siderophore essential for the virulence and survival of M. tuberculosis. Co-crystal structures of MbtI with members of a first generation inhibitor library revealed large inhibitor-induced rearrangements within the active site of the enzyme. This plasticity of the MbtI active site was probed via the preparation of a library of inhibitors based on a 2,3-dihydroxybenzoate scaffold with a range of substituted phenylacrylate side chains appended to the C3 position. Most compounds exhibited moderate inhibitory activity against the enzyme, with inhibition constants in the micromolar range, while several dimethyl ester variants possessed promising anti-tubercular activity in vitro. PMID:23108268

  3. Computer simulation of the active site of human serum cholinesterase

    SciTech Connect

    Kefang Jiao; Song Li; Zhengzheng Lu

    1996-12-31

    The first 3D-structure of acetylchelinesterase from Torpedo California electric organ (T.AChE) was published by JL. Sussman in 1991. We have simulated 3D-structure of human serum cholinesterase (H.BuChE) and the active site of H.BuChE. It is discovered by experiment that the residue of H.BuChE is still active site after a part of H.BuChE is cut. For example, the part of 21KD + 20KD is active site of H.BuChE. The 20KD as it is. Studies on these peptides by Hemelogy indicate that two active peptides have same negative electrostatic potential maps diagram. These negative electrostatic areas attached by acetyl choline with positive electrostatic potency. We predict that 147...236 peptide of AChE could be active site because it was as 20KD as with negative electrostatic potential maps. We look forward to proving from other ones.

  4. N-methyl-substituted fluorescent DAG-indololactone isomers exhibit dramatic differences in membrane interactions and biological activity

    PubMed Central

    Gal, Noga; Kolusheva, Sofiya; Kedei, Noemi; Telek, Andrea; Naeem, Taiyabah A.; Lewin, Nancy E.; Lim, Langston; Mannan, Poonam; Garfield, Susan H.; Kazzouli, Säid El; Sigano, Dina M.; Marquez, Victor E.; Blumberg, Peter M.

    2013-01-01

    N-methyl substituted diacylglycerol-indololactones (DAG-indololactones) are newly-synthesized effectors of protein kinase C (PKC) isoforms and exhibit substantial selectivity between RasGRP3 and PKC alpha. We present a comprehensive analysis of membrane interactions and biological activities of several DAG-indololactones. Translocation and binding activity assays underline significant variations between the PKC translocation characteristics affected by the ligands as compared to their binding activities. In parallel, the fluorescent properties of the ligands were employed for analysis of their membrane association profiles. Specifically, we find that a slight change in the linkage to the indole ring resulted in significant differences in membrane binding and association of the DAG-indololactones with lipid bilayers. Our analysis shows that seemingly small structural modifications of the hydrophobic regions of these biomimetic PKC effectors contribute to pronounced modulation of membrane interactions of the ligands PMID:23106081

  5. 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.

  6. Structure-Activity study of retinoid agonists bearing substituted dicarba-closo-dodecaborane. Relation between retinoidal activity and conformation of two aromatic nuclei.

    PubMed

    Endo, Y; Iijima, T; Yaguchi, K; Kawachi, E; Inoue, N; Kagechika, H; Kubo, A; Itai, A

    2001-05-21

    We have investigated the structure activity relationships of the potent retinoid agonist, 4-[4-(2-propyl-1,2-dicarba-closo-dodecaboran-l-yl)phenylamino]benzoic acid (BR403), which we have previously reported. Substitution of a methyl group on the aromatic nucleus or a methyl group on the nitrogen atom, or replacement of the amino group with ether, methylene, carboxyl or 1,1-ethylene greatly decreased the activity. The relatively planar conformation at the phenyl-N-phenyl moiety seems to play a critical role in the appearance of the biological activity.

  7. Epidermolytic palmoplantar keratoderma caused by activation of a cryptic splice site in KRT9.

    PubMed

    Fuchs-Telem, D; Padalon-Brauch, G; Sarig, O; Sprecher, E

    2013-03-01

    Epidermolytic palmoplantar keratoderma (EPPK) is caused by mutations in KRT9 and less often, KRT1. All known mutations in KRT9 have been found in regions of the gene encoding the conserved central α-helix rod domain. In the present study, we investigated the molecular basis of EPPK in a patient of Ashkenazi Jewish origin. The patient was found to carry a novel missense mutation in KRT9, resulting in the substitution of a poorly conserved leucine for valine at position 11 of the amino acid sequence. Despite its unusual location, the mutation was shown to be pathogenic through activation of a cryptic donor splice site, resulting in the deletion of 162 amino acids. The present data indicate the need to screen keratin genes in their entirety, as mutations altering domains of lesser functional importance may exert their deleterious effect at the transcriptional level.

  8. 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.

  9. 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

  10. 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.

  11. Multi-site Phosphorylation Regulates Bim Stability and Apoptotic Activity

    PubMed Central

    Hübner, Anette; Barrett, Tamera; Flavell, Richard A.; Davis, Roger J.

    2008-01-01

    The pro-apoptotic BH3-only protein Bim is established to be an important mediator of signaling pathways that induce cell death. Multi-site phosphorylation of Bim by several members of the MAP kinase group is implicated as a regulatory mechanism that controls the apoptotic activity of Bim. To test the role of Bim phosphorylation in vivo, we constructed mice with a series of mutant alleles that express phosphorylation-defective Bim proteins. We show that mutation of the phosphorylation site Thr-112 causes decreased binding of Bim to the anti-apoptotic protein Bcl2 and can increase cell survival. In contrast, mutation of the phosphorylation sites Ser-55, Ser-65, and Ser-73 can cause increased apoptosis because of reduced proteasomal degradation of Bim. Together, these data indicate that phosphorylation can regulate Bim by multiple mechanisms and that the phosphorylation of Bim on different sites can contribute to the sensitivity of cellular apoptotic responses. PMID:18498746

  12. 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

  13. 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.

  14. Activation of phenylalanine hydroxylase by phenylalanine does not require binding in the active site.

    PubMed

    Roberts, Kenneth M; Khan, Crystal A; Hinck, Cynthia S; Fitzpatrick, Paul F

    2014-12-16

    Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein's regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The k(cat)/K(phe) value is down 10⁴ for the mutant enzyme, and the K(m) value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain.

  15. 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.

  16. Binding of Mn-deoxyribonucleoside triphosphates to the active site of the DNA polymerase of bacteriophage T7

    PubMed Central

    Akabayov, Barak; Richardson, Charles C.

    2013-01-01

    Divalent metal ions are crucial as cofactors for a variety of intracellular enzymatic activities. Mg2+, 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 Mg2+ to an active site because Mg2+ is spectroscopically silent and Mg2+ binds with low affinity to the active site of an enzyme. Therefore, we substituted Mg2+ with Mn2+:Mn2+ 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 Mn2+ 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 Mn2+ that is free in solution and Mn2+ 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. PMID:23761703

  17. 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…

  18. 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.

  19. 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

  20. Site-specific conformational determination in thermal unfolding studies of helical peptides using vibrational circular dichroism with isotopic substitution

    PubMed Central

    Silva, R. A. G. D.; Kubelka, Jan; Bour, Petr; Decatur, Sean M.; Keiderling, Timothy A.

    2000-01-01

    Understanding the detailed mechanism of protein folding requires dynamic, site-specific stereochemical information. The short time response of vibrational spectroscopies allows evaluation of the distribution of populations in rapid equilibrium as the peptide unfolds. Spectral shifts associated with isotopic labels along with local stereochemical sensitivity of vibrational circular dichroism (VCD) allow determination of the segment sequence of unfolding. For a series of alanine-rich peptides that form α-helices in aqueous solution, we used isotopic labeling and VCD to demonstrate that the α-helix noncooperatively unwinds from the ends with increasing temperature. For these blocked peptides, the C-terminal is frayed at 5°C. Ab initio level theoretical simulations of the IR and VCD band shapes are used to analyze the spectra and to confirm the conformation of the labeled components. The VCD signals associated with the labeled residues are amplified by coupling to the nonlabeled parts of the molecule. Thus small labeled segments are detectable and stereochemically defined in moderately large peptides in this report of site-specific peptide VCD conformational analysis. PMID:10880566

  1. Conformational Transitions in Human AP Endonuclease 1 and Its Active Site Mutant during Abasic Site Repair†

    PubMed Central

    Kanazhevskaya, Lyubov Yu.; Koval, Vladimir V.; Zharkov, Dmitry O.; Strauss, Phyllis R.; Fedorova, Olga S.

    2010-01-01

    AP endonuclease 1 (APE 1) is a crucial enzyme of the base excision repair pathway (BER) in human cells. APE1 recognizes apurinic/apyrimidinic (AP) sites and makes a nick in the phosphodiester backbone 5′ to them. The conformational dynamics and presteady-state kinetics of wild-type APE1 and its active site mutant, Y171F-P173L-N174K, have been studied. To observe conformational transitions occurring in the APE1 molecule during the catalytic cycle, we detected intrinsic tryptophan fluorescence of the enzyme under single turnover conditions. DNA duplexes containing a natural AP site, its tetrahydrofuran analogue, or a 2′-deoxyguanosine residue in the same position were used as specific substrates or ligands. The stopped-flow experiments have revealed high flexibility of the APE1 molecule and the complexity of the catalytic process. The fluorescent traces indicate that wild-type APE1 undergoes at least four conformational transitions during the processing of abasic sites in DNA. In contrast, nonspecific interactions of APE1 with undamaged DNA can be described by a two-step kinetic scheme. Rate and equilibrium constants were extracted from the stopped-flow and fluorescence titration data for all substrates, ligands, and products. A replacement of three residues at the enzymatic active site including the replacement of tyrosine 171 with phenylalanine in the enzyme active site resulted in a 2 × 104-fold decrease in the reaction rate and reduced binding affinity. Our data indicate the important role of conformational changes in APE1 for substrate recognition and catalysis. PMID:20575528

  2. Role of arginine-304 in the diphosphate-triggered active site closure mechanism of trichodiene synthase.

    PubMed

    Vedula, L Sangeetha; Cane, David E; Christianson, David W

    2005-09-27

    The X-ray crystal structures of R304K trichodiene synthase and its complexes with inorganic pyrophosphate (PP(i)) and aza analogues of the bisabolyl carbocation intermediate are reported. The R304K substitution does not cause large changes in the overall structure in comparison with the wild-type enzyme. The complexes with (R)- and (S)-azabisabolenes and PP(i) bind three Mg2+ ions, and each undergoes a diphosphate-triggered conformational change that caps the active site cavity. This conformational change is only slightly attenuated compared to that of the wild-type enzyme complexed with Mg2+(3)-PP(i), in which R304 donates hydrogen bonds to PP(i) and D101. In R304K trichodiene synthase, K304 does not engage in any hydrogen bond interactions in the unliganded state and it donates a hydrogen bond to only PP(i) in the complex with (R)-azabisabolene; K304 makes no hydrogen bond contacts in its complex with PP(i) and (S)-azabisabolene. Thus, although the R304-D101 hydrogen bond interaction stabilizes diphosphate-triggered active site closure, it is not required for Mg2+(3)-PP(i) binding. Nevertheless, since R304K trichodiene synthase generates aberrant cyclic terpenoids with a 5000-fold reduction in kcat/KM, it is clear that a properly formed R304-D101 hydrogen bond is required in the enzyme-substrate complex to stabilize the proper active site contour, which in turn facilitates cyclization of farnesyl diphosphate for the exclusive formation of trichodiene. Structural analysis of the R304K mutant and comparison with the monoterpene cyclase (+)-bornyl diphosphate synthase suggest that the significant loss in activity results from compromised activation of the PP(i) leaving group. PMID:16171386

  3. Role of Arginine-304 in the Diphosphate-Triggered Active Site Closure Mechanism of Trichodiene Synthase

    SciTech Connect

    Vedula,L.; Cane, D.; Christianson, D.

    2005-01-01

    The X-ray crystal structures of R304K trichodiene synthase and its complexes with inorganic pyrophosphate (PPi) and aza analogues of the bisabolyl carbocation intermediate are reported. The R304K substitution does not cause large changes in the overall structure in comparison with the wild-type enzyme. The complexes with (R)- and (S)-azabisabolenes and PPi bind three Mg2+ ions, and each undergoes a diphosphate-triggered conformational change that caps the active site cavity. This conformational change is only slightly attenuated compared to that of the wild-type enzyme complexed with Mg{sup 2+}{sub 3-}PP{sub i}, in which R304 donates hydrogen bonds to PP{sub i} and D101. In R304K trichodiene synthase, K304 does not engage in any hydrogen bond interactions in the unliganded state and it donates a hydrogen bond to only PP{sub i} in the complex with (R)-azabisabolene; K304 makes no hydrogen bond contacts in its complex with PP{sub i} and (S)-azabisabolene. Thus, although the R304-D101 hydrogen bond interaction stabilizes diphosphate-triggered active site closure, it is not required for Mg{sup 2+}{sub 3-}PP{sub i} binding. Nevertheless, since R304K trichodiene synthase generates aberrant cyclic terpenoids with a 5000-fold reduction in kcat/KM, it is clear that a properly formed R304-D101 hydrogen bond is required in the enzyme-substrate complex to stabilize the proper active site contour, which in turn facilitates cyclization of farnesyl diphosphate for the exclusive formation of trichodiene. Structural analysis of the R304K mutant and comparison with the monoterpene cyclase (+)-bornyl diphosphate synthase suggest that the significant loss in activity results from compromised activation of the PP{sub i} leaving group.

  4. N-methyl-D-aspartate recognition site ligands modulate activity at the coupled glycine recognition site.

    PubMed

    Hood, W F; Compton, R P; Monahan, J B

    1990-03-01

    In synaptic plasma membranes from rat forebrain, the potencies of glycine recognition site agonists and antagonists for modulating [3H]1-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) binding and for displacing strychnine-insensitive [3H]glycine binding are altered in the presence of N-methyl-D-aspartate (NMDA) recognition site ligands. The NMDA competitive antagonist, cis-4-phosphonomethyl-2-piperidine carboxylate (CGS 19755), reduces [3H]glycine binding, and the reduction can be fully reversed by the NMDA recognition site agonist, L-glutamate. Scatchard analysis of [3H]glycine binding shows that in the presence of CGS 19755 there is no change in Bmax (8.81 vs. 8.79 pmol/mg of protein), but rather a decrease in the affinity of glycine (KD of 0.202 microM vs. 0.129 microM). Similar decreases in affinity are observed for the glycine site agonists, D-serine and 1-aminocyclopropane-1-carboxylate, in the presence of CGS 19755. In contrast, the affinity of glycine antagonists, 1-hydroxy-3-amino-2-pyrrolidone and 1-aminocyclobutane-1-carboxylate, at this [3H]glycine recognition site increases in the presence of CGS 19755. The functional consequence of this change in affinity was addressed using the modulation of [3H]TCP binding. In the presence of L-glutamate, the potency of glycine agonists for the stimulation of [3H]TCP binding increases, whereas the potency of glycine antagonists decreases. These data are consistent with NMDA recognition site ligands, through their interactions at the NMDA recognition site, modulating activity at the associated glycine recognition site.

  5. Intragenic suppression of an active site mutation in the human apurinic/apyrimidinic endonuclease.

    PubMed

    Izumi, T; Malecki, J; Chaudhry, M A; Weinfeld, M; Hill, J H; Lee, J C; Mitra, S

    1999-03-19

    The apurinic/apyrimidinic endonucleases (APE) contain several highly conserved sequence motifs. The glutamic acid residue in a consensus motif, LQE96TK98 in human APE (hAPE-1), is crucial because of its role in coordinating Mg2+, an essential cofactor. Random mutagenesis of the inactive E96A mutant cDNA, followed by phenotypic screening in Escherichia coli, led to isolation of an intragenic suppressor with a second site mutation, K98R. Although the Km of the suppressor mutant was about sixfold higher than that of the wild-type enzyme, their kcat values were similar for AP endonuclease activity. These results suggest that the E96A mutation affects only the DNA-binding step, but not the catalytic step of the enzyme. The 3' DNA phosphoesterase activities of the wild-type and the suppressor mutant were also comparable. No global change of the protein conformation is induced by the single or double mutations, but a local perturbation in the structural environment of tryptophan residues may be induced by the K98R mutation. The wild-type and suppressor mutant proteins have similar Mg2+ requirement for activity. These results suggest a minor perturbation in conformation of the suppressor mutant enabling an unidentified Asp or Glu residue to substitute for Glu96 in positioning Mg2+ during catalysis. The possibility that Asp70 is such a residue, based on its observed proximity to the metal-binding site in the wild-type protein, was excluded by site-specific mutation studies. It thus appears that another acidic residue coordinates with Mg2+ in the mutant protein. These results suggest a rather flexible conformation of the region surrounding the metal binding site in hAPE-1 which is not obvious from the X-ray crystallographic structure. PMID:10074406

  6. Properties of a recombinant human hemoglobin with aspartic acid 99(beta), an important intersubunit contact site, substituted by lysine.

    PubMed Central

    Yanase, H.; Cahill, S.; Martin de Llano, J. J.; Manning, L. R.; Schneider, K.; Chait, B. T.; Vandegriff, K. D.; Winslow, R. M.; Manning, J. M.

    1994-01-01

    Site-directed mutagenesis of an important subunit contact site, Asp-99(beta), by a Lys residue (D99K(beta)) was proven by sequencing the entire beta-globin gene and the mutant tryptic peptide. Oxygen equilibrium curves of the mutant hemoglobin (Hb) (2-15 mM in heme) indicated that it had an increased oxygen affinity and a lowered but significant amount of cooperativity compared to native HbA. However, in contrast to normal HbA, oxygen binding of the recombinant mutant Hb was only marginally affected by the allosteric regulators 2,3-diphosphoglycerate or inositol hexaphosphate and was not at all responsive to chloride. The efficiency of oxygen binding by HbA in the presence of allosteric regulators was limited by the mutant Hb. At concentrations of 0.2 mM or lower in heme, the mutant D99K(beta) Hb was predominantly a dimer as demonstrated by gel filtration, haptoglobin binding, fluorescence quenching, and light scattering. The purified dimeric recombinant Hb mutant exists in 2 forms that are separable on isoelectric focusing by about 0.1 pH unit, in contrast to tetrameric hemoglobin, which shows 1 band. These mutant forms, which were present in a ratio of 60:40, had the same masses for their heme and globin moieties as determined by mass spectrometry. The elution positions of the alpha- and beta-globin subunits on HPLC were identical. Circular dichroism studies showed that one form of the mutant Hb had a negative ellipticity at 410 nm and the other had positive ellipticity at this wavelength. The findings suggest that the 2 D99K(beta) recombinant mutant forms have differences in their heme-protein environments. PMID:7987216

  7. 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.

  8. Studies of Antimicrobial Activities of some 4-Thiazolidinone Fused Pyrimidines, [1,5]-Benzodiazepines and their Oxygen Substituted Hydroxylamine Derivatives.

    PubMed

    Singh, Bhawani; Maheshwari, A; Dak, G; Sharma, K; Talesara, G L

    2010-09-01

    Thiazolidin-4-one fused pyrimidines, [1,5]-benzodiazepines and their oxygen substituted hydroxylamine derivatives have been screened for antibacterial, antifungal and antimalarial activity. Bacillus subtilis, Escherichia coli, Proteus mirabilis and Salmonella typhi were used for antibacterial screening. Aspergillus fumigatus and Candida albicans were used for antifungal screening and Plasmodium species were used for antimalarial screening. The antibacterial and antifungal activities are expressed in terms of zone of inhibition and antimalarial activity is expressed in IC(50) value. Fifteen compounds 2Xa, 2Xb, 2Xc, 2Xs, 3IV, 3Va, 3Vc, 3VIIIa, 3VIIIh, 3IXa, 3IXb, 3IXc, 3Xa, 4IXa and 4Xa were tested for antibacterial as well as antifungal activity and seven compounds 2IXb, 2Xb, 3VIIIc, 3Xc, 4IXa, 4Xa and 4IXw were tested for antimalarial activity. Streptomycin, griseofulvin and chloroquine were taken as standard drugs in antibacterial, antifungal and antimalarial activity, respectively. The compound 2Xs was found significant antimicrobial against Bacillus subtilis, E. coli, Aspergillus fumigatus and Candida albicans as well as compound 3Xa was significant antimicrobial against Bacillus subtilis, E. coli, Salmonella typhi, Aspergillus fumigatus and Candida albicans. The compound 2Xb showed significant antimalarial activity.

  9. Control of active sites in selective flocculation: I -- Mathematical model

    SciTech Connect

    Behl, S.; Moudgil, B.M.; Prakash, T.S. . Dept. of Materials Science and Engineering)

    1993-12-01

    Heteroflocculation has been determined to be another major reason for loss in selectivity for flocculation process. In a mathematical model developed earlier, conditions for controlling heteroflocculation were discussed. Blocking active sites to control selective adsorption of a flocculant oil a desirable solid surface is discussed. It has been demonstrated that the lower molecular weight fraction of a flocculant which is incapable of flocculating the particles is an efficient site blocking agent. The major application of selective flocculation has been in mineral processing but many potential uses exist in biological and other colloidal systems. These include purification of ceramic powders, separating hazardous solids from chemical waste, and removal of deleterious components from paper pulp.

  10. 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.

  11. The site of activation of factor X by cancer procoagulant.

    PubMed

    Gordon, S G; Mourad, A M

    1991-12-01

    Cancer procoagulant (CP) is a cysteine proteinase found in a variety of malignant cells and tissues and in human amnion-chorion tissue. It initiates coagulation by activating factor X. However, the amino acid sequence of the substrate protein that determines the cleavage site of cysteine proteinases is different from that of the serine proteinases that normally activate factor X, such as factor IXa, VIIa and Russell's Viper Venom (RVV). Therefore, it was of interest to determine the site of cleavage of human factor X by CP. Purified CP was incubated with purified factor X and the reaction mixture was electrophoresed on a 10% Tris-tricine SDS-PAGE gel. The proteins were electroeluted on to a polyvinylidene difluoride (PVDF) membrane, and stained with Coomassie blue. The heavy chain of activated factor X was cut out of the PVDF membrane and sequenced with an Applied Biosystems 477A with on-line HPLC. The primary cleavage sequence was Asp-Ala-Ala-Asp-Leu-Asp-Pro-; two other secondary sequences Ser-Ile-Thr-Trp-Lys-Pro- and Glu-Asn-Pro-Phe-Asp-Leu were found. The penultimate amino acid on the carbonyl side of the hydrolysed amide bond plays a critical role for the recognition of the cleavage site of cysteine proteinases. These data indicate that the penultimate amino acid for the primary cleavage site of factor X by CP is proline-20 and for the secondary sites, proline-13 and proline-28. This is in contrast to arginine-52 that determines the specificity of the cleavage by normal serine proteinase activation.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. NO donors, part 8 [1]: synthesis and vasodilating activities of substituted benzylnitrates compared to cyclohexylmethylnitrate and GTN.

    PubMed

    Wessler, Christoph; Homann, Alexander; Fricke, Uwe; Lehmann, Jochen

    2003-06-01

    A series of substituted benzylnitrates (1) and the formally but not chemically similar cyclohexylmethylnitrate (CHMN) have been synthesised. Vasodilating activities were measured on endothelium-intact and N(G)-nitro-L-arginine (L-NNA)-blocked porcine right coronary arteries, precontracted with prostaglandin F(2alpha) (PGF(2alpha)). Glyceroltrinitrate (GTN) was used as reference. In intact coronary arteries the vasodilating activities of all benzylnitrates are lower compared with GTN, but higher compared with CHMN. However, blocking the function of the endothelium by L-NNA, the activity of all benzylnitrates increased, whereas that of CHMN and GTN remained nearly unaffected. Under these conditions, the mononitrates 4-nitro-benzylnitrate (1c) and 4-nitrooxymethyl-benzonitrile (1h) even showed higher vasodilator activities than the trinitrate GTN and in general, vasorelaxation by the benzylnitrates as defined by the concentrations for half maximal effects (EC(50) values) was found to be 2-3 orders of magnitude higher than that induced by CHMN. The study demonstrates that the in vitro activities of organic nitrates do not correlate with the number of nitrate groups within the molecule nor to the lipophilicity of the molecules. Instead, vasodilator activity is highly sensitive to the structure and the type of the substituents in the molecular carrier of the nitrate group.

  19. 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.

  20. Functional constituents of the active site of human neutrophil collagenase.

    PubMed

    Mookhtiar, K A; Wang, F; Van Wart, H E

    1986-05-01

    A series of chemical modification reactions has been carried out to identify functional constituents of the active site of human neutrophil collagenase. The enzyme is reversibly inhibited by the transition metal chelating agent 1,10-phenanthroline, and inhibition is fully reversed by zinc. Removal of weakly bound metal ions by gel filtration inactivates collagenase, and activity is fully restored on immediate readdition of calcium. The enzyme is unaffected by reagents that modify serine, cysteine, and arginine residues. However, reaction with the carboxyl reagents cyclohexylmorpholinocarbodiimide and Woodward's Reagent K lowers the activity of the enzyme substantially. Acetylimidazole inactivates the enzyme, but activity is completely restored on addition of hydroxylamine. The enzyme is also inactivated by tetranitromethane, indicating that it contains an essential tyrosine residue. Acylation of collagenase with diethyl pyrocarbonate, diketene, acetic anhydride, or trinitrobenzenesulfonate inactivates the enzyme, and activity is not restored on addition of hydroxylamine, indicating the presence of an essential lysine residue.

  1. [Aluminum coordination and active sites on aluminas, Y-zeolites and pillared layered silicates]. Progress report

    SciTech Connect

    Fripiat, J.J.

    1994-02-01

    This report is organized in four sections. In the first the authors will outline structural features which are common to all fine grained alumina, as well as to non-framework alumina in zeolites. This section will be followed by a study of the surface vs. bulk coordination of aluminum. The third section will deal with measurement of the number of acid sites and the scaling of their strength. The fourth and last section will describe three model reactions: the isomerization of 1-butene and of 2 cis-butene; the isomerization and disproportionation of oxtho-xylene; and the transformation of trichloroethane into vinyl chloride followed by the polymerization of the vinyl chloride. The relationship between chemical activity and selectivity and what is known of the local structure of the active catalytic sites will be underlined. Other kinds of zeolites besides Y zeolite have been studied. Instead of the aluminum pillared silicates they found it more interesting to study the substitution of silicon by aluminum in a layered structure containing a permanent porosity (aluminated sepiolite).

  2. 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

  3. 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.

  4. 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

    Casey, Ashley K; Hicks, Michael A; Johnson, Jordyn L; Babbitt, Patricia C; Frantom, Patrick A

    2014-05-13

    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.

  5. 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.

  6. 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

  7. 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.

  8. Active Sites Environmental Monitoring Program: FY 1991 report

    SciTech Connect

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

    1992-11-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) from April 1991 through September 1991. The ASEMP was established in 1989 by Solid Waste Operations (SWO) and the Environmental Sciences Division, both of Oak Ridge National Laboratory, 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. A new set of action levels was developed on the basis of a statistical analysis of background contamination. These new action levels have been used to evaluate results in this report. Results of ASEMP monitoring continue to demonstrate that no LLW (except [sup 3]H) is being leached from the storage vaults on the tumulus pads. Loading of vaults on Tumulus II, which began in early FY 1991, was >90% complete at the end of September 1991. Results of sampling of groundwater and surface waters is presented.

  9. Inhibition and active-site modelling of prolidase.

    PubMed

    King, G F; Crossley, M J; Kuchel, P W

    1989-03-15

    Consideration of the active-site model of prolidase led us to examine azetidine, pyrrolidine and piperidine substrate analogs as potential in vivo inhibitors of the enzyme. One of these, N-benzyloxycarbonyl-L-proline, was shown to be a potent competitive inhibitor of porcine kidney prolidase (Ki = 90 microM); its rapid protein-mediated permeation of human and sheep erythrocytes suggests that it may be effective in vivo. The higher homolog, N-benzyloxycarbonyl-L-pipecolic acid, was also a potent inhibitor of the enzyme while the antihypertensive drugs, captopril and enalaprilat, were shown to have mild and no inhibitory effects, respectively. Analysis of inhibitor action and consideration of X-ray crystallographic data of relevant Mn2+ complexes allowed the active-site model of prolidase to be further refined; a new model is presented in which the substrate acts as a bidentate ligand towards the active-site manganous ion. Various aspects of the new model help to explain why Mn2+ has been 'chosen' by the enzyme in preference to other biologically available metal ions. PMID:2924773

  10. 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…

  11. 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

    2014-06-30

    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.

  12. Preparation and root growth-inhibitory activity of N-substituted 2-(2-chloroacetamido)-3-(furan-2-yl)propanamides.

    PubMed

    Kitagawa, Tokujiro; Asada, Makoto

    2005-09-01

    A series of N-substituted 2-(2-chloroacetamido)-3-(furan-2-yl)propanamides (16--18) was prepared through the reaction of chloroacetyl chloride with N-substituted 2-amino-3-(furan-2-yl)propanamides (15), which were obtained via condensation of 2-(tert-butoxycarbonylamido)-3-(furan-2-yl)propanoic acid (Boc-furylalanine) (8) with amines (9, 11, 13), followed by hydrolysis of the resultant N-substituted Boc-furylalanine acid amides (10, 12, 14) in the presence of HCl/dioxane. The biological activity of the prepared 16, 17 and 18 as root growth inhibitors was examined by germination assay using rape seed. At the concentration of 5.0 x10 (-5) M, the most active compound, 2-(2-chloroacetamido)-N-(2,6-diethylphenyl)-3-(furan-2-yl)propanamide (16 n), showed potent root growth-inhibitory activity of 76% towards rape seedlings.

  13. 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.

  14. 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

  15. Structure-In Vitro Activity Relationships of Pentamidine Analogues and Dication-Substituted Bis-Benzimidazoles as New Antifungal Agents

    PubMed Central

    Del Poeta, Maurizio; Schell, Wiley A.; Dykstra, Christine C.; Jones, Susan; Tidwell, Richard R.; Czarny, Agnieszka; Bajic, Miroslav; Bajic, Marina; Kumar, Arvind; Boykin, David; Perfect, John R.

    1998-01-01

    Twenty analogues of pentamidine, 7 primary metabolites of pentamidine, and 30 dicationic substituted bis-benzimidazoles were screened for their inhibitory and fungicidal activities against Candida albicans and Cryptococcus neoformans. A majority of the compounds had MICs at which 80% of the strains were inhibited (MIC80s) comparable to those of amphotericin B and fluconazole. Unlike fluconazole, many of these compounds were found to have potent fungicidal activity. The most potent compound against C. albicans had an MIC80 of ≤0.09 μg/ml, and the most potent compound against C. neoformans had an MIC80 of 0.19 μg/ml. Selected compounds were also found to be active against Aspergillus fumigatus, Fusarium solani, Candida species other than C. albicans, and fluconazole-resistant strains of C. albicans and C. neoformans. It is clear from the data presented here that further studies on the structure-activity relationships, mechanisms of action and toxicities, and in vivo efficacies of these compounds are warranted to determine their clinical potential. PMID:9756747

  16. 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

  17. Synthesis and biological activity of some 5-substituted aminomethyl-8-hydroxyquinoline-7-sulphonic acids.

    PubMed

    Yanni, A S; Mohharam, A M

    1990-01-01

    5-Aryl (or alkyl)-8-hydroxyquinoline-7-sulphonic acids have been prepared by the Mannich reaction of 8-hydroxyquinoline-7-sulphonic acid with primary and secondary amines. Their bactericidal activities have been determined.

  18. 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

  19. Catechol oxidase activity of di-Cu2+-substituted aminopeptidase from Streptomyces griseus.

    PubMed

    da Silva, Giordano F Z; Ming, Li-June

    2005-11-30

    Streptomyces griseus aminopeptidase exhibits activities toward the hydrolyses of peptides and bis(p-nitrophenyl)phosphate (40 billion fold) and catechol oxidation reported herein with catalytic efficiency (kcat/Km) only about 10 times smaller than that of gypsywort catechol oxidase. The multifunctionality of this enzyme suggests that it is a unique system for further exploration of protein structure and function and a template for design of enzymes of diverse activities. PMID:16305209

  20. The loss of a polymorphic glycosylation site caused by Thr-927 [yields] Ile is linked to a second polymorphic Val-816 [yields] Ile substitution in lysosomal [alpha]-glucosidase of American blacks

    SciTech Connect

    Hermans, M.M.P.; Oostra, B.A.; Reuser, A.J.J. ); Svetkey, L.P.; Chen, Y.T. )

    1993-04-01

    Inherited deficiency of the lysosomal hydrolase [alpha]-glucosidase (acid maltase) causes lysosomal accumulation of glycogen, a condition known as glycogen storage disease type II (GSD II). The clinical phenotype is heterogeneous, and heterogeneity also exists at the molecular level. Point mutations were recently identified in two cases of severe infantile and one case of adult GSD II. In addition to mutations with a deleterious effect on enzyme function, several polymorphisms have been documented. Of note are those polymorphisms that relate to racial differences, but they can pose a problem when mutant alleles are being analyzed. An example of this was an American black patient with an adult form of GSD II (GM1935). Mutation analysis revealed three amino acid substitutions that were not encountered in the Caucasian population. The three substitutions were Asp-645 [r arrow] Glu (exon 14), Val-816 [r arrow] Ile (exon 17), and Thr-927 [r arrow] Ile (exon 19), and they were linked to the same allele . The Thr-927 [r arrow] Ile substitution deleting the most carboxyl-terminal N-linked glycosylation site was found to have no effect on enzyme function, in agreement with the studies on the role of the individual glycosylation sites. There were different opinions, however, with respect to the effect of the other two substitutions. The Val-816 [r arrow] Ile substitution was reported by Martiniuk et al. to cause the lysosomal [alpha]-glucosidase deficiency, but the authors concluded on the basis of more extensive studies that the Asp-645[r arrow] Glu substitution was actually the deleterious mutation and the Val-816 [r arrow] Ile substitution a polymorphism. 8 refs., 1 fig.

  1. 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

  2. Structure activity relationship of C-2 ether substituted 1,5-naphthyridine analogs of oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents (Part-5).

    PubMed

    Singh, Sheo B; Kaelin, David E; Meinke, Peter T; Wu, Jin; Miesel, Lynn; Tan, Christopher M; Olsen, David B; Lagrutta, Armando; Fukuda, Hideyuki; Kishii, Ryuta; Takei, Masaya; Takeuchi, Tomoko; Takano, Hisashi; Ohata, Kohei; Kurasaki, Haruaki; Nishimura, Akinori; Shibata, Takeshi; Fukuda, Yasumichi

    2015-09-01

    Oxabicyclooctane linked novel bacterial topoisomerase inhibitors (NBTIs) are new class of recently reported broad-spectrum antibacterial agents. They target bacterial DNA gyrase and topoisomerase IV and bind to a site different than quinolones. They show no cross-resistance to known antibiotics and provide opportunity to combat drug-resistant bacteria. A structure activity relationship of the C-2 substituted ether analogs of 1,5-naphthyridine oxabicyclooctane-linked NBTIs are described. Synthesis and antibacterial activities of a total of 63 analogs have been summarized representing alkyl, cyclo alkyl, fluoro alkyl, hydroxy alkyl, amino alkyl, and carboxyl alkyl ethers. All compounds were tested against three key strains each of Gram-positive and Gram-negative bacteria as well as for hERG binding activities. Many key compounds were also tested for the functional hERG activity. Six compounds were evaluated for efficacy in a murine bacteremia model of Staphylococcus aureus infection. Significant tolerance for the ether substitution (including polar groups such as amino and carboxyl) at C-2 was observed for S. aureus activity however the same was not true for Enterococcus faecium and Gram-negative strains. Reduced clogD generally showed reduced hERG activity and improved in vivo efficacy but was generally associated with decreased overall potency. One of the best compounds was hydroxy propyl ether (16), which mainly retained the potency, spectrum and in vivo efficacy of AM8085 associated with the decreased hERG activity and improved physical property.

  3. Preparation and electrochemical properties of Zr-site substituted Li7La3(Zr2-xMx)O12 (M = Ta, Nb) solid electrolytes

    NASA Astrophysics Data System (ADS)

    Huang, Mian; Shoji, Mao; Shen, Yang; Nan, Ce-Wen; Munakata, Hirokazu; Kanamura, Kiyoshi

    2014-09-01

    Li7La3Zr2O12 (LLZ) solid electrolytes with Zr site partially substituted by Ta and Nb elements were prepared via the conventional solid-state reaction. All the compositions could lead to the cubic garnet-type structure after sintering at 1150 °C. The use of γ-Al2O3 as a sintering aid in the preparation of doped LLZ was studied. It was shown that Al could help to improve the micro-structure for Nb doping, but not necessary for Ta doping. The Ta and Nb doping enhanced the ionic conductivity at 25 °C to 4.09 × 10-4 S cm-1 and 4.50 × 10-4 S cm-1, respectively. A conductivity as high as 1.23 × 10-3 S cm-1 was obtained when measured at 50 °C in air for the Nb-doped LLZ. All-solid-state batteries with LLZTa and LLZNb solid electrolytes were assembled and tested. The cyclic voltammetry (CV) measurement indicated the successful working of the batteries.

  4. Tuning optical absorption and photoexcited recombination dynamics in La1-xSrxFeO3-δ through A-site substitution and oxygen vacancies

    NASA Astrophysics Data System (ADS)

    Smolin, Sergey; Scafetta, Mark; Choquette, Amber; Sfeir, Matthew; Baxter, Jason; May, Steven

    We study optical absorption and recombination dynamics in La1-xSrxFeO3-δ thin films, uncovering the effects of tuning nominal Fe valence via A-site substitution and oxygen stoichiometry. Variable angle spectroscopic ellipsometry was used to measure static optical properties, revealing a linear increase in absorption coefficient at 1.25 eV and a red-shifting of the optical absorption edge with increasing Sr fraction. The absorption spectra can be similarly tuned through the introduction of oxygen vacancies, indicating the critical role that nominal Fe valence plays in optical absorption. Dynamic optoelectronic properties were studied with ultrafast transient reflectance spectroscopy, revealing similar nanosecond photoexcited carrier lifetimes for oxygen deficient and stoichiometric films with the same nominal Fe valence. These results demonstrate that while the static optical absorption is strongly dependent on Fe valence tuned through cation or anion stoichiometry, oxygen vacancies do not appear to play a significantly detrimental role in the recombination kinetics. Nsf: ECCS-1201957, MRI DMR-0922929, MRI DMR-1040166. This research used resources of the Center for Functional Nanomaterials, which is a U.S. DOE Office of Science Facility, at Brookhaven National Laboratory under Contract No. DE-SC0012704.

  5. Druggability analysis and classification of protein tyrosine phosphatase active sites

    PubMed Central

    Ghattas, Mohammad A; Raslan, Noor; Sadeq, Asil; Al Sorkhy, Mohammad; Atatreh, Noor

    2016-01-01

    Protein tyrosine phosphatases (PTP) play important roles in the pathogenesis of many diseases. The fact that no PTP inhibitors have reached the market so far has raised many questions about their druggability. In this study, the active sites of 17 PTPs were characterized and assessed for its ability to bind drug-like molecules. Consequently, PTPs were classified according to their druggability scores into four main categories. Only four members showed intermediate to very druggable pocket; interestingly, the rest of them exhibited poor druggability. Particularly focusing on PTP1B, we also demonstrated the influence of several factors on the druggability of PTP active site. For instance, the open conformation showed better druggability than the closed conformation, while the tight-bound water molecules appeared to have minimal effect on the PTP1B druggability. Finally, the allosteric site of PTP1B was found to exhibit superior druggability compared to the catalytic pocket. This analysis can prove useful in the discovery of new PTP inhibitors by assisting researchers in predicting hit rates from high throughput or virtual screening and saving unnecessary cost, time, and efforts via prioritizing PTP targets according to their predicted druggability. PMID:27757011

  6. 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.

  7. Discovery of novel N-substituted carbazoles as neuroprotective agents with potent anti-oxidative activity.

    PubMed

    Zhu, Daqian; Chen, Meihui; Li, Min; Luo, Bingling; Zhao, Yang; Huang, Peng; Xue, Fengtian; Rapposelli, Simona; Pi, Rongbiao; Wen, Shijun

    2013-10-01

    Carbazole moiety is an important scaffold with a variety of biological applications, for example, anti-oxidative stress. Our previous synthesized carbazoles were screened for their neuroprotective properties against two individual oxidative stresses. Some of the new carbazole derivatives were observed with modest to good neuroprotective effects on neuronal cells HT22 against cell injury induced by glutamate or homocysteic acid (HCA). Substituents introduced to the carbazole ring system play crucial roles in their biological activities. In particular, a bulky group favors the neuroprotective activity of the compounds. One of the new compounds, 6, showed the best neuroprotective effects, which might result from its anti-oxidative activity with a GSH-independent mechanism. These findings might provide an alternative strategy for the development of novel carbazole derivatives for the treatment of CNS diseases such as Alzheimer's disease.

  8. 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

  9. 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.

  10. Toxicity of substituted anilines to Pseudokirchneriella subcapitata and quantitative structure-activity relationship analysis for polar narcotics.

    PubMed

    Chen, Chung-Yuan; Ko, Chia-Wen; Lee, Po-I

    2007-06-01

    This study evaluated the toxic effects of substituted anilines on Pseudokirchneriella subcapitata with the use of a closed algal toxicity testing technique with no headspace. Two response endpoints (i.e., dissolved oxygen production [DO] and algal growth rate) were used to evaluate the toxicity of anilines. Both DO and growth rate endpoints revealed similar sensitivity to the effects of anilines. However, trichloroanilines showed stronger inhibitory effects on microalgal photosynthetic reactions than that on algal growth. For various aquatic organisms, the relative sensitivity relationship for anilines is Daphnia magna > luminescent bacteria (Microtox) > or = Pocelia reticulata > or = Pseudokirchneriella subcapitata > or = fathead minnow > Tetrahymena pyriformis. The susceptibility of P. subcapitata to anilines is similar to fish, but P. subcapitata is apparently less sensitive than the water flea. The lack of correlation between the toxicity revealed by different aquatic organisms (microalgae, D. magna, luminescent bacteria, and P. reticulata) suggests that anilines might have different metabolic routes in these organisms. Both hydrogen bonding donor capacity (the lowest unoccupied molecular orbital energy, Elumo) and hydrophobicity (1-octanol:water partition coefficient, Kow) were found to provide satisfactory descriptions for the toxicity of polar narcotics (substituted anilines and chlorophenols). Quantitative structure-activity relationships (QSARs) based on Elumo, log Kow, or both values were established with r2 values varying from 0.75 to 0.92. The predictive power for the QSAR models were found to be satisfactory through leave-one-out cross-validation. Such relationships could provide useful information for the estimation of toxicity for other polar narcotic compounds.

  11. Catalysis of hydrolysis and nucleophilic substitution at the P-N bond of phosphoimidazolide-activated nucleotides in phosphate buffers.

    PubMed

    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). PMID:11538282

  12. 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).

  13. Optimization of antiproliferative activity of substituted phenyl 4-(2-oxoimidazolidin-1-yl) benzenesulfonates: QSAR and CoMFA analyses.

    PubMed

    Masand, Vijay H; Mahajan, Devidas T; Alafeefy, Ahmed M; Bukhari, Syed Nasir Abbas; Elsayed, Nahed N

    2015-09-18

    Multiple separate quantitative structure-activity relationships (QSARs) models were built for the antiproliferative activity of substituted Phenyl 4-(2-Oxoimidazolidin-1-yl)-benzenesulfonates (PIB-SOs). A variety of descriptors were considered for PIB-SOs through QSAR model building. Genetic algorithm (GA), available in QSARINS, was employed to select optimum number and set of descriptors to build the multi-linear regression equations for a dataset of PIB-SOs. The best three parametric models were subjected to thorough internal and external validation along with Y-randomization using QSARINS, according to the OECD principles for QSAR model validation. The models were found to be statistically robust with high external predictivity. The best three parametric model, based on steric, 3D- and finger print descriptors, was found to have R(2)=0.91, R(2)ex=0.89, and CCCex=0.94. The CoMFA model, which is based on a combination of steric and electrostatic effects and graphically inferred using contour plots, gave F=229.34, R(2)CV=0.71 and R(2)=0.94. Steric repulsion, frequency of occurrence of carbon and nitrogen at topological distance of seven, and internal electronic environment of the molecule were found to have correlation with the anti-tumor activity of PIB-SOs. PMID:26066412

  14. 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.

  15. 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.

  16. Interactions with polynucleotides and antitumor activity of amidino and imidazolinyl substituted 2-phenylbenzothiazole mesylates.

    PubMed

    Racané, Livio; Stojković, Ranko; Tralić-Kulenović, Vesna; Cerić, Helena; Đaković, Marijana; Ester, Katja; Krpan, Ana Mišir; Stojković, Marijana Radić

    2014-10-30

    Based on previously reported antiproliferative activity screening, four most promising disubstituted 2-phenylbenzothiazole hydrochlorides were chosen for detailed study. Water solubility, as well as liphophilicity/hydrophilicity balance of organic core were modified by conversion to mesylate salts. For purpose of structure/activity studies their structures were determined by X-ray structure analysis. Detailed analysis of interactions of new compounds with double stranded (ds-) DNA/RNA by UV/Vis and CD titrations, thermal melting and viscometry experiments revealed that most of studied compounds intercalate into ds-RNA but bind into minor groove of AT-DNA, and agglomerate along GC-DNA. Furthermore, compounds also interact with ss-RNA, but only amino-imidazolinyl 2-phenylbenzothiazole, 4b displayed well defined orientation and dominant binding mode (by induced CD signals) with poly A and poly G. Besides, in vitro investigations revealed moderate to high antiproliferative activity of benzothiazoles against seven human cancer cell lines, while in some cases (HTC 116, SW620, MIA PaCa-2) high correlation between the type of the amidino group and cytotoxic activity was observed.

  17. In Vitro Antifungal Activities of a Series of Dication-Substituted Carbazoles, Furans, and Benzimidazoles

    PubMed Central

    Del Poeta, Maurizio; Schell, Wiley A.; Dykstra, Christine C.; Jones, Susan K.; Tidwell, Richard R.; Kumar, Arvind; Boykin, David W.; Perfect, John R.

    1998-01-01

    Aromatic dicationic compounds possess antimicrobial activity against a wide range of eucaryotic pathogens, and in the present study an examination of the structures-functions of a series of compounds against fungi was performed. Sixty-seven dicationic molecules were screened for their inhibitory and fungicidal activities against Candida albicans and Cryptococcus neoformans. The MICs of a large number of compounds were comparable to those of the standard antifungal drugs amphotericin B and fluconazole. Unlike fluconazole, potent inhibitory compounds in this series were found to have excellent fungicidal activities. The MIC of one of the most potent compounds against C. albicans was 0.39 μg/ml, and it was the most potent compound against C. neoformans (MIC, ≤0.09 μg/ml). Selected compounds were also found to be active against Aspergillus fumigatus, Fusarium solani, Candida species other than C. albicans, and fluconazole-resistant strains of C. albicans and C. neoformans. Since some of these compounds have been safely given to animals, these classes of molecules have the potential to be developed as antifungal agents. PMID:9756748

  18. Diastereoselective synthesis of substituted hexahydrobenzo[de]isochromanes and evaluation of their antileishmanial activity.

    PubMed

    Saikia, Anil K; Sultana, Sabera; Devi, Ngangbam Renubala; Deka, Manash J; Tiwari, Kartikeya; Dubey, Vikash K

    2016-01-21

    Hexahydrobenzo[de]isochromanes and hexahydropyrano[3,4,5-ij]isoquinolines can be efficiently synthesized via Friedel Crafts and oxa Pictet-Spengler reaction of acrylyl enol ethers mediated by triflic acid in good yields. The reaction is highly stereoselective. Two of the hexahydrobenzo[de]isochromanes are found to have moderate antileishmanial activity. PMID:26625982

  19. Electrostatic fields in the active sites of lysozymes.

    PubMed

    Sun, D P; Liao, D I; Remington, S J

    1989-07-01

    Considerable experimental evidence is in support of several aspects of the mechanism that has been proposed for the catalytic activity of lysozyme. However, the enzymatically catalyzed hydrolysis of polysaccharides proceeds over 5 orders of magnitude faster than that of model compounds that mimic the configuration of the substrate in the active site of the enzyme. Although several possible explanations for this rate enhancement have been discussed elsewhere, a definitive mechanism has not emerged. Here we report striking results obtained by classical electrodynamics, which suggest that bond breakage and the consequent separation of charge in lysozyme is promoted by a large electrostatic field across the active site cleft, produced in part by a very asymmetric distribution of charged residues on the enzyme surface. Lysozymes unrelated in amino acid sequence have similar distributions of charged residues and electric fields. The results reported here suggest that the electrostatic component of the rate enhancement is greater than 9 kcal.mol-1. Thus, electrostatic interactions may play a more important role in the enzymatic mechanism than has generally been appreciated.

  20. Synthesis and in vitro antitumor activity of substituted quinazoline and quinoxaline derivatives: search for anticancer agent.

    PubMed

    Noolvi, Malleshappa N; Patel, Harun M; Bhardwaj, Varun; Chauhan, Ankit

    2011-06-01

    The synthesis of some 2-furano-4(3H)-quinazolinones, diamides (open ring quinazolines), quinoxalines and their biological evaluation as antitumor agents using National Cancer Institute (NCI) disease oriented antitumor screen protocol are investigated. Among the synthesize compounds, seventeen compounds were granted NSC code and screened at National Cancer Institute (NCI), USA for anticancer activity at a single high dose (10(-5) M) in full NCI 60 cell panel. Among the selected compounds, 3-(2-chloro benzylideneamine)-2-(furan-2-yl) quinazoline-4(3h)-one 21 was found to be the most active candidate of the series at five dose level screening against Ovarian OVCAR-4 and Non-small cell lung cancer NCI-H522 with GI50 1.82 & 2.14 μM respectively. Rational approach and QSAR techniques enabled the understanding of the pharmacophoric requirement for quinazoline, diamides and quinoxaline derivatives. PMID:21458891

  1. Synthesis and in vitro antitumor activity of substituted quinazoline and quinoxaline derivatives: search for anticancer agent.

    PubMed

    Noolvi, Malleshappa N; Patel, Harun M; Bhardwaj, Varun; Chauhan, Ankit

    2011-06-01

    The synthesis of some 2-furano-4(3H)-quinazolinones, diamides (open ring quinazolines), quinoxalines and their biological evaluation as antitumor agents using National Cancer Institute (NCI) disease oriented antitumor screen protocol are investigated. Among the synthesize compounds, seventeen compounds were granted NSC code and screened at National Cancer Institute (NCI), USA for anticancer activity at a single high dose (10(-5) M) in full NCI 60 cell panel. Among the selected compounds, 3-(2-chloro benzylideneamine)-2-(furan-2-yl) quinazoline-4(3h)-one 21 was found to be the most active candidate of the series at five dose level screening against Ovarian OVCAR-4 and Non-small cell lung cancer NCI-H522 with GI50 1.82 & 2.14 μM respectively. Rational approach and QSAR techniques enabled the understanding of the pharmacophoric requirement for quinazoline, diamides and quinoxaline derivatives.

  2. [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

  3. [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.

  4. Synthesis, structure and antibiotic activity of aryl-substituted LpxC inhibitors

    PubMed Central

    Zhao, Jinshi; Toone, Eric J.; Zhou, Pei

    2013-01-01

    The zinc-dependent deacetylase LpxC catalyzes the committed step of lipid A biosynthesis in Gram-negative bacteria and is a validated target for development of novel antibiotics to combat multidrug-resistant Gram-negative infections. Many potent LpxC inhibitors contain an essential threonyl-hydroxamate head group for high-affinity interaction with LpxC. We report the synthesis, antibiotic activity, and structural and enzymatic characterization of novel LpxC inhibitors containing an additional aryl-group in the threonyl-hydroxamate moiety, which expands the inhibitor-binding surface in LpxC. These compounds display enhanced potency against LpxC in enzymatic assays and superior antibiotic activity against F. novicida in cell culture. Comparison of the antibiotic activities of these compounds against a leaky E. coli strain and the wild-type strain reveals the contribution of the formidable outer membrane permeability barrier that reduces the compound efficacy in cell culture and emphasizes the importance of maintaining a balanced hydrophobicity and hydrophilicity profile in developing effective LpxC-targeting antibiotics. PMID:23914798

  5. 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

  6. 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

  7. 2'-deoxy cyclic adenosine 5'-diphosphate ribose derivatives: importance of the 2'-hydroxyl motif for the antagonistic activity of 8-substituted cADPR derivatives.

    PubMed

    Zhang, Bo; Wagner, Gerd K; Weber, Karin; Garnham, Clive; Morgan, Anthony J; Galione, Antony; Guse, Andreas H; Potter, Barry V L

    2008-03-27

    The structural features needed for antagonism at the cyclic ADP-ribose (cADPR) receptor are unclear. Chemoenzymatic syntheses of novel 8-substituted 2'-deoxy-cADPR analogues, including 8-bromo-2'-deoxy-cADPR 7, 8-amino-2'-deoxy-cADPR 8, 8- O-methyl-2'-deoxy-cADPR 9, 8-phenyl-2'-deoxy-cADPR 10 and its ribose counterpart 8-phenyl-cADPR 5 are reported, including improved syntheses of established antagonists 8-amino-cADPR 2 and 8-bromo-cADPR 3. Aplysia californica ADP-ribosyl cyclase tolerates even the bulky 8-phenyl-nicotinamide adenine 5'-dinucleotide as a substrate. Structure-activity relationships of 8-substituted cADPR analogues in both Jurkat T-lymphocytes and sea urchin egg homogenate (SUH) were investigated. 2'-OH Deletion decreased antagonistic activity (at least for the 8-amino series), showing it to be an important motif. Some 8-substituted 2'-deoxy analogues showed agonist activity at higher concentrations, among which 8-bromo-2'-deoxy-cADPR 7 was, unexpectedly, a weak but almost full agonist in SUH and was membrane-permeant in whole eggs. Classical antagonists 2 and 3 also showed previously unobserved agonist activity at higher concentrations in both systems. The 2'-OH group, without effect on the Ca (2+)-mobilizing ability of cADPR itself, is an important motif for the antagonistic activities of 8-substituted cADPR analogues. PMID:18303825

  8. Antimicrobial activity of quinoxaline 1,4-dioxide with 2- and 3-substituted derivatives.

    PubMed

    Vieira, Mónica; Pinheiro, Cátia; Fernandes, Rúben; Noronha, João Paulo; Prudêncio, Cristina

    2014-04-01

    Quinoxaline is a chemical compound that presents a structure that is similar to quinolone antibiotics. The present work reports the study of the antimicrobial activity of quinoxaline N,N-dioxide and some derivatives against bacterial and yeast strains. The compounds studied were quinoxaline-1,4-dioxide (QNX), 2-methylquinoxaline-1,4-dioxide (2MQNX), 2-methyl-3-benzoylquinoxaline-1,4-dioxide (2M3BenzoylQNX), 2-methyl-3-benzylquinoxaline-1,4-dioxide (2M3BQNX), 2-amino-3-cyanoquinoxaline-1,4-dioxide (2A3CQNX), 3-methyl-2-quinoxalinecarboxamide-1,4-dioxide (3M2QNXC), 2-hydroxyphenazine-N,N-dioxide (2HF) and 3-methyl-N-(2-methylphenyl)quinoxalinecarboxamide-1,4-dioxide (3MN(2MF)QNXC). The prokaryotic strains used were Staphylococcus aureus ATCC 6538, S. aureus ATCC 6538P, S. aureus ATCC 29213, Escherichia coli ATCC 25922, E. coli S3R9, E. coli S3R22, E. coli TEM-1 CTX-M9, E. coli TEM-1, E. coli AmpC Mox-2, E. coli CTX-M2 e E. coli CTX-M9. The Candida albicans ATCC 10231 and Saccharomyces cerevisiae PYCC 4072 were used as eukaryotic strains. For the compounds that presented activity using the disk diffusion method, the minimum inhibitory concentration (MIC) was determined. The alterations of cellular viability were evaluated in a time-course assay. Death curves for bacteria and growth curves for S. cerevisiae PYCC 4072 were also accessed. The results obtained suggest potential new drugs for antimicrobial activity chemotherapy since the MIC's determined present low values and cellular viability tests show the complete elimination of the bacterial strain. Also, the cellular viability tests for the eukaryotic model, S. cerevisiae, indicate low toxicity for the compounds tested.

  9. 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

  10. Structure-activity studies of 5-substituted pyridopyrimidines as adenosine kinase inhibitors.

    PubMed

    Cowart, M; Lee, C H; Gfesser, G A; Bayburt, E K; Bhagwat, S S; Stewart, A O; Yu, H; Kohlhaas, K L; McGaraughty, S; Wismer, C T; Mikusa, J; Zhu, C; Alexander, K M; Jarvis, M F; Kowaluk, E A

    2001-01-01

    The synthesis and SAR of a novel series of non-nucleoside pyridopyrimidine inhibitors of the enzyme adenosine kinase (AK) are described. It was found that pyridopyrimidines with a broad range of medium and large non-polar substituents at the 5-position potently inhibited AK activity. A narrower range of analogues was capable of potently inhibiting adenosine phosphorylation in intact cells indicating an enhanced ability of these analogues to penetrate cell membranes. Potent AK inhibitors were found to effectively reduce nociception in animal models of thermal hyperalgesia and persistent pain.

  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. Implications of binding mode and active site flexibility for inhibitor potency against the salicylate synthase from Mycobacterium tuberculosis.

    PubMed

    Chi, Gamma; Manos-Turvey, Alexandra; O'Connor, Patrick D; Johnston, Jodie M; Evans, Genevieve L; Baker, Edward N; Payne, Richard J; Lott, J Shaun; Bulloch, Esther M M

    2012-06-19

    MbtI is the salicylate synthase that catalyzes the first committed step in the synthesis of the iron chelating compound mycobactin in Mycobacterium tuberculosis. We previously developed a series of aromatic inhibitors against MbtI based on the reaction intermediate for this enzyme, isochorismate. The most potent of these inhibitors had hydrophobic substituents, ranging in size from a methyl to a phenyl group, appended to the terminal alkene of the enolpyruvyl group. These compounds exhibited low micromolar inhibition constants against MbtI and were at least an order of magnitude more potent than the parental compound for the series, which carries a native enolpyruvyl group. In this study, we sought to understand how the substituted enolpyruvyl group confers greater potency, by determining cocrystal structures of MbtI with six inhibitors from the series. A switch in binding mode at the MbtI active site is observed for inhibitors carrying a substituted enolpyruvyl group, relative to the parental compound. Computational studies suggest that the change in binding mode, and higher potency, is due to the effect of the substituents on the conformational landscape of the core inhibitor structure. The crystal structures and fluorescence-based thermal shift assays indicate that substituents larger than a methyl group are accommodated in the MbtI active site through significant but localized flexibility in the peptide backbone. These findings have implications for the design of improved inhibitors of MbtI, as well as other chorismate-utilizing enzymes from this family. PMID:22607697

  13. Trichodiene synthase. Identification of active site residues by site-directed mutagenesis.

    PubMed

    Cane, D E; Shim, J H; Xue, Q; Fitzsimons, B C; Hohn, T M

    1995-02-28

    Derivatization of 5,5'-dithiobis(2-nitrobenzoic acid)-treated trichodiene synthase with [methyl-14C]methyl methanethiosulfonate and analysis of the derived tryptic peptides suggested the presence of two cysteine residues at the active site. The corresponding C146A and C190A mutants were constructed by site-directed mutagenesis. The C190A mutant displayed partial but significantly reduced activity, with a reduction in kcat/Km of 3000 compared to the wild-type trichodiene synthase, while the C146A mutant was essentially inactive. A hybrid trichodiene synthase, constructed from amino acids 1-309 of the Fusarium sporotrichioides enzyme and amino acids 310-383 of the Gibberella pulicaris cyclase, had steady state kinetic parameters nearly identical to those of the wild-type F. sporotrichioides enzyme. From this parent hybrid, a series of mutants was constructed by site-directed mutagenesis in which the amino acids in the base-rich region, 302-306 (DRRYR), were systematically modified. Three of these mutants were overexpressed and purified to homogeneity. The importance of Arg304 for catalysis was established by the observation that the R304K mutant showed a more than 25-fold increase in Km, as well as a 200-fold reduction in kcat. In addition, analysis of the incubation products of the R304K mutant by gas chromatography-mass spectrometry (GC-MS) indicated that farnesyl diphosphate was converted not only to trichodiene but to at least two additional C15H24 hydrocarbons, mle 204. Replacement of the Tyr305 residue of trichodiene synthase with Phe had little effect on kcat, while increasing the Km by a factor of ca. 7-8.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7873527

  14. 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

  15. Structure-activity relationships of substituted 1H-indole-2-carboxamides as CB1 receptor allosteric modulators.

    PubMed

    Nguyen, Thuy; German, Nadezhda; Decker, Ann M; Li, Jun-Xu; Wiley, Jenny L; Thomas, Brian F; Kenakin, Terry P; Zhang, Yanan

    2015-05-01

    A series of substituted 1H-indole-2-carboxamides structurally related to compounds Org27569 (1), Org29647 (2) and Org27759 (3) were synthesized and evaluated for CB1 allosteric modulating activity in calcium mobilization assays. Structure-activity relationship studies showed that the modulation potency of this series at the CB1 receptor was enhanced by the presence of a diethylamino group at the 4-position of the phenyl ring, a chloro or fluoro group at the C5 position and short alkyl groups at the C3 position on the indole ring. The most potent compound (45) had an IC₅₀ value of 79 nM which is ∼2.5 and 10 fold more potent than the parent compounds 3 and 1, respectively. These compounds appeared to be negative allosteric modulators at the CB1 receptor and dose-dependently reduced the Emax of agonist CP55,940. These analogs may provide the basis for further optimization and use of CB1 allosteric modulators.

  16. Synthesis of new 2-substituted 6-bromo-3-methylthiazolo[3,2-alpha]-benzimidazole derivatives and their biological activities.

    PubMed

    Abdel-Aziz, Hatem A; Hamdy, Nehal A; Gamal-Eldeen, Amira M; Fakhr, Issa M I

    2011-01-01

    1-(6-Bromo-3-methyl-1,3-thiazolo[3,2-alpha]benzimidazol-2-yl)ethanone (2) was prepared by bromination at ambient temperature of 1-(3-methylthiazolo[3,2-alpha]benzimidazol-2-yl)ethanone (1). The structure of 2 was determined by single-crystal X-ray diffraction. The precursor 5 was synthesized by heating a mixture of acetyl 2 and bromine. Various 2-substituted 6-bromo-3-methylthiazolo[3,2-alpha]benzimidazoles containing 1,3-thiazole, 1,4-benzothiazine, quinoxaline or imidazo[1,2-alpha]pyridine moieties were prepared starting from bromoacetyl 5. Taken together from the biological investigations, 2, 5, and 7a were potent immunosuppressors against both macrophages and T-lymphocytes, and 7b, 11b, and 14 were potent immunostimulators towards both types of immune cells. The results also revealed that, among others, 2 and 14 were the most significant inhibitors of LPS-stimulated NO generation, and that 5, 7a, and 7b had a weak radical scavenging activity against DPPH radicals. Moreover, 2, 5, and 7a had a concomitant strong cytotoxicity against colon carcinoma, hepatocellular carcinoma, and lymphoblastic leukemia cells. Collectively, compounds 2, 5, and 7a are multipotent compounds with promising biological activities.

  17. Optically active microspheres constructed by helical substituted polyacetylene and used for adsorption of organic compounds in aqueous systems.

    PubMed

    Liang, Junya; Song, Ci; Deng, Jianping

    2014-11-12

    This article reports optically active microspheres consisting of chiral helical substituted polyacetylene and β-cyclodextrin-derivative (β-CD-A). The microspheres showed remarkable adsorption toward various organic compounds in water. To prepare the microspheres, an acetylenic-derived helical macro-monomer was synthesized and then underwent aqueous suspension copolymerization with octadecyl acrylate and butyl acrylate by using azobis(isobutyronitrile) as initiator and β-CD-A simultaneously as comonomer and cross-linking agent. The helical macro-monomer chains enabled the microspheres to exhibit desirable enantio-differentiating adsorption capacity toward chiral compounds respectively dissolved in organic solvent, dispersed in water, and dissolved in water. The saturated absorbency toward (R)-(+)- and (S)-(-)-1-phenylethylamine was 29 and 12 mg · g(-1), respectively. The microspheres also showed large oil absorbency (e.g., 22 g · g(-1) CCl4) and a large adsorption toward methyl red (as a model for organic dyes) dispersed in water. The presence of β-CD-A moieties improved the adsorption performance of the microspheres. The present optically active microspheres open a new approach for preparing adsorbents particularly chiral adsorbents with potentials for wastewater treatment. PMID:25290256

  18. Comparative 1-substituted imidazole inhibition of cytochrome p450 isozyme-selective activities in human and mouse hepatic microsomes.

    PubMed

    Franklin, Michael R; Constance, Jonathan E

    2007-01-01

    Inhibition of cytochrome P450(CYP)-selective reactions in a single human and a single mouse hepatic microsome preparation by fourteen 1-substituted imidazoles provides a simultaneous ranking of reaction susceptibility to a specific imidazole and the relative inhibitory potency of the imidazoles for a given reaction. CYP3A4/5 activity was inhibited (IC(50) <5 microM) by the greatest number of imidazoles, followed closely by CYP2C9. Seven imidazoles exhibited IC(50) values for CYP3A4/5 <0.3 microM (none for CYP2C9) and were exclusively above 300 MW. Nafimidone (MW, 236) exhibited an IC(50) value <0.3 microM towards CYP2D6 and CYP1A2 reactions. CYP2E1 and CYP2A6 were exclusively inhibited (IC(50) <5 microM) by imidazoles with MWs below approximately 200. In general, mouse activities exhibited lower IC(50) values than in human microsomes. PMID:17786623

  19. 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

  20. Activation of muscarinic acetylcholine receptors via their allosteric binding sites.

    PubMed Central

    Jakubík, J; Bacáková, L; Lisá, V; el-Fakahany, E E; Tucek, S

    1996-01-01

    Ligands that bind to the allosteric-binding sites on muscarinic acetylcholine receptors alter the conformation of the classical-binding sites of these receptors and either diminish or increase their affinity for muscarinic agonists and classical antagonists. It is not known whether the resulting conformational change also affects the interaction between the receptors and the G proteins. We have now found that the muscarinic receptor allosteric modulators alcuronium, gallamine, and strychnine (acting in the absence of an agonist) alter the synthesis of cAMP in Chinese hamster ovary (CHO) cells expressing the M2 or the M4 subtype of muscarinic receptors in the same direction as the agonist carbachol. In addition, most of their effects on the production of inositol phosphates in CHO cells expressing the M1 or the M3 muscarinic receptor subtypes are also similar to (although much weaker than) those of carbachol. The agonist-like effects of the allosteric modulators are not observed in CHO cells that have not been transfected with the gene for any of the subtypes of muscarinic receptors. The effects of alcuronium on the formation of cAMP and inositol phosphates are not prevented by the classical muscarinic antagonist quinuclidinyl benzilate. These observations demonstrate for the first time that the G protein-mediated functional responses of muscarinic receptors can be evoked not only from their classical, but also from their allosteric, binding sites. This represents a new mechanism of receptor activation. PMID:8710935

  1. Cp*Co(III) Catalyzed Site-Selective C-H Activation of Unsymmetrical O-Acyl Oximes: Synthesis of Multisubstituted Isoquinolines from Terminal and Internal Alkynes.

    PubMed

    Sun, Bo; Yoshino, Tatsuhiko; Kanai, Motomu; Matsunaga, Shigeki

    2015-10-26

    The synthesis of isoquinolines by site-selective C-H activation of O-acyl oximes with a Cp*Co(III) catalyst is described. In the presence of this catalyst, the C-H activation of various unsymmetrically substituted O-acyl oximes selectively occurred at the sterically less hindered site, and reactions with terminal as well as internal alkynes afforded the corresponding products in up to 98 % yield. Whereas the reactions catalyzed by the Cp*Co(III) system proceeded with high site selectivity (15:1 to 20:1), use of the corresponding Cp*Rh(III) catalysts led to low selectivities and/or yields when unsymmetrical O-acyl oximes and terminal alkynes were used. Deuterium labeling studies indicate a clear difference in the site selectivity of the C-H activation step under Cp*Co(III) and Cp*Rh(III) catalysis.

  2. Radiation inactivation study of aminopeptidase: probing the active site

    NASA Astrophysics Data System (ADS)

    Jamadar, V. K.; Jamdar, S. N.; Mohan, Hari; Dandekar, S. P.; Harikumar, P.

    2004-04-01

    Ionizing radiation inactivated purified chicken intestinal aminopeptidase in media saturated with gases in the order N 2O>N 2>air. The D 37 values in the above conditions were 281, 210 and 198 Gy, respectively. OH radical scavengers such as t-butanol and isopropanol effectively nullified the radiation-induced damage in N 2O. The radicals (SCN) 2•-, Br 2•- and I 2•- inactivated the enzyme, pointing to the involvement of aromatic amino acids and cysteine in its catalytic activity. The enzyme exhibited fluorescence emission at 340 nm which is characteristic of tryptophan. The radiation-induced loss of activity was accompanied by a decrease in the fluorescence of the enzyme suggesting a predominant influence on tryptophan residues. The enzyme inhibition was associated with a marked increase in the Km and a decrease in the Vmax and kcat values, suggesting an irreversible alteration in the catalytic site. The above observations were confirmed by pulse radiolysis studies.

  3. 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

  4. 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.

  5. 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

  6. Synthesis and Herbicidal Activity of α-(Substituted Phenoxybutyryloxy or Valeryloxy)alkylphosphonates and 2-(Substituted Phenoxybutyryloxy)alkyl-5,5-dimethyl-1,3,2-dioxaphosphinan-2-one.

    PubMed

    Wang, Wei; Zhang, Sha-Sha; Zhou, Yuan; Peng, Hao; He, Hong-Wu; Lu, Xing-Tao

    2016-09-21

    On the basis of our work on the modification of alkylphosphonates 1, a series of α-(substituted phenoxybutyryloxy or valeryloxy)alkylphosphonates (4-5) and 2-(substituted phenoxybutyryloxy)alkyl-5,5-dimethyl-1,3,2-dioxaphosphinan-2-one (6) were designed and synthesized. The bioassay results indicated that 14 of title compounds 4 exhibited significant postemergence herbicidal activity against velvetleaf, common amaranth, and false daisy at 150 g ai/ha. Compounds 5 were inactive against all tested weeds. Compounds 6 exhibited moderate to good inhibitory effect against the tested dicotyledonous weeds. Structure-activity relationship (SAR) analyses showed that the length of the carbon chain as linking bridge had a great effect on the herbicidal activity. Broad-spectrum tests of compounds 4-1, 4-2, 4-9, 4-30, and 4-36 were carried out at 75 g ai/ha. Especially, 4-1 exhibited 100% inhibition activity against the tested dicotyledonous weeds, which was higher than that of glyphosate.

  7. Synthesis and Herbicidal Activity of α-(Substituted Phenoxybutyryloxy or Valeryloxy)alkylphosphonates and 2-(Substituted Phenoxybutyryloxy)alkyl-5,5-dimethyl-1,3,2-dioxaphosphinan-2-one.

    PubMed

    Wang, Wei; Zhang, Sha-Sha; Zhou, Yuan; Peng, Hao; He, Hong-Wu; Lu, Xing-Tao

    2016-09-21

    On the basis of our work on the modification of alkylphosphonates 1, a series of α-(substituted phenoxybutyryloxy or valeryloxy)alkylphosphonates (4-5) and 2-(substituted phenoxybutyryloxy)alkyl-5,5-dimethyl-1,3,2-dioxaphosphinan-2-one (6) were designed and synthesized. The bioassay results indicated that 14 of title compounds 4 exhibited significant postemergence herbicidal activity against velvetleaf, common amaranth, and false daisy at 150 g ai/ha. Compounds 5 were inactive against all tested weeds. Compounds 6 exhibited moderate to good inhibitory effect against the tested dicotyledonous weeds. Structure-activity relationship (SAR) analyses showed that the length of the carbon chain as linking bridge had a great effect on the herbicidal activity. Broad-spectrum tests of compounds 4-1, 4-2, 4-9, 4-30, and 4-36 were carried out at 75 g ai/ha. Especially, 4-1 exhibited 100% inhibition activity against the tested dicotyledonous weeds, which was higher than that of glyphosate. PMID:27530975

  8. 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

  9. An active-site lysine in avian liver phosphoenolpyruvate carboxykinase

    SciTech Connect

    Guidinger, P.F.; Nowak, T. )

    1991-09-10

    The participation of lysine in the catalysis by avian liver phosphoenolpyruvate carboxykinase was studied by chemical modification and by a characterization of the modified enzyme. The rate of inactivation by 2,4-pentanedione is pseudo-first-order and linearly dependent on reagent concentration with a second-order rate constant of 0.36 {plus minus} 0.025 M{sup {minus}1} min{sup {minus}1}. Inactivation by pyridoxal 5{prime}-phosphate of the reversible reaction catalyzed by phosphoenolpyruvate carboxykinase follows bimolecular kinetics with a second-order rate constant of 7,700 {plus minus} 860 m{sup {minus}1} min{sup {minus}1}. Treatment of the enzyme or one lysine residue modified concomitant with 100% loss in activity. A stoichiometry of 1:1 is observed when either the reversible or the irreversible reactions catalyzed by the enzyme are monitored. A study of k{sub obs} vs pH suggests this active-site lysine has a pK{sub a} of 8.1 and a pH-independent rate constant of inactivation of 47,700 m{sup {minus}1} min{sup {minus}1}. Proton relaxation rate measurements suggest that pyridoxal 5{prime}-phosphate modification alters binding of the phosphate-containing substrates. {sup 31}P NMR relaxation rate measurements show altered binding of the substrates in the ternary enzyme {center dot}Mn{sup 2+}{center dot}substrate complex. Circular dichroism studies show little change in secondary structure of pyridoxal 5{prime}-phosphate modified phosphoenolpyruvate carboxykinase. These results indicate that avian liver phosphoenolpyruvate carboxykinase has one reactive lysine at the active site and it is involved in the binding and activation of the phosphate-containing substrates.

  10. Interaction of fluoroethane chlorofluorocarbon (CFC) substitutes with microsomal cytochrome P450. Stimulation of P450 activity and chlorodifluoroethene metabolism.

    PubMed

    Wang, Y; Olson, M J; Baker, M T

    1993-07-01

    The abilities of halothane and the fluoroethane chlorofluorocarbon (CFC) substitutes, FC-123, FC-133a, FC-124, FC-134a and FC-125, to stimulate cytochrome P450 activities and 2-chloro-1,1-difluoroethene (CDE) defluorination in hepatic microsomes from phenobarbital-treated rabbits were compared. At 1% (v/v) each, halothane and FC-123 similarly increased the consumption of NADPH and O2 by 300 and 100%, respectively, over that in microsomes without substrate. FC-133a and FC-124 were less effective, increasing NADPH and O2 consumption by 150-200 and 70%. FC-134a and FC-125 were the least effective, increasing NADPH and O2 consumption by only 70 and 50%, respectively. No metabolism of any fluoroethane could be detected under the incubation conditions used. Halothane and FC-123 were most effective in stimulating CDE metabolism with increases of CDE defluorination ranging from 1.5- to 2-fold. FC-133a and FC-124 enhanced CDE oxidation 89 and 74%, respectively, and FC-134a and FC-125 had no effect. While CDE metabolism was enhanced in the presence of the fluoroethanes, no additional NADPH or O2 was consumed when halothane or FC-124 was incubated with CDE compared with incubations containing only halothane or FC-124. Log-log plots of NADPH consumption and CDE metabolism with the olive oil/gas partition coefficients of each fluoroethane showed linear relationships. These data demonstrate that the activity of the fluoroethanes in stimulating P450 activity and CDE metabolism is a function of their lipid solubility, and fluoroethane-enhanced CDE metabolism is related to the ability of these compounds to increase uncoupled P450 activity.

  11. 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

  12. Selection of orally active antifungal agents from 3,5-substituted isoxazolidine derivatives based on acute efficacy-safety profiles.

    PubMed

    Palmer, G C; Ordy, M J; Simmons, R D; Strand, J C; Radov, L A; Mullen, G B; Kinsolving, C R; St Georgiev, V; Mitchell, J T; Allen, S D

    1989-06-01

    Routine in vitro screening of a new synthetic series of 3,5-substituted 2-methylisoxazolidines revealed that three imidazole analogs (PR 967-248, PR 967-234, and PR 969-566) and, to a lesser extent, a triazole analog (PR 988-399) exerted rather potent antifungal activity against three systemic and four dermatophytic classes of fungi. When tested in vivo for ability to eradicate Candida vaginitis in the rat, the triazole derivative, PR 988-399, was effective after oral administration. In this in vivo test for efficacy, PR 967-234 and PR 969-566 reduced but did not eradicate the infection, while PR 967-248 was inactive. PR 988-399 was, moreover, 4- to 13-fold less potent than the three imidazoles in inhibiting testosterone synthesis in isolated rat Leydig cells. After oral or intravenous administration, PR 988-399 and PR 969-566 elicited the fewest cardiovascular and behavioural side effects in conscious dogs. The rat safety study consisted of oral dosing followed by evaluation of the exploratory motor activity of the naive animals in a novel environment. Motor activity was suppressed least by PR 988-399 and most by PR 969-566. In a battery of mouse behavioural-neuromuscular-drug interaction tests, PR 988-399 and PR 969-566 produced the fewest central-behavioural-neuromuscular signs. These efficacy-safety evaluations were performed with ketoconazole as a positive reference standard. The sequence of drug testing with respect to efficacy-safety considerations appears to be a suitable approach for early detection of orally active antifungal agents such as PR 988-399 for more advanced development.

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. Intramolecular electron transfer in sulfite oxidizing enzymes: elucidating the role of a conserved active site arginine†

    PubMed Central

    Emesh, Safia; Rapson, Trevor D.; Rajapakshe, S. Asha; Kappler, Ulrike; Bernhardt, Paul V.; Tollin, Gordon; Enemark, John H.

    2009-01-01

    All reported sulfite oxidizing enzymes have a conserved arginine in their active site which hydrogen bonds to the equatorial oxygen ligand on the Mo atom. Previous studies on the pathogenic R160Q mutant of human sulfite oxidase (HSO) have shown that Mo-heme intramolecular electron transfer (IET) is dramatically slowed when positive charge is lost at this position. In order to better understand the function that this conserved positively charged residue plays in IET, we have studied the equivalent uncharged substitutions, R55Q and R55M, as well as the positively charged substitution, R55K, in bacterial sulfite dehydrogenase (SDH). The heme and molybdenum cofactor (Moco) subunits are tightly associated in SDH, which makes it an ideal system for increasing the understanding of residue function in IET without the added complexity of the inter-domain movement that occurs in HSO. Unexpectedly, the uncharged SDH variants (R55Q and R55M) showed increased IET rate constants relative to the wildtype (3–4 fold) when studied by laser flash photolysis. The gain in function observed in SDHR55Q and SDHR55M suggests that the reduction of IET seen in HSOR160Q is not due to a required role of this residue in the IET pathway itself, but to the fact that it plays an important role in heme orientation during the inter-domain movement necessary for IET in HSO (as seen in viscosity experiments). The pH profiles of SDHwt, SDHR55M, and SDHR55Q show that the arginine substitution also alters the behavior of the Mo-heme IET equilibrium (Keq) and rate constants (ket) of both variants with respect to SDHWT enzyme. SDHWT has a ket that is independent of pH and a Keq that increases as pH decreases, whereas both SDHR55M and SDHR55Q have a ket that increases as pH decreases, and SDHR55M has a Keq that is pH independent. IET in the SDHR55Q variant is inhibited by sulfate in laser flash photolysis experiments, a behavior that differs from SDHWT, but which also occurs in HSO. IET in SDHR55K is

  18. Zinc site redesign in T4 gene 32 protein: structure and stability of cobalt(II) complexes formed by wild-type and metal ligand substitution mutants.

    PubMed

    Guo, J; Giedroc, D P

    1997-01-28

    Phage T4 gene 32 protein (gp32) is a zinc metalloprotein which binds cooperatively and preferentially to single-stranded nucleic acids and functions as a replication and recombination accessory protein. Zn(II) coordination by gp32 employs a His-Cys3 metal ligand donor set derived from the His64-X12-Cys77-X9-Cys87-X2-Cys90 sequence in the ssDNA-binding core domain of the molecule. Crystallographic studies reveal that His64 and Cys77 are derived from two independent beta-strands within a distorted three-stranded beta-sheet and are relatively more buried from solvent than are Cys87 and Cys90, which are positioned immediately before and within, respectively, an alpha-helix. In an effort to understand the origin of the stability of the metal complex, we have employed an anaerobic optical spectroscopic, competitive metal binding assay to determine the coordination geometry and association constants (Ka) for the binding of Co(II) to wild-type gp32 and a series of zinc ligand substitution mutants. At pH 7.5, 25 degrees C, wild-type gp32 binds Co(II) with a Ka approximately 1 x 10(9) M-1. Competition experiments reveal that Ka for Zn(II) is 3.0 (+/-1.0) x 10(11) M-1. We find that all non-native metal complexes retain tetrahedral or distorted tetrahedral coordination geometry but are greatly destabilized in a manner essentially of whether a new protein-derived coordination bond is formed (e.g., in H64C gp32) or not. Co(II) binding isotherms obtained for three His64 substitution mutants, H64C, H64D, and H64N gp32s, suggest that each mutant forms a dimeric Cys4 tetrathiolate intermediate complex at limiting [Co(II)]f, each then rearranges at high [Co(II)]f to form a monomolecular site of the expected geometry and Ka approximately 1 x 10(4) M-1. Like the His64 mutants, C77A gp32 appears to form at least two types of complexes over the course of a Co(II) titration: one with octahedral coordination geometry formed at low [Co(II)]f, with a second tetrahedral or five

  19. Synthesis and biological evaluation of substituted 2-benzoylpyridine thiosemicarbazones: novel structure-activity relationships underpinning their anti-proliferative and chelation efficacy.

    PubMed

    Lukmantara, Adeline Y; Kalinowski, Danuta S; Kumar, Naresh; Richardson, Des R

    2013-02-15

    The 2-benzoylpyridine thiosemicarbazone (BpT) chelators demonstrate potent anti-proliferative effects against tumor cells. To understand their structure-activity relationships, BpT analogues incorporating electron-donating substituents on the pyridine and phenyl rings of the BpT scaffold were designed and represent the first attempts to modify the pyridine ring of these thiosemicarbazones. Eight analogues showed significantly (p <0.001) greater anti-proliferative activity than the 'gold-standard' chelator, desferrioxamine. Structure-activity analysis revealed that mono- or di-methoxy substitution at the phenyl ring resulted in lower anti-proliferative activity, while methoxy substitutions at the phenyl ring enhanced iron chelation efficacy. These important findings facilitate the design of thiosemicarbazones with greater anti-tumor activity.

  20. 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.

  1. 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.

  2. Three novel acetylation sites in the Foxp3 transcription factor regulate the suppressive activity of regulatory T cells

    PubMed Central

    Kwon, Hye-Sook; Lim, Hyung W.; Wu, Jessica; Schnoelzer, Martina; Verdin, Eric; Ott, Melanie

    2012-01-01

    The Foxp3 transcription factor is the master regulator of regulatory T cell (Treg) differentiation and function. Its activity is regulated by reversible acetylation. Using mass spectrometry of immunoprecipitated proteins, we identify three novel acetylation sites in murine Foxp3 (K31, K262, and K267) and the corresponding sites in human FoxP3 proteins. Newly raised modification-specific antibodies against acetylated K31 and K267 confirm acetylation of these residues in murine Tregs. Mutant Foxp3 proteins carrying arginine substitutions at the three acetylation sites (3KR) accumulate in T cells to higher levels than wildtype Foxp3 and exert better suppressive activity in co-culture experiments. Acetylation and stability of wildtype, but not mutant, Foxp3 is enhanced when cells are treated with Ex-527, an inhibitor of the NAD+-dependent deacetylase SIRT1. Treatment with Ex-527 promotes Foxp3 expression during induced Treg differentiation, enhances Foxp3 levels in natural Tregs, and prevents loss of Foxp3 expression in adoptively transferred Tregs in mice. Our data identify SIRT1 as a negative regulator of Treg function via deacetylation of three novel target sites in Foxp3. SIRT1 inhibitors strengthen the suppressive activity of Tregs and may be useful in enhancing Treg-based therapeutic approaches to autoimmune diseases or graft rejections. PMID:22312127

  3. 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.

  4. 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.

  5. 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.

  6. Structural mutations that probe the interactions between the catalytic and dianion activation sites of triosephosphate isomerase.

    PubMed

    Zhai, Xiang; Amyes, Tina L; Wierenga, Rik K; Loria, J Patrick; Richard, John P

    2013-08-27

    Triosephosphate isomerase (TIM) catalyzes the isomerization of dihydroxyacetone phosphate to form d-glyceraldehyde 3-phosphate. The effects of two structural mutations in 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- and 80-fold decreases in (kcat/Km)E and (kcat/Km)E·HPi, respectively, for deprotonation of GA catalyzed by free TIM and by the TIM·HPO(3)(2-) complex. The mutation has little effect on the observed and intrinsic phosphite dianion binding energy or 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 with 208-YGGS. 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 provides 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.

  7. 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

  8. 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

  9. 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.

  10. 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

  11. Synthesis and analgesic activity of 1,3-dihydro-3-(substituted phenyl)imidazo[4,5-b]pyridin-2-ones and 3-(substituted phenyl)-1,2,3-triazolo[4,5-b]pyridines.

    PubMed

    Clark, R L; Pessolano, A A; Shen, T Y; Jacobus, D P; Jones, H; Lotti, V J; Flataker, L M

    1978-09-01

    In a study of nonsteroidal antiinflammatory and analgesic agents, a series of 1,3-dihydro-3-(substituted phenyl)imidazo[4,5-b]pyridin-2-ones-and 3-(substituted phenyl)triazolo[4,5-b]pyridines was prepared. Many of the imidazolones were alkylated on the free nitrogen. In a modified Randall-Selitto analgesic assay, the pain thresholds of both the inflamed and normal foot were elevated. This is not commonly observed with nonsteroidal antiinflammatory agents. The most active compounds were 1,3-dihydro-3[3,4-(methylenedioxy)phenyl]imidazo[4,5-b]pyridin-2-one (I-15) and its N-allyl (I-21) and N-isopropyl (I-121) derivatives. In the triazole series the 3-(2-fluoro- and 2,4-difluorophenyl)triazolo[4,5-b]pyridines (T-1 and T-8) were the best. The imidazole compounds were somewhat superior in analgesic activity to codeine and d-propoxyphene without showing any narcotic characteristics. Some of the compounds also possessed activity against carrageenan-induced foot edema in the rat, so these compounds represent a new class of nonnarcotic analgesic antiinflammatories, capable of producing a greater degree of analgesia than that obtainable with other nonsteroidal antiinflammatory agents.

  12. 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.

  13. Probing the active center of benzaldehyde lyase with substitutions and the pseudosubstrate analogue benzoylphosphonic acid methyl ester.

    PubMed

    Brandt, Gabriel S; Nemeria, Natalia; Chakraborty, Sumit; McLeish, Michael J; Yep, Alejandra; Kenyon, George L; Petsko, Gregory A; Jordan, Frank; Ringe, Dagmar

    2008-07-22

    Benzaldehyde lyase (BAL) catalyzes the reversible cleavage of ( R)-benzoin to benzaldehyde utilizing thiamin diphosphate and Mg (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 A (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.

  14. 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.

  15. 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

  16. Active site loop conformation regulates promiscuous activity in a lactonase from Geobacillus kaustophilus HTA426.

    PubMed

    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.

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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.

  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. Catch-and-Hold Activation of Muscle Acetylcholine Receptors Having Transmitter Binding Site Mutations

    PubMed Central

    Purohit, Prasad; Bruhova, Iva; Gupta, Shaweta; Auerbach, Anthony

    2014-01-01

    Agonists turn on receptors because their target sites have a higher affinity in the active versus resting conformation of the protein. We used single-channel electrophysiology to measure the lower-affinity (LA) and higher-affinity (HA) equilibrium dissociation constants for acetylcholine in adult-type muscle mouse nicotinic receptors (AChRs) having mutations of agonist binding site amino acids. For a series of agonists and for all mutations of αY93, αG147, αW149, αY190, αY198, εW55, and δW57, the change in LA binding energy was approximately half that in HA binding energy. The results were analyzed as a linear free energy relationship between LA and HA agonist binding, the slope of which (κ) gives the fraction of the overall binding chemical potential where the LA complex is established. The linear correlation between LA and HA binding energies suggests that the overall binding process is by an integrated mechanism (catch-and-hold). For the agonist and the above mutations, κ ∼ 0.5, but side-chain substitutions of two residues had a slope that was significantly higher (0.90; αG153) or lower (0.25; εP121). The results suggest that backbone rearrangements in loop B, loop C, and the non-α surface participate in both LA binding and the LA ↔ HA affinity switch. It appears that all of the intermediate steps in AChR activation comprise a single, energetically coupled process. PMID:24988344

  4. Amino acid substitutions in an alpha-helical antimicrobial arachnid peptide affect its chemical properties and biological activity towards pathogenic bacteria but improves its therapeutic index.

    PubMed

    Rodríguez, A; Villegas, E; Satake, H; Possani, L D; Corzo, Gerardo

    2011-01-01

    Four variants of the highly hemolytic antimicrobial peptide Pin2 were chemically synthesized with the aim to investigate the role of the proline residue in this peptide, by replacing it with the motif glycine-valine-glycine [GVG], which was found to confer low hemolytic activity in a spider antimicrobial peptide. The proline residue in position 14 of Pin2 was substituted by [V], [GV], [VG] and [GVG]. Only the peptide variant with the proline substituted for [GVG] was less hemolytic compared to that of all other variants. The peptide variant [GVG] kept its antimicrobial activity in Muller-Hilton agar diffusion assays, whereas the other three variants were less effective. However, all Pin2 antimicrobial peptide variants, were active when challenged against a Gram-positive bacteria in Muller-Hilton broth assays suggesting that chemical properties of the antimicrobial peptides such as hydrophobicity is an important indication for antimicrobial activity in semi-solid environments.

  5. 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

  6. Substitutions at the opening of the Rubisco central solvent channel affect holoenzyme stability and CO2/O 2 specificity but not activation by Rubisco activase.

    PubMed

    Esquivel, M Gloria; Genkov, Todor; Nogueira, Ana S; Salvucci, Michael E; Spreitzer, Robert J

    2013-12-01

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the initial step of carbon metabolism in photosynthesis. The holoenzyme comprises eight large subunits, arranged as a tetramer of dimers around a central solvent channel that defines a fourfold axis of symmetry, and eight small subunits, arranged as two tetramers at the poles of the axis. The phylogenetically divergent small-subunit loops between β-strands A and B form the entrance to the solvent channel. In the green alga Chlamydomonas reinhardtii, Ile-58 from each of the four small-subunit βA-βB loops defines the minimal diameter of the channel opening. To understand the role of the central solvent channel in Rubisco function, directed mutagenesis and transformation of Chlamydomonas were employed to replace Ile-58 with Ala, Lys, Glu, Trp, or three Trp residues (I58W3) to close the entrance to the channel. The I58E, I58K, and I58W substitutions caused only small decreases in photosynthetic growth at 25 and 35 °C, whereas I58W3 had a substantial effect at both temperatures. The mutant enzymes had decreased carboxylation rates, but the I58W3 enzyme had decreases in both carboxylation and CO2/O2 specificity. The I58E, I58W, and I58W3 enzymes were inactivated at lower temperatures than wild-type Rubisco, and were degraded at slower rates under oxidative stress. However, these mutant enzymes were activated by Rubisco activase at normal rates, indicating that the structural transition required for carboxylation is not affected by altering the solvent channel opening. Structural dynamics alone may not be responsible for these distant effects on the Rubisco active site.

  7. 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

  8. HIV-1 trans-activator of transcription substitutes for oxidative signaling in activation-induced T cell death.

    PubMed

    Gülow, Karsten; Kaminski, Marcin; Darvas, Katalin; Süss, Dorothee; Li-Weber, Min; Krammer, Peter H

    2005-05-01

    Termination of an immune response requires elimination of activated T lymphocytes by activation-induced cell death (AICD). In AICD, CD95 (Apo-1/Fas) ligand (L) triggers apoptosis of CD95-positive activated T lymphocytes. In AIDS patients, AICD is strongly enhanced and accelerated. We and others have previously shown that HIV-1 trans-activator of transcription (HIV-1 Tat) sensitizes T cells toward CD95-mediated apoptosis and up-regulates CD95L expression by affecting the cellular redox balance. In this study, we show that it is hydrogen peroxide (H(2)O(2)) that functions as an essential second messenger in TCR signaling. The H(2)O(2) signal combined with simultaneous calcium (Ca(2+)) influx into the cytosol constitutes the minimal requirement for induction of CD95L expression. Either signal alone is insufficient. We further show that HIV-1 Tat interferes with TCR signaling and induces a H(2)O(2) signal. H(2)O(2) generated by HIV-1 Tat combines with CD4-dependent calcium influx and causes massive T cell apoptosis. Thus, our data provide an explanation for CD4(+) T lymphocyte depletion during progression of AIDS.

  9. Metals in the active site of native protein phosphatase-1.

    PubMed

    Heroes, Ewald; Rip, Jens; Beullens, Monique; Van Meervelt, Luc; De Gendt, Stefan; Bollen, Mathieu

    2015-08-01

    Protein phosphatase-1 (PP1) is a major protein Ser/Thr phosphatase in eukaryotic cells. Its activity depends on two metal ions in the catalytic site, which were identified as manganese in the bacterially expressed phosphatase. However, the identity of the metal ions in native PP1 is unknown. In this study, total reflection X-ray fluorescence (TXRF) was used to detect iron and zinc in PP1 that was purified from rabbit skeletal muscle. Metal exchange experiments confirmed that the distinct substrate specificity of recombinant and native PP1 is determined by the nature of their associated metals. We also found that the iron level associated with native PP1 is decreased by incubation with inhibitor-2, consistent with a function of inhibitor-2 as a PP1 chaperone. PMID:25890482

  10. 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

  11. Zymogen Activation and Subcellular Activity of Subtilisin Kexin Isozyme 1/Site 1 Protease*

    PubMed Central

    da Palma, Joel Ramos; Burri, Dominique Julien; Oppliger, Joël; Salamina, Marco; Cendron, Laura; de Laureto, Patrizia Polverino; Seidah, Nabil Georges; Kunz, Stefan; Pasquato, Antonella

    2014-01-01

    The proprotein convertase subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P) plays crucial roles in cellular homeostatic functions and is hijacked by pathogenic viruses for the processing of their envelope glycoproteins. Zymogen activation of SKI-1/S1P involves sequential autocatalytic processing of its N-terminal prodomain at sites B′/B followed by the herein newly identified C′/C sites. We found that SKI-1/S1P autoprocessing results in intermediates whose catalytic domain remains associated with prodomain fragments of different lengths. In contrast to other zymogen proprotein convertases, all incompletely matured intermediates of SKI-1/S1P showed full catalytic activity toward cellular substrates, whereas optimal cleavage of viral glycoproteins depended on B′/B processing. Incompletely matured forms of SKI-1/S1P further process cellular and viral substrates in distinct subcellular compartments. Using a cell-based sensor for SKI-1/S1P activity, we found that 9 amino acid residues at the cleavage site (P1–P8) and P1′ are necessary and sufficient to define the subcellular location of processing and to determine to what extent processing of a substrate depends on SKI-1/S1P maturation. In sum, our study reveals novel and unexpected features of SKI-1/S1P zymogen activation and subcellular specificity of activity toward cellular and pathogen-derived substrates. PMID:25378398

  12. 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

  13. Multiple Transport-Active Binding Sites Are Available for a Single Substrate on Human P-Glycoprotein (ABCB1)

    PubMed Central

    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, [125I]-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 substrate

  14. 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.

  15. 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

  16. 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

  17. Hemoglobin substitutes.

    PubMed

    Anbari, Kevin K; Garino, Jonathan P; Mackenzie, Colin F

    2004-10-01

    Orthopaedic patients frequently require blood transfusions to treat peri-operative anemia. Research in the area of hemoglobin substitutes has been of great interest since it holds the promise of reducing the reliance on allogeneic blood transfusions. The three categories of hemoglobin substitutes are (1) cell-free, extracellular hemoglobin preparations made from human or bovine hemoglobin (hemoglobin-based oxygen carriers or HBOCs); (2) fluorine-substituted linear or cyclic carbon chains with a high oxygen-carrying capacity (perfluorocarbons); and (3) liposome-encapsulated hemoglobin. Of the three, HBOCs have been the most extensively studied and tested in preclinical and clinical trials that have shown success in diminishing the number of blood transfusions as well as an overall favorable side-effect profile. This has been demonstrated in vascular, cardiothoracic, and orthopaedic patients. HBOC-201, which is a preparation of cell-free bovine hemoglobin, has been approved for clinical use in South Africa. These products may well become an important tool for physicians treating peri-operative anemia in orthopaedic patients.

  18. A single amino acid substitution confers high cinchonidine oxidation activity comparable with that of rabbit to monkey aldehyde oxidase 1.

    PubMed

    Fukiya, Kensuke; Itoh, Kunio; Yamaguchi, Satoshi; Kishiba, Akiko; Adachi, Mayuko; Watanabe, Nobuaki; Tanaka, Yorihisa

    2010-02-01

    Aldehyde oxidase 1 (AOX1) is a major member of the xanthine oxidase family belonging to the class of complex molybdo-flavoenzymes and plays an important role in the nucleophilic oxidation of N-heterocyclic aromatic compounds and various aldehydes. The enzyme has been well known to show remarkable species differences. Comparing the rabbit and monkey enzymes, the former showed extremely high activity toward cinchonidine and methotrexate, but the latter exhibited only marginal activities. In contrast, monkey had several times greater activity than did rabbit toward zonisamide and (+)-4-(4-cyanoanilino)-5,6-dihydro-7-hydroxy-7H-cyclopenta[d]-pyrimidine [(S)-RS-8359]. In this report, we tried to confer high cinchonidine oxidation activity comparable with that of rabbit AOX1 to monkey AOX1. The chimera proteins prepared by restriction enzyme digestion and recombination methods between monkey and rabbit AOX1s indicated that the sequences from Asn993 to Ala1088 of rabbit AOX1 are essential for the activity. The kinetic parameters were then measured using monkey AOX1 mutants prepared by site-directed mutagenesis. The monkey V1085A mutant acquired the high cinchonidine oxidation activity. Inversely, the reciprocal rabbit A1081V mutant lost the activity entirely: amino acid 1081 of rabbit AOX1 corresponding to amino acid 1085 of monkey AOX1. Thus, cinchonidine oxidation activity was drastically changed by mutation of a single residue in AOX1. However, this might be true for bulky substrates such as cinchonidine but not for small substrates. The mechanism of substrate-dependent species differences in AOX1 activity toward bulky substrates is discussed.

  19. 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

  20. Effective charge on acetylcholinesterase active sites determined from the ionic strength dependence of association rate constants with cationic ligands.

    PubMed

    Nolte, H J; Rosenberry, T L; Neumann, E

    1980-08-01

    The reaction of the specific fluorescent cationic ligand N-methylacridinium with the active site of 11S acetylcholinesterase from electric eel was monitored by temperature-jump relaxation kinetics at a variety of ionic strengths. The ionic strength dependence of the bimolecular association rate constant is analyzed with a Brønsted-Debye-Hückel expression and leads to estimates of the association rate constant at zero ionic strength of K120 = 1.1 X 10(10) M-1 S-1 at 25 degrees C and the net charge number of the enzyme active site of ZE = -6.3. The ionic strength dependence of the second-order hydrolysis rate constant kcat/Kapp for acetylthiocholine under steady-state conditions is also very pronounced and indicates a value of ZE = -9. Thus, a large effective negative charge on the enzyme active site appears to be a general characteristic of its interaction with cationic ligands. The ionic strength dependence of Kcat/Kapp is identical with that of sodium chloride, sodium phosphate, and sodium citrate, thus ruling out any possibility that the phenomena arise from a specific, partially competitive binding of Na+ to the enzyme active site. Substitution of the calculated electrostatic parameters into theoretical equations indicates that the most significant effect of these ZE values is a 2-3 order of magnitude reduction in the rate constant for dissociation of the initial ligand-enzyme encounter complex; this decrease renders the bimolecular reaction diffusion controlled. The high value of k120 and the space requirements of six to nine charged groups suggest that regions of the enzyme surface area larger than the catalytic sites themselves are effective in trapping cationic ligands.

  1. 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

  2. Design, synthesis and in vitro antitumor activity of novel N-substituted-4-phenyl/benzylphthalazin-1-ones.

    PubMed

    Eldehna, Wagdy M; Ibrahim, Hany S; Abdel-Aziz, Hatem A; Farrag, Noha N; Youssef, Mohieldin M

    2015-01-01

    A novel series of N-substituted-4-phenylphthalazin-1-ones 14a-g bearing different anilines at the N-2 of phthalazin-1-one scaffold via acetyl-flexible linker was designed and synthesized for the development of potential anticancer agents. Compounds 19a-g were synthesized by insertion of methylene (CH2) bridge at C4-position of 14a-g to provide a flexibility for the phenyl group. The newly synthesized compounds 14a-g and 19a-g were evaluated for their anti-proliferative activity against three human tumor cell lines HepG2 hepatocellular carcinoma, HT-29 colon cancer and MCF-7 breast cancer. In particular, HepG2 and HT-29 cancer cell lines were more susceptible to the synthesized derivatives. Compound 19d (IC50 = 1.2 ± 0.09 μM) was found to be the most potent derivative against HepG2 as it was 2.9 times more active than doxorubicin (IC50 = 3.45 ± 0.54) and sorafenib (IC50 = 3.5 ± 1.04 μM). Compounds 14e, 14g, 19d and 19g with IC50 = (3.29 ± 0.45), (3.50 ± 0.846), (1.20 ± 0.09) and (3.52 ± 0.70) μM, respectively, were found to be active candidates against HepG2 cancer cells. Compounds 14e, 14g, 19d and 19g were able to induce apoptosis in HepG2, this was assured by; the significant increase in the percentage of annexin V-FITC-positive apoptotic cells (UR + LR), the down-regulation of the anti-apoptotic protein Bcl-2 and the up-regulation of the pro-apoptotic protein Bax, in addition to boosting caspase-3 levels. Moreover, cytotoxicity evaluation of the newly synthesized compounds in HT-29 revealed that compounds 14e, 14f, 19e and 19f (IC50 = 3.05 ± 0.78, 4.02 ± 1.18, 3.68 ± 0.79 and 2.98 ± 0.47 μM, respectively) were more potent than doxorubicin (IC50 = 7.70 ± 1.78 μM).

  3. 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.

  4. Evaluation of substituted methyl cyclohexanone hybrids for anti-tubercular, anti-bacterial and anti-fungal activity: Facile syntheses under catalysis by ionic liquids.

    PubMed

    Ghatole, Ajay M; Lanjewar, Kushal R; Gaidhane, Mahesh K; Hatzade, Kishor M

    2015-01-01

    An library of unresolved racemic Mannich bases incorporating two stereogenic carbon centers was evaluated for antibacterial activity against clinically isolated Gram-positive bacteria i.e. Staphylococcus aureus, Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa and for antifungal activity against Candida albicans strains. Additionally, the susceptibility of microorganisms to Mannich bases prompted us to evaluate the potential for anti-tubercular activity against clinically isolated Mycobacterium tuberculosis and virulent H37Rv strains. All compounds showed potent activity against M. tuberculosis strains at MIC ranging from 50 μg/mL to 6.25 μg/mL of concentration. Facile one pot Mannich type syntheses of rac-(2S)-2-[(R)-[(4-substituted phenyl){[4-(4-substituted phenyl)-1,3-thiazol-2-yl]amino}methyl]cyclohexanone derivatives were achieved by reactions of various 2-amino-4-aryl-thiazoles, appropriately substituted aromatic aldehydes and cyclohexanone in Bronsted acidic quaternary ammonium sulfated ionic liquids serving as dual solvents and catalysts.

  5. Evolution of a new function in an esterase: simple amino acid substitutions enable the activity present in the larger paralog, BioH.

    PubMed

    Flores, Humberto; Lin, Steven; Contreras-Ferrat, Gabriel; Cronan, John E; Morett, Enrique

    2012-08-01

    Gene duplication and divergence are essential processes for the evolution of new activities. Divergence may be gradual, involving simple amino acid residue substitutions, or drastic, such that larger structural elements are inserted, deleted or rearranged. Vast protein sequence comparisons, supported by some experimental evidence, argue that large structural modifications have been necessary for certain catalytic activities to evolve. However, it is not clear whether these activities could not have been attained by gradual changes. Interestingly, catalytic promiscuity could play a fundamental evolutionary role: a preexistent secondary activity could be increased by simple amino acid residue substitutions that do not affect the enzyme's primary activity. The promiscuous profile of the enzyme may be modified gradually by genetic drift, making a pool of potentially useful activities that can be selected before duplication. In this work, we used random mutagenesis and in vivo selection to evolve the Pseudomonas aeruginosa PAO1 carboxylesterase PA3859, a small protein, to attain the function of BioH, a much larger paralog involved in biotin biosynthesis. BioH was chosen as a target activity because it provides a highly sensitive selection for evolved enzymatic activities by auxotrophy complementation. After only two cycles of directed evolution, mutants with the ability to efficiently complement biotin auxotrophy were selected. The in vivo and in vitro characterization showed that the activity of one of our mutant proteins was similar to that of the wild-type BioH enzyme. Our results demonstrate that it is possible to evolve enzymatic activities present in larger proteins by discrete amino acid substitutions.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. Important role for phylogenetically invariant PP2Acalpha active site and C-terminal residues revealed by mutational analysis in Saccharomyces cerevisiae.

    PubMed Central

    Evans, D R; Hemmings, B A

    2000-01-01

    PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acalpha functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acalpha Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acalpha catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acalpha C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acalpha catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo. PMID:10978272

  12. Characterization of the active site of chloroperoxidase using physical techniques

    SciTech Connect

    Hall, K.S.

    1986-01-01

    Chloroperoxidase (CPO) and Cytochrome P-450, two very different hemeproteins, have been shown to have similar active sites by several techniques. Recent work has demonstrated thiolate ligation from a cysteine residue to the iron in P-450. A major portion of this research has been devoted to obtaining direct evidence that CPO also has a thiolate 5th ligand from a cysteine residue. This information will provide the framework for a detailed analysis of the structure-function relationships between peroxidases, catalase and cytochrome P-450 hemeproteins. To determine whether the 5th ligand is a cysteine, methionine or a unique amino acid, specific isotope enrichment experiments were used. Preliminary /sup 1/H-NMR studies show that the carbon monoxide-CPO complex has a peak in the upfield region corresponding to alpha-protons of a thiolate amino acid. C. fumago was grown on 95% D/sub 2/O media with a small amount of /sup 1/H-cysteine added. Under these conditions C. fumago slows down the biosynthesis of cysteine by at least 50% and utilizes the exogenous cysteine in the media. GC-MS was able to show that the methylene protons next to the sulfur atom in cysteine are 80-90% protonated while these positions in methionine are approximately 73% deuterated. Comparison of the /sup 1/H-NMR spectra of CO-CPO and CO-CPO indicate the presence of a cysteine ligand in chloroperoxidase.

  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. 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.

  15. 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

  16. Active Site Characterization of Proteases Sequences from Different Species of Aspergillus.

    PubMed

    Morya, V K; Yadav, Virendra K; Yadav, Sangeeta; Yadav, Dinesh

    2016-09-01

    A total of 129 proteases sequences comprising 43 serine proteases, 36 aspartic proteases, 24 cysteine protease, 21 metalloproteases, and 05 neutral proteases from different Aspergillus species were analyzed for the catalytically active site residues using MEROPS database and various bioinformatics tools. Different proteases have predominance of variable active site residues. In case of 24 cysteine proteases of Aspergilli, the predominant active site residues observed were Gln193, Cys199, His364, Asn384 while for 43 serine proteases, the active site residues namely Asp164, His193, Asn284, Ser349 and Asp325, His357, Asn454, Ser519 were frequently observed. The analysis of 21 metalloproteases of Aspergilli revealed Glu298 and Glu388, Tyr476 as predominant active site residues. In general, Aspergilli species-specific active site residues were observed for different types of protease sequences analyzed. The phylogenetic analysis of these 129 proteases sequences revealed 14 different clans representing different types of proteases with diverse active site residues.

  17. 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

  18. Cu substitution effects on the local magnetic properties of Ba(Fe(1-x)Cu(x))(2)As(2): a site-selective (75)As and (63)Cu NMR study.

    PubMed

    Takeda, Hikaru; Imai, Takashi; Tachibana, Makoto; Gaudet, Jonathan; Gaulin, Bruce D; Saparov, Bayrammurad I; Sefat, Athena S

    2014-09-12

    We take advantage of the site-selective nature of the ^{75}As and ^{63}Cu NMR techniques to probe the Cu substitution effects on the local magnetic properties of the FeAs planes in Ba(Fe_{1-x}Cu_{x})_{2}As_{2}. We show that the suppression of antiferromagnetic Fe spin fluctuations induced by Cu substitution is weaker than a naive expectation based on a simple rigid band picture, in which each Cu atom would donate three electrons to the FeAs planes. Comparison between ^{63}Cu and ^{75}As NMR data indicates that spin fluctuations are suppressed at the Cu and their neighboring Fe sites in the tetragonal phase, suggesting the strongly local nature of the Cu substitution effects. We attribute the absence of a large superconducting dome in the phase diagram of Ba(Fe_{1-x}Cu_{x})_{2}As_{2} to the emergence of a nearly magnetically ordered FeAs plane under the presence of orthorhombic distortion.

  19. Structure-activity relationship of prenyl-substituted polyphenols from Artocarpus heterophyllus as inhibitors of melanin biosynthesis in cultured melanoma cells.

    PubMed

    Arung, Enos Tangke; Shimizu, Kuniyoshi; Kondo, Ryuichiro

    2007-09-01

    A series of prenylated, flavone-based polyphenols, compounds 1-8, were isolated from the wood of Artocarpus heterophyllus. These compounds, which have previously been shown not to inhibit tyrosinase activity, were found to be active inhibitors of the in vivo melanin biosynthesis in B16 melanoma cells, with little or no cytotoxicity. To clarify the structural requirement for inhibition, some structure-activity relationships were studied, in comparison with related compounds lacking prenyl side chains. Our experiments indicate that both prenyl and OH groups, as well as the type of substitution pattern, are crucial for the inhibition of melanin production in B16 melanoma cells.

  20. Self-assembly and enhanced visible-light-driven photocatalytic activities of Bi2MoO6 by tungsten substitution

    NASA Astrophysics Data System (ADS)

    Yu, Hongguang; Zhu, Zhenfeng; Zhou, Jianhong; Wang, Jing; Li, Junqi; Zhang, Yanli

    2013-01-01

    The different compositions of Bi2MoO6 by tungsten substitution have been successfully synthesized via a facile hydrothermal process in the absence of a surfactant. All W-doped Bi2MoO6 samples were composed of nanosheets with similar orthorhombic Aurivillius layered structures. Bi2MoO6 samples show different phases varied with W content. For the Bi2MoO6 samples with the different nw values, their band gaps have changed obviously compared with Bi2MoO6 (2.69 eV) and Bi2WO6 (2.75 eV). The Bi2MoO6 by tungsten substitution exhibited good photocatalytic activity in degradation of Rhodamine-B under 500 W Xe lamp light irradiation. When nw value is 0.5, the sample has the highest photocatalytic activity for RhB photodecomposition under visible light irradiation. It shows that photocatalytic activities of Bi2MoO6 samples by W substitution are relevant to nanosheet morphology and size, the intrinsic layered structure, band gap and the W content.

  1. 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

  2. Chelate control of diiron(I) dithiolates relevant to the [Fe-Fe]- hydrogenase active site.

    PubMed

    Justice, Aaron K; Zampella, Giuseppe; De Gioia, Luca; Rauchfuss, Thomas B; van der Vlugt, Jarl Ivar; Wilson, Scott R

    2007-03-01

    The reaction of Fe2(S2C2H4)(CO)6 with cis-Ph2PCH=CHPPh2 (dppv) yields Fe2(S2C2H4)(CO)4(dppv), 1(CO)4, wherein the dppv ligand is chelated to a single iron center. NMR analysis indicates that in 1(CO)4, the dppv ligand spans axial and basal coordination sites. In addition to the axial-basal isomer, the 1,3-propanedithiolate and azadithiolate derivatives exist as dibasal isomers. Density functional theory (DFT) calculations indicate that the axial-basal isomer is destabilized by nonbonding interactions between the dppv and the central NH or CH2 of the larger dithiolates. The Fe(CO)3 subunit in 1(CO)4 undergoes substitution with PMe3 and cyanide to afford 1(CO)3(PMe3) and (Et4N)[1(CN)(CO)3], respectively. Kinetic studies show that 1(CO)4 reacts faster with donor ligands than does its parent Fe2(S2C2H4)(CO)6. The rate of reaction of 1(CO)4 with PMe3 was first order in each reactant, k = 3.1 x 10(-4) M(-1) s(-1). The activation parameters for this substitution reaction, DeltaH = 5.8(5) kcal/mol and DeltaS = -48(2) cal/deg.mol, indicate an associative pathway. DFT calculations suggest that, relative to Fe2(S2C2H4)(CO)6, the enhanced electrophilicity of 1(CO)4 arises from the stabilization of a "rotated" transition state, which is favored by the unsymmetrically disposed donor ligands. Oxidation of MeCN solutions of 1(CO)3(PMe3) with Cp2FePF6 yielded [Fe2(S2C2H4)(mu-CO)(CO)2(dppv)(PMe3)(NCMe)](PF6)2. Reaction of this compound with PMe3 yielded [Fe2(S2C2H4)(mu-CO)(CO)(dppv)(PMe3)2(NCMe)](PF6)2.

  3. 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...

  4. 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...

  5. 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...

  6. 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...

  7. 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...

  8. 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.

  9. GAS HYDRATES AT TWO SITES OF AN ACTIVE CONTINENTAL MARGIN.

    USGS Publications Warehouse

    Kvenvolden, K.A.

    1985-01-01

    Sediment containing gas hydrates from two distant Deep Sea Drilling Project sites (565 and 568), located about 670 km apart on the landward flank of the Middle America Trench, was studied to determine the geochemical conditions that characterize the occurrence of gas hydrates. Site 565 was located in the Pacific Ocean offshore the Nicoya Peninsula of Costa Rica in 3,111 m of water. The depth of the hole at this site was 328 m, and gas hydrates were recovered from 285 and 319 m. Site 568 was located about 670 km to the northwest offshore Guatemala in 2,031 m of water. At this site the hole penetrated to 418 m, and gas hydrates were encountered at 404 m.

  10. Control of active sites in selective flocculation: III -- Mechanism of site blocking

    SciTech Connect

    Behl, S.; Moudgil, B.M. . Dept. of Materials Science and Engineering)

    1993-12-01

    It has been shown in Parts I and II of this paper that heteroflocculation can be controlled by poisoning the sites for flocculant adsorption using a site blocking agent (SBA). An efficient SBA was determined to be the lower molecular weight fraction of the flocculant. In this paper, the underlying mechanism of SBA action is described. Also, the mathematical model detailed in Part I is used to determine the effect of different SBAs on apatite-dolomite separation efficiency. It has been demonstrated that the depression in flocculation is directly related to the site blocking parameter ([bar [Phi

  11. Dynamically achieved active site precision in enzyme catalysis.

    PubMed

    Klinman, Judith P

    2015-02-17

    CONSPECTUS: The grand challenge in enzymology is to define and understand all of the parameters that contribute to enzymes' enormous rate accelerations. The property of hydrogen tunneling in enzyme reactions has moved the focus of research away from an exclusive focus on transition state stabilization toward the importance of the motions of the heavy atoms of the protein, a role for reduced barrier width in catalysis, and the sampling of a protein conformational landscape to achieve a family of protein substates that optimize enzyme-substrate interactions and beyond. This Account focuses on a thermophilic alcohol dehydrogenase for which the chemical step of hydride transfer is rate determining across a wide range of experimental conditions. The properties of the chemical coordinate have been probed using kinetic isotope effects, indicating a transition in behavior below 30 °C that distinguishes nonoptimal from optimal C-H activation. Further, the introduction of single site mutants has the impact of either enhancing or eliminating the temperature dependent transition in catalysis. Biophysical probes, which include time dependent hydrogen/deuterium exchange and fluorescent lifetimes and Stokes shifts, have also been pursued. These studies allow the correlation of spatially resolved transitions in protein motions with catalysis. It is now possible to define a long-range network of protein motions in ht-ADH that extends from a dimer interface to the substrate binding domain across to the cofactor binding domain, over a distance of ca. 30 Å. The ongoing challenge to obtaining spatial and temporal resolution of catalysis-linked protein motions is discussed.

  12. Monoclonal antibody against the active site of caeruloplasmin and the ELISA system detecting active caeruloplasmin.

    PubMed

    Hiyamuta, S; Ito, K

    1994-04-01

    Serum caeruloplasmin deficiency is a characteristic biochemical abnormality found in patients with Wilson's disease, but the mechanism of this disease is unknown. Although the phenylenediamine oxidase activity of serum caeruloplasmin is markedly low in patients with Wilson's disease, mRNA of caeruloplasmin exists to some extent. To investigate the deficiency of caeruloplasmin oxidase activity in Wilson's disease, we generated 14 monoclonal antibodies (MAbs) and selected ID1, which had the strongest reactivity, and ID2, which had neutralizing ability. We also established a system to measure active caeruloplasmin specifically using these MAbs. These MAbs and the system will be useful tools in analyzing the active site of caeruloplasmin in patients with Wilson's disease.

  13. Evidence for a Dual Role of an Active Site Histidine in [alpha]-Amino-[beta]-carboxymuconate-[epsilon]-semialdehyde Decarboxylase

    SciTech Connect

    Huo, Lu; Fielding, Andrew J.; Chen, Yan; Li, Tingfeng; Iwaki, Hiroaki; Hosler, Jonathan P.; Chen, Lirong; Hasegawa, Yoshie; Que, Jr., Lawrence; Liu, Aimin

    2012-10-09

    The previously reported crystal structures of {alpha}-amino-{beta}-carboxymuconate-{epsilon}-semialdehyde decarboxylase (ACMSD) show a five-coordinate Zn(II)(His){sub 3}(Asp)(OH{sub 2}) active site. The water ligand is H-bonded to a conserved His228 residue adjacent to the metal center in ACMSD from Pseudomonas fluorescens (PfACMSD). Site-directed mutagenesis of His228 to tyrosine and glycine in this study results in a complete or significant loss of activity. Metal analysis shows that H228Y and H228G contain iron rather than zinc, indicating that this residue plays a role in the metal selectivity of the protein. As-isolated H228Y displays a blue color, which is not seen in wild-type ACMSD. Quinone staining and resonance Raman analyses indicate that the blue color originates from Fe(III)-tyrosinate ligand-to-metal charge transfer. Co(II)-substituted H228Y ACMSD is brown in color and exhibits an electron paramagnetic resonance spectrum showing a high-spin Co(II) center with a well-resolved {sup 59}Co (I = 7/2) eight-line hyperfine splitting pattern. The X-ray crystal structures of as-isolated Fe-H228Y (2.8 {angstrom}) and Co-substituted (2.4 {angstrom}) and Zn-substituted H228Y (2.0 {angstrom} resolution) support the spectroscopic assignment of metal ligation of the Tyr228 residue. The crystal structure of Zn-H228G (2.6 {angstrom}) was also determined. These four structures show that the water ligand present in WT Zn-ACMSD is either missing (Fe-H228Y, Co-H228Y, and Zn-H228G) or disrupted (Zn-H228Y) in response to the His228 mutation. Together, these results highlight the importance of His228 for PfACMSD's metal specificity as well as maintaining a water molecule as a ligand of the metal center. His228 is thus proposed to play a role in activating the metal-bound water ligand for subsequent nucleophilic attack on the substrate.

  14. Determination of the protease cleavage site repertoire—The RNase H but not the RT domain is essential for foamy viral protease activity

    SciTech Connect

    Spannaus, Ralf; Bodem, Jochen

    2014-04-15

    In contrast to orthoretroviruses, the foamy virus protease is only active as a protease-reverse transcriptase fusion protein and requires viral RNA for activation. Maturation of foamy viral proteins seems to be restricted to a single cleavage site in Gag and Pol. We provide evidence that unprocessed Gag is required for optimal infectivity, which is unique among retroviruses. Analyses of the cleavage site sequences of the Gag and Pol cleavage sites revealed a high similarity compared to those of Lentiviruses. We show that positions P2' and P2 are invariant and that Gag and Pol cleavage sites are processed with similar efficiencies. The RNase H domain is essential for protease activity, but can functionally be substituted by RNase H domains of other retroviruses. Thus, the RNase H domain might be involved in the stabilization of the protease dimer, while the RT domain is essential for RNA dependent protease activation. - Highlights: • Unprocessed Gag is required for optimal infectivity of foamy viruses. • Positions P2 and P2' are invariant in the foamy viral cleavage sites. • The RNaseH domain is essential for protease activity. • The RNaseH domains of other retroviruses support foamy viral protease activity.

  15. Synthesis, spectral characterization and biological activity of zinc(II) complexes with 3-substituted phenyl-4-amino-5-hydrazino-1, 2, 4-triazole Schiff bases

    NASA Astrophysics Data System (ADS)

    Singh, A. K.; Pandey, O. P.; Sengupta, S. K.

    New Zn(II) complexes have been synthesized by the reactions of zinc(II) acetate with Schiff bases derived from 3-substituted phenyl-4-amino-5-hydrazino-1, 2, 4-triazole and benzaldehyde, 2-hydroxyacetophenone or indoline-2,3-dione. All these complexes are soluble in DMF and DMSO; low molar conductance values indicate that they are non-electrolytes. Elemental analyses suggest that the complexes have 1:1 stoichiometry of the type [ZnL(H 2O) 2], [ZnL'(OAc) 2(H 2O) 2] (L = dianionic Schiff bases derived from 3-(substituted phenyl)-4-amino-5-hydrazino-1, 2, 4-triazole and 2-hydroxyacetophenone or indoline-2,3-dione; L' = neutral Schiff bases derived from 3-(substituted phenyl)-4-amino-5-hydrazino-1, 2, 4-triazole and benzaldehyde) and they were characterized by FT-IR, 1H NMR, 13C NMR and FAB mass. All these Schiff bases and their complexes have also been screened for their antibacterial activities against Bacillus subtilis, Escherichia coli and antifungal activities against Colletotrichum falcatum, Aspergillus niger, Fusarium oxysporium and Carvularia pallescence by petriplates methods.

  16. Robotics and Automation Activities at the Savannah River Site: A Site Report for SUBWOG 39F

    SciTech Connect

    Teese, G.D.

    1995-09-28

    The Savannah River Site has successfully used robots, teleoperators, and remote video to reduce exposure to ionizing radiation, improve worker safety, and improve the quality of operations. Previous reports have described the use of mobile teleoperators in coping with a high level liquid waste spill, the removal of highly contaminated equipment, and the inspection of nuclear reactor vessels. This report will cover recent applications at the Savannah River, as well as systems which SRS has delivered to other DOE site customers.

  17. Control of active sites in selective flocculation: II -- Role of site blocking agents

    SciTech Connect

    Behl, S.; Moudgil, B.M. . Dept. of Materials Science and Engineering)

    1993-12-01

    Control of heteroflocculation using a lower molecular weight fraction of the flocculant as a site blocking agent is demonstrated in the apatite-dolomite-polyethylene oxide system. The most effective SBA (site blocking agent) was determined to be the highest molecular weight fraction of the flocculant itself which was not capable of flocculating any of the components of the mixture. In the presence of the SBA, flocculant adsorption decreased significantly on apatite particles, thereby inhibiting coflocculation.

  18. Interspecies differences in the metabolism of methotrexate: An insight into the active site differences between human and rabbit aldehyde oxidase.

    PubMed

    Choughule, Kanika V; Joswig-Jones, Carolyn A; Jones, Jeffrey P

    2015-08-01

    Several drug compounds have failed in clinical trials due to extensive biotransformation by aldehyde oxidase (AOX) (EC 1.2.3.1). One of the main reasons is the difficulty in scaling clearance for drugs metabolised by AOX, from preclinical species to human. Using methotrexate as a probe substrate, we evaluated AOX metabolism in liver cytosol from human and commonly used laboratory species namely guinea pig, monkey, rat and rabbit. We found that the metabolism of methotrexate in rabbit liver cytosol was several orders of magnitude higher than any of the other species tested. The results of protein quantitation revealed that the amount of AOX1 in human liver was similar to rabbit liver. To understand if the observed differences in activity were due to structural differences, we modelled rabbit AOX1 using the previously generated human AOX1 homology model. Molecular docking of methotrexate into the active site of the enzyme led to the identification of important residues that could potentially be involved in substrate binding and account for the observed differences. In order to study the impact of these residue changes on enzyme activity, we used site directed mutagenesis to construct mutant AOX1 cDNAs by substituting nucleotides of human AOX1 with relevant ones of rabbit AOX1. AOX1 mutant proteins were expressed in Escherichia coli. Differences in the kinetic properties of these mutants have been presented in this study.

  19. Effect of substituting arginine and lysine with alanine on antimicrobial activity and the mechanism of action of a cationic dodecapeptide (CL(14-25)), a partial sequence of cyanate lyase from rice.

    PubMed

    Taniguchi, Masayuki; Takahashi, Nobuteru; Takayanagi, Tomohiro; Ikeda, Atsuo; Ishiyama, Yohei; Saitoh, Eiichi; Kato, Tetsuo; Ochiai, Akihito; Tanaka, Takaaki

    2014-01-01

    The antimicrobial activity of analogs obtained by substituting arginine and lysine in CL(14-25), a cationic α-helical dodecapeptide, with alanine against Porphyromonas gingivalis, a periodontal pathogen, varied significantly depending on the number and position of cationic amino acids. The alanine-substituted analogs had no hemolytic activity, even at a concentration of 1 mM. The antimicrobial activities of CL(K20A) and CL(K20A, K25A) were 3.8-fold and 9.1-fold higher, respectively, than that of CL(14-25). The antimicrobial activity of CL(R15A) was slightly lower than that of CL(14-25), suggesting that arginine at position 15 is not essential but is important for the antimicrobial activity. The experiments in which the alanine-substituted analogs bearing the replacement of arginine at position 24 and/or lysine at position 25 were used showed that arginine at position 24 was crucial for the antimicrobial activity whenever lysine at position 25 was substituted with alanine. Helical wheel projections of the alanine-substituted analogs indicate that the hydrophobicity in the vicinity of leucine at position 16 and alanines at positions 18 and/or 21 increased by substituting lysine at positions 20 and 25 with alanine, respectively. The degrees of diSC3 -5 release from P. gingivalis cells and disruption of GUVs induced by the alanine-substituted analogs with different positive charges were not closely related to their antimicrobial activities. The enhanced antimicrobial activities of the alanine-substituted analogs appear to be mainly attributable to the changes in properties such as hydrophobicity and amphipathic propensity due to alanine substitution and not to their extents of positive charge (cationicity).

  20. Vitreous Substitutes

    PubMed Central

    Foster, William Joseph

    2008-01-01

    Modern vitreoretinal surgery is a young science. While tremendous developments have occurred in instrument design and technique since Machemer first described vitrectomy surgery in 1973[1], the application of advanced materials concepts to the development of intra-ocular compounds is a particularly exciting area of research. To date, the development of vitreous substitutes has played a significant role in enabling the dramatic and progressive improvement in surgical outcome, but perhaps no other area of research has the potential to further improve the treatment of retinal detachment and other retinal disorders. While prior research has focused solely upon the ability of a compound to re-attach the retina, future research should seek to enable the surgeon to inhibit the development of proliferative vitreoretinopathy and re-detachment, the integration of stem-cell therapies with surgical retina, long-term delivery of medications to the posterior segment, and the promotion of more rapid and complete visual rehabilitation. PMID:19343097

  1. Blood substitutes.

    PubMed

    Palmer, Andre F; Intaglietta, Marcos

    2014-07-11

    The toxic side effects of early generations of red blood cell substitutes have stimulated development of more safe and efficacious high-molecular-weight polymerized hemoglobins, poly(ethylene glycol)-conjugated hemoglobins, and vesicle-encapsulated hemoglobins. Unfortunately, the high colloid osmotic pressure and blood plasma viscosity of these new-generation materials limit their application to blood concentrations that, in general, are not sufficient for full restoration of oxygen-carrying and -delivery capacity. However, these materials may serve as oxygen therapeutics for treating tissues affected by ischemia and trauma, particularly when the therapeutics are coformulated with antioxidants. These new oxygen therapeutics also possess additional beneficial effects owing to their optimal plasma expansion properties, which induce systemic supraperfusion that increases endothelial nitric oxide production and improves tissue washout of metabolic wastes, further contributing to their therapeutic role.

  2. Inhibition of trace element release during Fe(II)-activated recrystallization of Al-, Cr-, and Sn-substituted goethite and hematite.

    PubMed

    Frierdich, Andrew J; Scherer, Michelle 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 (1-8 mol %) 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 cosubstituted into the structure, and the total amount of release decreases exponentially with increasing cosubstituent. 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. PMID:22924460

  3. Inhibition of trace element release during Fe(II)-activated recrystallization of Al-, Cr-, and Sn-substituted goethite and hematite.

    PubMed

    Frierdich, Andrew J; Scherer, Michelle 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 (1-8 mol %) 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 cosubstituted into the structure, and the total amount of release decreases exponentially with increasing cosubstituent. 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.

  4. Structure-activity relationships of N-substituted 4-(trifluoromethoxy)benzamidines with affinity for GluN2B-containing NMDA receptors.

    PubMed

    Beinat, Corinne; Banister, Samuel D; Hoban, Jane; Tsanaktsidis, John; Metaxas, Athanasios; Windhorst, Albert D; Kassiou, Michael

    2014-02-01

    GluN2B subtype-selective NMDA antagonists represent promising therapeutic targets for the symptomatic treatment of multiple CNS pathologies. A series of N-benzyl substituted benzamidines were synthesised and the benzyl ring was further replaced with various polycyclic moieties. Compounds were evaluated for activity at GluN2B containing NMDA receptors where analogues 9, 12, 16 and 18 were the most potent of the series, replacement of the benzyl ring with polycycles resulted in a complete loss of activity. PMID:24412068

  5. The Influence of the Anionic Counter-Ion on the Activity of Ammonium Substituted Hoveyda-Type Olefin Metathesis Catalysts in Aqueous Media

    NASA Astrophysics Data System (ADS)

    Gułajski, Łukasz; Grela, Karol

    Polar olefin metathesis catalysts, bearing an ammonium group are presented. The electron withdrawing ammonium group not only activates the catalysts electronically, but at the same time makes the catalysts more hydrophilic. Catalysts can be therefore efficiently used not only in traditional media, such as methylene chloride and toluene, but also in technical-grade alcohols, alcohol— water mixtures and in neat water. Finally, in this overview the influence of the anionic counter-ion on the activity of ammonium substituted Hoveyda-type olefin metathesis catalysts in aqueous media is presented.

  6. Design, synthesis and biological assessment of novel N-substituted 3-(phthalimidin-2-yl)-2,6-dioxopiperidines and 3-substituted 2,6-dioxopiperidines for TNF-α inhibitory activity.

    PubMed

    Luo, Weiming; Yu, Qian-sheng; Salcedo, Isidro; Holloway, Harold W; Lahiri, Debomoy K; Brossi, Arnold; Tweedie, David; Greig, Nigel H

    2011-07-01

    Eight novel 2-(2,6-dioxopiperidin-3-yl)phthalimidine EM-12 dithiocarbamates 9 and 10, N-substituted 3-(phthalimidin-2-yl)-2,6-dioxopiperidines 11-14 and 3-substituted 2,6-dioxopiperidines 16 and 18 were synthesized as tumor necrosis factor-α (TNF-α) synthesis inhibitors. Synthesis involved utilization of a novel condensation approach, a one-pot reaction involving addition, iminium rearrangement and elimination, to generate the phthalimidine ring required for the creation of compounds 9-14. Agents were, thereafter, quantitatively assessed for their ability to suppress the synthesis on TNF-α in a lipopolysaccharide (LPS)-challenged mouse macrophage-like cellular screen, utilizing cultured RAW 264.7 cells. Whereas compounds 9, 14 and 16 exhibited potent TNF-α lowering activity, reducing TNF-α by up to 48% at 30 μM, compounds 12, 17 and 18 presented moderate TNF-α inhibitory action. The TNF-α lowering properties of these analogs proved more potent than that of revlimid (3) and thalidomide (1). In particular, N-dithiophthalimidomethyl-3-(phthalimidin-2-yl)-2,6-dioxopiperidine 14 not only possessed the greatest potency of the analogs to reduce TNF-α synthesis, but achieved this with minor cellular toxicity at 30 μM. The pharmacological focus of the presented compounds is towards the development of well-tolerated agents to ameliorate the neuroinflammation, that is, commonly associated with neurodegenerative disorders, epitomized by Alzheimer's disease and Parkinson's disease. PMID:21658960

  7. Quantitative structure-activity relationship analysis of substituted arylazo pyridone dyes in photocatalytic system: Experimental and theoretical study.

    PubMed

    Dostanić, J; Lončarević, D; Zlatar, M; Vlahović, F; Jovanović, D M

    2016-10-01

    A series of arylazo pyridone dyes was synthesized by changing the type of the substituent group in the diazo moiety, ranging from strong electron-donating to strong electron-withdrawing groups. The structural and electronic properties of the investigated dyes was calculated at the M062X/6-31+G(d,p) level of theory. The observed good linear correlations between atomic charges and Hammett σp constants provided a basis to discuss the transmission of electronic substituent effects through a dye framework. The reactivity of synthesized dyes was tested through their decolorization efficiency in TiO2 photocatalytic system (Degussa P-25). Quantitative structure-activity relationship analysis revealed a strong correlation between reactivity of investigated dyes and Hammett substituent constants. The reaction was facilitated by electron-withdrawing groups, and retarded by electron-donating ones. Quantum mechanical calculations was used in order to describe the mechanism of the photocatalytic oxidation reactions of investigated dyes and interpret their reactivities within the framework of the Density Functional Theory (DFT). According to DFT based reactivity descriptors, i.e. Fukui functions and local softness, the active site moves from azo nitrogen atom linked to benzene ring to pyridone carbon atom linked to azo bond, going from dyes with electron-donating groups to dyes with electron-withdrawing groups.

  8. Structure-activity relationships for interaction with multidrug resistance protein 2 (ABCC2/MRP2): the role of torsion angle for a series of biphenyl-substituted heterocycles.

    PubMed

    Lai, Yurong; Xing, Li; Poda, Gennadiy I; Hu, Yiding

    2007-06-01

    Multidrug resistance protein 2 (ABCC2/MRP2) is an ATP-binding cassette transporter involved in the absorption, distribution, and excretion of drugs and xenobiotics. Identifying compounds that are ABCC2/MRP2 substrates and/or inhibitors and understanding their structure-activity relationships (SARs) are important considerations in the selection and optimization of drug candidates. In the present study, the interactions between ABCC2/MRP2 and a series of biphenyl-substituted heterocycles were evaluated using Caco-2 cells and human ABCC2/MRP2 gene-transfected Madin-Darby canine kidney cells. It was observed that ABCC2/MRP2 transport and/or inhibition profile, both in nature and in magnitude, depends strongly on the substitution patterns of the biphenyl system. In particular, different ortho-substitutions cause various degrees of twisting between the two-phenyl rings, resulting in changing interactions between the ligands and ABCC2/MRP2. The compounds with small ortho functions (hydrogen, fluorine, and oxygen) and, thus, the ones displaying the smallest torsion angles of biphenyl (37-45 degrees) are neither substrates nor inhibitors of human ABCC2/MRP2. The transporter interactions increase as the steric bulkiness of the ortho-substitutions increase. When the tested compounds are 2-methyl substituted biphenyls, they exhibit moderate torsion angles (54-65 degrees) and behave as ABCC2/MRP2 substrates as well as mild inhibitors [10-40% compared with 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethyl-sulfanyl)methylsulfanyl] propionic acid (MK571)]. For the 2,2'-dimethyl substituted biphenyls, the torsions are enhanced (78-87 degrees) and so is the inhibition of ABCC2/MRP2. This class of compounds behaves as strong inhibitors of ABCC2/MRP2. These results can be used to define the three-dimensional structural requirements of ABCC2/MRP2 interaction with their substrates and inhibitors, as well as to provide SAR guidance to support drug discovery.

  9. A novel phosphorylation site at Ser130 adjacent to the pseudosubstrate domain contributes to the activation of protein kinase C-δ

    PubMed Central

    Gong, Jianli; Holewinski, Ronald J.; Van Eyk, Jennifer E.; Steinberg, Susan F.

    2016-01-01

    Protein kinase C-δ (PKCδ) is a signalling kinase that regulates many cellular responses. Although most studies focus on allosteric mechanisms that activate PKCδ at membranes, PKCδ also is controlled via multi-site phosphorylation [Gong et al. (2015) Mol. Cell. Biol. 35, 1727–1740]. The present study uses MS-based methods to identify PKCδ phosphorylation at Thr50 and Ser645 (in resting and PMA-treated cardiomyocytes) as well as Thr37, Thr38, Ser130, Thr164, Thr211, Thr215, Ser218, Thr295, Ser299 and Thr656 (as sites that increase with PMA). We focused on the consequences of phosphorylation at Ser130 and Thr141 (sites just N-terminal to the pseudosubstrate domain).We show that S130D and T141E substitutions</