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Sample records for active site substitution

  1. 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.

  2. Single-site substitutions improve cold activity and increase thermostability of the dehairing alkaline protease (DHAP).

    PubMed

    Zhao, Hong-Yan; Wu, Li-Ying; Liu, Gang; Feng, Hong

    2016-12-01

    To engineer dehairing alkaline protease (DHAP) variants to improve cold activity and increase thermostability so these variants are suitable for the leather processing industry. Based on previous studies with bacterial alkaline proteases, double-site mutations (W106K/V149I and W106K/M124L) were introduced into the DHAP from Bacillus pumilus. Compared with the wild-type DHAP hydrolytic activity, the double-site variant W106K/V149I showed an increase in specific hydrolytic activity at 15 °C by 2.3-fold toward casein in terms of hydrolytic rate and 2.7-fold toward the synthetic peptide AAPF-pN by means of kcat/Km value. The thermostability of the variant (W106K/V149I) was improved with the half-life at 60 and 70 °C increased by 2.7- and 5.0-fold, respectively, when compared with the thermostability of the wild-type DHAP. Conclusively, an increase in the cold activity and thermostability of a bacterial alkaline protease was achieved by protein engineering.

  3. New hypotheses for the binding mode of 4- and 7-substituted indazoles in the active site of neuronal nitric oxide synthase.

    PubMed

    Lohou, Elodie; Sopkova-de Oliveira Santos, Jana; Schumann-Bard, Pascale; Boulouard, Michel; Stiebing, Silvia; Rault, Sylvain; Collot, Valérie

    2012-09-01

    Taking into account the potency of 4- and 7-nitro and haloindazoles as nNOS inhibitors previously reported in the literature by our team, a multidisciplinary study, described in this article, has recently been carried out to elucidate their binding mode in the enzyme active site. Firstly, nitrogenous fastening points on the indazole building block have been investigated referring to molecular modeling hypotheses and thanks to the in vitro biological evaluation of N(1)- and N(2)-methyl and ethyl-4-substituted indazoles on nNOS. Secondly, we attempted to confirm the importance of the substitution in position 4 or 7 by a hydrogen bond acceptor group thanks to the synthesis and the in vitro biological evaluation of a new analogous 4-substituted derivative, the 4-cyanoindazole. Finally, by opposition to previous hypotheses describing NH function in position 1 of the indazole as a key fastening point, the present work speaks in favour of a crucial role of nitrogen in position 2.

  4. 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.

  5. Two synthetic Sp1-binding sites functionally substitute for the 21-base-pair repeat region to activate simian virus 40 growth in CV-1 cells.

    PubMed Central

    Lednicky, J; Folk, W R

    1992-01-01

    The 21-bp repeat region of simian virus 40 (SV40) activates viral transcription and DNA replication and contains binding sites for many cellular proteins, including Sp1, LSF, ETF, Ap2, Ap4, GT-1B, H16, and p53, and for the SV40 large tumor antigen. We have attempted to reduce the complexity of this region while maintaining its growth-promoting capacity. Deletion of the 21-bp repeat region from the SV40 genome delays the expression of viral early proteins and DNA replication and reduces virus production in CV-1 cells. Replacement of the 21-bp repeat region with two copies of DNA sequence motifs bound with high affinities by Sp1 promotes SV40 growth in CV-1 cells to nearly wild-type levels, but substitution by motifs bound less avidly by Sp1 or bound by other activator proteins does not restore growth. This indicates that Sp1 or a protein with similar sequence specificity is primarily responsible for the function of the 21-bp repeat region. We speculate about how Sp1 activates both SV40 transcription and DNA replication. Images PMID:1328672

  6. Action at a distance: amino acid substitutions that affect binding of the phosphorylated CheY response regulator and catalysis of dephosphorylation can be far from the CheZ phosphatase active site.

    PubMed

    Freeman, Ashalla M; Mole, Beth M; Silversmith, Ruth E; Bourret, Robert B

    2011-09-01

    Two-component regulatory systems, in which phosphorylation controls the activity of a response regulator protein, provide signal transduction in bacteria. For example, the phosphorylated CheY response regulator (CheYp) controls swimming behavior. In Escherichia coli, the chemotaxis phosphatase CheZ stimulates the dephosphorylation of CheYp. CheYp apparently binds first to the C terminus of CheZ and then binds to the active site where dephosphorylation occurs. The phosphatase activity of the CheZ(2) dimer exhibits a positively cooperative dependence on CheYp concentration, apparently because the binding of the first CheYp to CheZ(2) is inhibited compared to the binding of the second CheYp. Thus, CheZ phosphatase activity is reduced at low CheYp concentrations. The CheZ21IT gain-of-function substitution, located far from either the CheZ active site or C-terminal CheY binding site, enhances CheYp binding and abolishes cooperativity. To further explore mechanisms regulating CheZ activity, we isolated 10 intragenic suppressor mutations of cheZ21IT that restored chemotaxis. The suppressor substitutions were located along the central portion of CheZ and were not allele specific. Five suppressor mutants tested biochemically diminished the binding of CheYp and/or the catalysis of dephosphorylation, even when the suppressor substitutions were distant from the active site. One suppressor mutant also restored cooperativity to CheZ21IT. Consideration of results from this and previous studies suggests that the binding of CheYp to the CheZ active site (not to the C terminus) is rate limiting and leads to cooperative phosphatase activity. Furthermore, amino acid substitutions distant from the active site can affect CheZ catalytic activity and CheYp binding, perhaps via the propagation of structural or dynamic perturbations through a helical bundle.

  7. Substitution of Feline Leukemia Virus Long Terminal Repeat Sequences into Murine Leukemia Virus Alters the Pattern of Insertional Activation and Identifies New Common Insertion Sites

    PubMed Central

    Johnson, Chassidy; Lobelle-Rich, Patricia A.; Puetter, Adriane; Levy, Laura S.

    2005-01-01

    The recombinant retrovirus, MoFe2-MuLV (MoFe2), was constructed by replacing the U3 region of Moloney murine leukemia virus (M-MuLV) with homologous sequences from the FeLV-945 LTR. NIH/Swiss mice neonatally inoculated with MoFe2 developed T-cell lymphomas of immature thymocyte surface phenotype. MoFe2 integrated infrequently (0 to 9%) near common insertion sites (CISs) previously identified for either parent virus. Using three different strategies, CISs in MoFe2-induced tumors were identified at six loci, none of which had been previously reported as CISs in tumors induced by either parent virus in wild-type animals. Two of the newly identified CISs had not previously been implicated in lymphoma in any retrovirus model. One of these, designated 3-19, encodes the p101 regulatory subunit of phosphoinositide-3-kinase-gamma. The other, designated Rw1, is predicted to encode a protein that functions in the immune response to virus infection. Thus, substitution of FeLV-945 U3 sequences into the M-MuLV long terminal repeat (LTR) did not alter the target tissue for M-MuLV transformation but significantly altered the pattern of CIS utilization in the induction of T-cell lymphoma. These observations support a growing body of evidence that the distinctive sequence and/or structure of the retroviral LTR determines its pattern of insertional activation. The findings also demonstrate the oligoclonal nature of retrovirus-induced lymphomas by demonstrating proviral insertions at CISs in subdominant populations in the tumor mass. Finally, the findings demonstrate the utility of novel recombinant retroviruses such as MoFe2 to contribute new genes potentially relevant to the induction of lymphoid malignancy. PMID:15596801

  8. 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

  9. Ambi-site substitution of Mn in lanthanum germanate apatites

    SciTech Connect

    Kendrick, E.; Knight, K.S.; Slater, P.R.

    2009-08-05

    A neutron diffraction study at 4 K of the Mn doped lanthanum germanate apatite-type oxide ion conductor of nominal starting composition 'La{sub 9.5}Mn{sub 0.5}(GeO{sub 4}){sub 6}O{sub 2.75}' is reported. The structure was refined in space group P6{sub 3}/m, although high thermal displacement parameters were observed for the oxide ion sites (particularly O3, and O4). Reduced thermal displacement parameters were obtained by splitting the O3 site, and allowing the O4 oxygen to move off site, which may indicate local regions of lower symmetry within the structure. In addition, the data suggested ambi-site substitution of Mn, with it being present on both the Ge site and the La site. Assuming no change in La:Mn:Ge ratio, a composition of La{sub 9.18}Mn{sub 0.28}(GeO{sub 4}){sub 5.8}(MnO{sub 4}){sub 0.2}O{sub 2} was determined. As such there are nominally no interstitial oxide ions, but rather cation vacancies on the La site. Therefore, the high conductivity for this sample is most likely related to the introduction of Frenkel-type defects at higher temperature, as previously proposed for other apatite-type systems containing vacancies on the La site.

  10. Following the Formation of Active Co(III) Sites in Cobalt Substituted Aluminophosphates Catalysts by In-Situ Combined UV-VIS/XAFS/XRD Technique

    SciTech Connect

    Sankar, Gopinathan; Fiddy, Steven; Harvey, Ian; Hayama, Shusaku; Bushnell-Wye, Graham; Beale, Andrew M.

    2007-02-02

    Cobalt substituted aluminophosphates, CoAlPO-34 (Chabazite structure) and DAF-8 (Phillipsite structure) were investigated by in situ combined XRD/EXAFS/UV-VIS technique. In-situ combined XRD, Co K-edge EXAFS and UV-Vis measurements carried out during the calcination process reveal that CoAlPO-34 containing 10 wt percent cobalt is stable and the cobalt ions are converted from Co(II) in the as synthesised form to Co(III); DAF-8 containing about 25 percent cobalt is not stable and does not show change in oxidation state.

  11. 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

    2016-05-04

    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).

  12. Active Sites of Reduced Epidermal Fluorescence1 (REF1) Isoforms Contain Amino Acid Substitutions That Are Different between Monocots and Dicots

    PubMed Central

    Missihoun, Tagnon D.; Kotchoni, Simeon O.; Bartels, Dorothea

    2016-01-01

    Plant aldehyde dehydrogenases (ALDHs) play important roles in cell wall biosynthesis, growth, development, and tolerance to biotic and abiotic stresses. The Reduced Epidermal Fluorescence1 is encoded by the subfamily 2C of ALDHs and was shown to oxidise coniferaldehyde and sinapaldehyde to ferulic acid and sinapic acid in the phenylpropanoid pathway, respectively. This knowledge has been gained from works in the dicotyledon model species Arabidopsis thaliana then used to functionally annotate ALDH2C isoforms in other species, based on the orthology principle. However, the extent to which the ALDH isoforms differ between monocotyledons and dicotyledons has rarely been accessed side-by-side. In this study, we used a phylogenetic approach to address this question. We have analysed the ALDH genes in Brachypodium distachyon, alongside those of other sequenced monocotyledon and dicotyledon species to examine traits supporting either a convergent or divergent evolution of the ALDH2C/REF1-type proteins. We found that B. distachyon, like other grasses, contains more ALDH2C/REF1 isoforms than A. thaliana and other dicotyledon species. Some amino acid residues in ALDH2C/REF1 isoforms were found as being conserved in dicotyledons but substituted by non-equivalent residues in monocotyledons. One example of those substitutions concerns a conserved phenylalanine and a conserved tyrosine in monocotyledons and dicotyledons, respectively. Protein structure modelling suggests that the presence of tyrosine would widen the substrate-binding pocket in the dicotyledons, and thereby influence substrate specificity. We discussed the importance of these findings as new hints to investigate why ferulic acid contents and cell wall digestibility differ between the dicotyledon and monocotyledon species. PMID:27798665

  13. 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.

  14. The I427T neuraminidase (NA) substitution, located outside the NA active site of an influenza A(H1N1)pdm09 variant with reduced susceptibility to NA inhibitors, alters NA properties and impairs viral fitness.

    PubMed

    Tu, Véronique; Abed, Yacine; Barbeau, Xavier; Carbonneau, Julie; Fage, Clément; Lagüe, Patrick; Boivin, Guy

    2017-01-01

    Emergence of pan neuraminidase inhibitor (NAI)-resistant variants constitutes a serious clinical concern. An influenza A(H1N1)pdm09 variant containing the I427T/Q313R neuraminidase (NA) substitutions was previously identified in a surveillance study. Although these changes are not part of the NA active site, the variant showed reduced susceptibility to many NAIs. In this study, we investigated the mechanism of resistance for the I427T/Q313R substitution and its impact on the NA enzyme and viral fitness. Recombinant wild-type (WT), I427T/Q313R and I427T A(H1N1)pdm09 viruses were generated by reverse genetics and tested for their drug susceptibilities, enzymatic properties and replication kinetics in vitro as well as their virulence in mice. Molecular dynamics (MD) simulations were performed for NA structural analysis. The I427T substitution, which was responsible for the resistance phenotype observed in the double (I427T/Q313R) mutant, induced 17-, 56-, 7-, and 14-fold increases in IC50 values against oseltamivir, zanamivir, peramivir and laninamivir, respectively. The I427T substitution alone or combined to Q313R significantly reduced NA affinity. The I427T/Q313R and to a lesser extent I427T recombinant viruses displayed reduced viral titers vs WT in vitro. In experimentally-infected mice, the mortality rates were 62.5%, 0% and 14.3% for the WT, I417T/Q313R and I427T viruses, respectively. There were about 2.5- and 2-Log reductions in mean lung viral titers on day 5 post-infection for the I427T/Q313R and I427T mutants, respectively, compared to WT. Results from simulations revealed that the I427T change indirectly altered the stability of the catalytic R368 residue of the NA enzyme causing its reduced binding to the substrate/inhibitor. This study demonstrates that the I427T/Q313R mutant, not only alters NAI susceptibility but also compromises NA properties and viral fitness, which could explain its infrequent detection in clinic.

  15. Evolution of lactate dehydrogenase-A homologs of barracuda fishes (genus Sphyraena) from different thermal environments: differences in kinetic properties and thermal stability are due to amino acid substitutions outside the active site.

    PubMed

    Holland, L Z; McFall-Ngai, M; Somero, G N

    1997-03-18

    Orthologous homologs of lactate dehydrogenase-A (LDH-A) (EC 1.1.1.27; NAD+:lactate oxidoreductase) of six barracuda species (genus Sphyraena) display differences in Michaelis-Menten constants (apparent Km) for substrate (pyruvate) and cofactor (NADH) that reflect evolution at different habitat temperatures. Significant increases in Km with increasing measurement temperature occur for all homologs, yet Km at normal body temperatures is similar among species because of the inverse relationship between adaptation temperature and Km. Thermal stabilities of the homologs also differ. To determine the amino acid substitutions responsible for differences in Km and thermal stability, peptide mapping of the LDH-As of all six species was first performed. Then, the amino acid sequences of the three homologs having the most similar peptide maps, those of the north temperate species, S. argentea, the subtropical species, S. lucasana, and the south temperate species, S. idiastes, were deduced from the respective cDNA sequences. At most, there were four amino acid substitutions between any pair of species, none of which occurred in the loop or substrate binding sites of the enzymes. The sequence of LDH-A from S. lucasana differs from that of S. idiastes only at position 8. The homolog of S. argentea differs from the other two sequences at positions 8, 61, 68, and 223. We used a full-length cDNA clone of LDH-A of S. lucasana to test, by site-directed mutagenesis, the importance of these sequence changes in establishing the observed differences in kinetics and thermal stability. Differences in sequence at sites 61 and/or 68 appear to account for the differences in Km between the LDH-As of S. argentea and S. lucasana. Differences at position 8 appear to account for the difference in thermal stability between the homologs of S. argentea and S. lucasana. Evolutionary adaptation of proteins to temperature thus may be achieved by minor changes in sequence at locations outside of active

  16. 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.

  17. List 9 - Active CERCLIS Sites:

    EPA Pesticide Factsheets

    The List 9 displays the sequence of activities undertaken at active CERCLIS sites. An active site is one at which site assessment, removal, remedial, enforcement, cost recovery, or oversight activities are being planned or conducted.

  18. Design of an adenosine phosphorylase by active-site modification of murine purine nucleoside phosphorylase. Enzyme kinetics and molecular dynamics simulation of Asn-243 and Lys-244 substitutions of purine nucleoside phosphorylase.

    PubMed

    Maynes, J T; Yam, W; Jenuth, J P; Gang Yuan, R; Litster, S A; Phipps, B M; Snyder, F F

    1999-12-01

    Our objective was to alter the substrate specificity of purine nucleoside phosphorylase such that it would catalyse the phosphorolysis of 6-aminopurine nucleosides. We modified both Asn-243 and Lys-244 in order to promote the acceptance of the C6-amino group of adenosine. The Asn-243-Asp substitution resulted in an 8-fold increase in K(m) for inosine from 58 to 484 microM and a 1000-fold decrease in k(cat)/K(m). The Asn-243-Asp construct catalysed the phosphorolysis of adenosine with a K(m) of 45 microM and a k(cat)/K(m) 8-fold that with inosine. The Lys-244-Gln construct showed only marginal reduction in k(cat)/K(m), 83% of wild type, but had no activity with adenosine. The Asn-243-Asp;Lys-244-Gln construct had a 14-fold increase in K(m) with inosine and 7-fold decrease in k(cat)/K(m) as compared to wild type. This double substitution catalysed the phosphorolysis of adenosine with a K(m) of 42 microM and a k(cat)/K(m) twice that of the single Asn-243-Asp substitution. Molecular dynamics simulation of the engineered proteins with adenine as substrate revealed favourable hydrogen bond distances between N7 of the purine ring and the Asp-243 carboxylate at 2.93 and 2.88 A, for Asn-243-Asp and the Asn-243-Asp;Lys-244-Gln constructs respectively. Simulation also supported a favourable hydrogen bond distance between the purine C6-amino group and Asp-243 at 2.83 and 2.88 A for each construct respectively. The Asn-243-Thr substitution did not yield activity with adenosine and simulation gave unfavourable hydrogen bond distances between Thr-243 and both the C6-amino group and N7 of the purine ring. The substitutions were not in the region of phosphate binding and the apparent S(0.5) for phosphate with wild type and the Asn-243-Asp enzymes were 1.35+/-0.01 and 1.84+/-0.06 mM, respectively. Both proteins exhibited positive co-operativity with phosphate giving Hill coefficients of 7.9 and 3.8 respectively.

  19. Design of an adenosine phosphorylase by active-site modification of murine purine nucleoside phosphorylase. Enzyme kinetics and molecular dynamics simulation of Asn-243 and Lys-244 substitutions of purine nucleoside phosphorylase.

    PubMed Central

    Maynes, J T; Yam, W; Jenuth, J P; Gang Yuan, R; Litster, S A; Phipps, B M; Snyder, F F

    1999-01-01

    Our objective was to alter the substrate specificity of purine nucleoside phosphorylase such that it would catalyse the phosphorolysis of 6-aminopurine nucleosides. We modified both Asn-243 and Lys-244 in order to promote the acceptance of the C6-amino group of adenosine. The Asn-243-Asp substitution resulted in an 8-fold increase in K(m) for inosine from 58 to 484 microM and a 1000-fold decrease in k(cat)/K(m). The Asn-243-Asp construct catalysed the phosphorolysis of adenosine with a K(m) of 45 microM and a k(cat)/K(m) 8-fold that with inosine. The Lys-244-Gln construct showed only marginal reduction in k(cat)/K(m), 83% of wild type, but had no activity with adenosine. The Asn-243-Asp;Lys-244-Gln construct had a 14-fold increase in K(m) with inosine and 7-fold decrease in k(cat)/K(m) as compared to wild type. This double substitution catalysed the phosphorolysis of adenosine with a K(m) of 42 microM and a k(cat)/K(m) twice that of the single Asn-243-Asp substitution. Molecular dynamics simulation of the engineered proteins with adenine as substrate revealed favourable hydrogen bond distances between N7 of the purine ring and the Asp-243 carboxylate at 2.93 and 2.88 A, for Asn-243-Asp and the Asn-243-Asp;Lys-244-Gln constructs respectively. Simulation also supported a favourable hydrogen bond distance between the purine C6-amino group and Asp-243 at 2.83 and 2.88 A for each construct respectively. The Asn-243-Thr substitution did not yield activity with adenosine and simulation gave unfavourable hydrogen bond distances between Thr-243 and both the C6-amino group and N7 of the purine ring. The substitutions were not in the region of phosphate binding and the apparent S(0.5) for phosphate with wild type and the Asn-243-Asp enzymes were 1.35+/-0.01 and 1.84+/-0.06 mM, respectively. Both proteins exhibited positive co-operativity with phosphate giving Hill coefficients of 7.9 and 3.8 respectively. PMID:10567244

  20. CLCAs - A Family of Metalloproteases of Intriguing Phylogenetic Distribution and with Cases of Substituted Catalytic Sites

    PubMed Central

    Lenart, Anna; Dudkiewicz, Małgorzata; Grynberg, Marcin; Pawłowski, Krzysztof

    2013-01-01

    The zinc-dependent metalloproteases with His-Glu-x-x-His (HExxH) active site motif, zincins, are a broad group of proteins involved in many metabolic and regulatory functions, and found in all forms of life. Human genome contains more than 100 genes encoding proteins with known zincin-like domains. A survey of all proteins containing the HExxH motif shows that approximately 52% of HExxH occurrences fall within known protein structural domains (as defined in the Pfam database). Domain families with majority of members possessing a conserved HExxH motif include, not surprisingly, many known and putative metalloproteases. Furthermore, several HExxH-containing protein domains thus identified can be confidently predicted to be putative peptidases of zincin fold. Thus, we predict zincin-like fold for eight uncharacterised Pfam families. Besides the domains with the HExxH motif strictly conserved, and those with sporadic occurrences, intermediate families are identified that contain some members with a conserved HExxH motif, but also many homologues with substitutions at the conserved positions. Such substitutions can be evolutionarily conserved and non-random, yet functional roles of these inactive zincins are not known. The CLCAs are a novel zincin-like protease family with many cases of substituted active sites. We show that this allegedly metazoan family has a number of bacterial and archaeal members. An extremely patchy phylogenetic distribution of CLCAs in prokaryotes and their conserved protein domain composition strongly suggests an evolutionary scenario of horizontal gene transfer (HGT) from multicellular eukaryotes to bacteria, providing an example of eukaryote-derived xenologues in bacterial genomes. Additionally, in a protein family identified here as closely homologous to CLCA, the CLCA_X (CLCA-like) family, a number of proteins is found in phages and plasmids, supporting the HGT scenario. PMID:23671590

  1. 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.

  2. 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.

  3. Syntheses and antimalarial activities of N-substituted 11-azaartemisinins.

    PubMed

    Torok, D S; Ziffer, H; Meshnick, S R; Pan, X Q; Ager, A

    1995-12-22

    A two-step reaction sequence between artemisinin and methanolic ammonia followed by treatment with Amberlyst 15 yielded 11-azaartemisinin in 65% yield. Substituting a variety of primary alkyl- and heteroaromatic amines for ammonia in the reaction sequence yields N-substituted 11-azaartemisinins in similar or greater yield. When Amberlyst 15 is replaced by a mixture of sulfuric acid/silica gel, both 11-azaartemisinin and the expected metabolite, 10-azadesoxyartemisinin, are formed in 45% and 15% yields, respectively. In vitro and in vivo test data for a number of novel N-substituted 11-azaartemisinins, against drug-resistant strains of Plasmodium falciparum, show they possess antimalarial activities equal to or greater than that of artemisinin. The most active derivative, N-(2'-acetaldehydo)-11-azaartemisinin, 17, was 26 times more active in vitro and 4 times more active in vivo than artemisinin.

  4. 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.

  5. Antimicrobial activity of various 4- and 5-substituted 1-phenylnaphthalenes

    PubMed Central

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

    2014-01-01

    Bacterial cell division occurs in conjunction with the formation of a cytokinetic Z-ring structure comprised of FtsZ subunits. Agents that can disrupt Z-ring formation have the potential, through this unique mechanism, to be effective against several of the newly emerging multi-drug resistant strains of infectious bacteria. 1- and 12-Aryl substituted benzo[c]phenanthridines have been identified as antibacterial agents that could exert their activity by disruption of Z-ring formation. Substituted 4- and 5-amino-1-phenylnaphthalenes represent substructures within the pharmacophore of these benzo[c]phenanthridines. Several 4- and 5-substituted 1-phenylnaphthalenes were synthesized and evaluated for antibacterial activity against Staphylococcus aureus and Enterococcus faecalis. The impact of select compounds on the polymerization dynamics of S. aureus FtsZ was also assessed. PMID:23314053

  6. 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)

  7. Chemical reactivity and antimicrobial activity of N-substituted maleimides.

    PubMed

    Salewska, Natalia; Boros-Majewska, Joanna; Lącka, Izabela; Chylińska, Katarzyna; Sabisz, Michał; Milewski, Sławomir; Milewska, Maria J

    2012-02-01

    Several N-substituted maleimides containing substituents of varying bulkiness and polarity were synthesised and tested for antimicrobial and cytostatic activity. Neutral maleimides displayed relatively strong antifungal effect minimum inhibitory concentrations (MICs in the 0.5-4 µg ml(-1) range); their antibacterial activity was structure dependent and all were highly cytostatic, with IC(50) values below 0.1 µg ml(-1). Low antimicrobial but high cytostatic activity was noted for basic maleimides containing tertiary aminoalkyl substituents. Chemical reactivity and lipophilicity influenced antibacterial activity of neutral maleimides but had little if any effect on their antifungal and cytostatic action. N-substituted maleimides affected biosynthesis of chitin and β(1,3)glucan, components of the fungal cell wall. The membrane enzyme, β(1,3)glucan synthase has been proposed as a putative primary target of N-ethylmaleimide and some of its analogues in Candida albicans cells.

  8. Missense substitutions reflecting regulatory control of transmitter phosphatase activity in two-component signalling.

    PubMed

    Huynh, TuAnh Ngoc; Noriega, Chris E; Stewart, Valley

    2013-05-01

    Negative control in two-component signal transduction results from sensor transmitter phosphatase activity for phospho-receiver dephosphorylation. A hypothetical mechanism for this reaction involves a catalytic residue in the H-box active-site region. However, a complete understanding of transmitter phosphatase regulation is hampered by the abundance of kinase-competent, phosphatase-defective missense substitutions (K(+) P(-) phenotype) outside of the active-site region. For the Escherichia coli NarX sensor, a model for the HisKA_3 sequence family, DHp domain K(+) P(-) mutants defined two classes. Interaction mutants mapped to the active site-distal base of the DHp helix 1, whereas conformation mutants were affected in the X-box region of helix 2. Thus, different types of perturbations can influence transmitter phosphatase activity indirectly. By comparison, K(+) P(-) substitutions in the HisKA sensors EnvZ and NtrB additionally map to a third region, at the active site-proximal top of the DHp helix 1, independently identified as important for DHp-CA domain interaction in this sensor class. Moreover, the NarX transmitter phosphatase activity was independent of nucleotides, in contrast to the activity for many HisKA family sensors. Therefore, distinctions involving both the DHp and the CA domains suggest functional diversity in the regulation of HisKA and HisKA_3 transmitter phosphatase activities.

  9. Preparation, characterization and biological activity of C8-substituted cytokinins.

    PubMed

    Zahajská, Lenka; Nisler, Jaroslav; Voller, Jiří; Gucký, Tomáš; Pospíšil, Tomáš; Spíchal, Lukáš; Strnad, Miroslav

    2017-03-01

    Naturally occurring cytokinins are adenine-based plant hormones. Although, the effect of various substituents at positions N1, C2, N3, N(6), N7, or N9 on the biological activity of cytokinins has been studied, the C8-substituted compounds have received little attention. Here, we report the synthesis and in vitro biological testing of thirty-one cytokinin derivatives substituted at the C8 position of the adenine skeleton and twenty-seven compounds which served as their N9-tetrahydropyranyl protected precursors. The cytokinin activity of all the compounds was determined in classical cytokinin biotests (wheat leaf senescence, Amaranthus and tobacco callus assays). With some exceptions, the compounds with a N9-tetrahydropyranyl group were generally less active than their de-protected analogs. The latter were further tested for their ability to activate the Arabidopsis cytokinin receptors AHK3 and CRE1/AHK4 in bacterial receptor activation assays. Using this approach, we identified derivatives bearing short aliphatic chains and retaining high cytokinin activity. Such compounds are suitable candidates for fluorescence labeling or as protein-affinity ligands. We further found that some C8-substituted cytokinins exhibited no or lower cytotoxicity toward tobacco cells when compared to their parent compound. Therefore, we also present and discuss the cytotoxicity of all the compounds against three normal human cell lines.

  10. 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.

  11. First principles calculations of the site substitution behavior in gamma prime phase in nickel based superalloys

    NASA Astrophysics Data System (ADS)

    Chaudhari, Mrunalkumar

    Nickel based superalloys have superior high temperature mechanical strength, corrosion and creep resistance in harsh environments and found applications in the hot sections as turbine blades and turbine discs in jet engines and gas generator turbines in the aerospace and energy industries. The efficiency of these turbine engines depends on the turbine inlet temperature, which is determined by the high temperature strength and behavior of these superalloys. The microstructure of nickel based superalloys usually contains coherently precipitated gamma prime (gamma') Ni3Al phase within the random solid solution of the gamma (gamma) matrix, with the gamma' phase being the strengthening phase of the superalloys. How the alloying elements partition into the gamma and gamma' phases and especially in the site occupancy behaviors in the strengthening gamma' phases play a critical role in their high temperature mechanical behaviors. The goal of this dissertation is to study the site substitution behavior of the major alloying elements including Cr, Co and Ti through first principles based calculations. Site substitution energies have been calculated using the anti-site formation, the standard defect formation formalism, and the vacancy formation based formalism. Elements such as Cr and Ti were found to show strong preference for Al sublattice, whereas Co was found to have a compositionally dependent site preference. In addition, the interaction energies between Cr-Cr, Co-Co, Ti-Ti and Cr-Co atoms have also been determined. Along with the charge transfer, chemical bonding and alloy chemistry associated with the substitutions has been investigated by examining the charge density distributions and electronic density of states to explain the chemical nature of the site substitution. Results show that Cr and Co atoms prefer to be close by on either Al sublattice or on a Ni-Al mixed lattice, suggesting a potential tendency of Cr and Co segregation in the gamma' phase.

  12. 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.

  13. Site-specific time heterogeneity of the substitution process and its impact on phylogenetic inference

    PubMed Central

    2011-01-01

    Background Model violations constitute the major limitation in inferring accurate phylogenies. Characterizing properties of the data that are not being correctly handled by current models is therefore of prime importance. One of the properties of protein evolution is the variation of the relative rate of substitutions across sites and over time, the latter is the phenomenon called heterotachy. Its effect on phylogenetic inference has recently obtained considerable attention, which led to the development of new models of sequence evolution. However, thus far focus has been on the quantitative heterogeneity of the evolutionary process, thereby overlooking more qualitative variations. Results We studied the importance of variation of the site-specific amino-acid substitution process over time and its possible impact on phylogenetic inference. We used the CAT model to define an infinite mixture of substitution processes characterized by equilibrium frequencies over the twenty amino acids, a useful proxy for qualitatively estimating the evolutionary process. Using two large datasets, we show that qualitative changes in site-specific substitution properties over time occurred significantly. To test whether this unaccounted qualitative variation can lead to an erroneous phylogenetic tree, we analyzed a concatenation of mitochondrial proteins in which Cnidaria and Porifera were erroneously grouped. The progressive removal of the sites with the most heterogeneous CAT profiles across clades led to the recovery of the monophyly of Eumetazoa (Cnidaria+Bilateria), suggesting that this heterogeneity can negatively influence phylogenetic inference. Conclusion The time-heterogeneity of the amino-acid replacement process is therefore an important evolutionary aspect that should be incorporated in future models of sequence change. PMID:21235782

  14. Transition Metal Ion Substitution into the Cu(1) Sites in the "123" High-Temperature Superconductors

    NASA Astrophysics Data System (ADS)

    Tang, I. M.; Leelaprute, S.

    The effects of substituting either non-magnetic or magnetic (trivalent) transition metal (TM) ions into the Cu(1) sites in the CuO chains occurring in the "123" YBa2Cu3O7-x superconductor are studied. It is assumed that pair condensation occurs in both the 2D CuO2 planes and the 1D CuO chains, that the exchange of the spin fluctuations (SF) is responsible for the pair formation and that the order parameters in the chains and planes are coupled together by pair tunneling. Just like the case for substitution into the Cu(2) sites, the depressions of Tc due to non-magnetic and magnetic TM ions substitution into the Cu(1) sites are caused by the different ways the two type of ions act on the Cooper pairs. The non-magnetic TM ions suppress the formation of the Cooper pairs through their suppression of the SF-induced pairing interaction while the magnetic TM ions break the Cooper pairs through their sping flipping of one of the carriers. For both types of ions, the suppression of superconductivity is enhanced by the normal scattering process.

  15. 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

  16. Design of protease-resistant myelin basic protein-derived peptides by cleavage site directed amino acid substitutions.

    PubMed

    Burster, Timo; Marin-Esteban, Viviana; Boehm, Bernhard O; Dunn, Shannon; Rotzschke, Olaf; Falk, Kirsten; Weber, Ekkehard; Verhelst, Steven H L; Kalbacher, Hubert; Driessen, Christoph

    2007-11-15

    Multiple Sclerosis (MS) is considered to be a T cell-mediated autoimmune disease. An attractive strategy to prevent activation of autoaggressive T cells in MS, is the use of altered peptide ligands (APL), which bind to major histocompatibility complex class II (MHC II) molecules. To be of clinical use, APL must be capable of resisting hostile environments including the proteolytic machinery of antigen presenting cells (APC). The current design of APL relies on cost- and labour-intensive strategies. To overcome these major drawbacks, we used a deductive approach which involved modifying proteolytic cleavage sites in APL. Cleavage site-directed amino acid substitution of the autoantigen myelin basic protein (MBP) resulted in lysosomal protease-resistant, high-affinity binding peptides. In addition, these peptides mitigated T cell activation in a similar fashion as conventional APL. The strategy outlined allows the development of protease-resistant APL and provides a universal design strategy to improve peptide-based immunotherapeutics.

  17. 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.

  18. 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.

  19. 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.

  20. Efficient red phosphor double-perovskite Ca3WO6 with A-site substitution of Eu3+.

    PubMed

    Zhao, Xin; Wang, Jiajia; Fan, Li; Ding, Yufeng; Li, Zhaosheng; Yu, Tao; Zou, Zhigang

    2013-10-07

    Luminescent properties of Eu(3+) activated double perovskite structure Ca3WO6 were investigated. It emits an ideal red color centered at the wavelength of 618 nm with suitable excitation from f-f transitions of Eu(3+) ions (360-550 nm) matching the near ultraviolet and blue LEDs. Charge compensation effect of Li(+), Na(+), and K(+) was investigated, and K(+) proved to be the best. The co-doping ion K(+) has a great effect on the lattice distortion of the host matrix Ca3WO6, which facilitates the red emission of Eu(3+). The substitution site for Ca in Ca3WO6 was analyzed in detail by Raman spectra and calculation results. A-site substitution is responsible for the red emission of Eu(3+) activated Ca3WO6. The integrated emission intensity of optimal Ca3WO6:K(+),Eu(3+) excited at 395 nm is about 3.5 times greater than that of Y2O2S:Eu(3+) commercial phosphors, which makes it a promising red phosphor for white LEDs.

  1. 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

  2. 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

  3. B-site substituted solid solutions on the base of sodium-bismuth titanate

    NASA Astrophysics Data System (ADS)

    Ishchuk, V. M.; Gusakova, L. G.; Kisel, N. G.; Kuzenko, D. V.; Spiridonov, N. A.; Sobolev, V. L.

    The paper presents results of studies of the formation of phases during the solid-state synthesis in the [(Na0.5Bi0.5)0.80Ba0.20](Ti1-yBy)O3 system of solid solutions with B-site substitutions. The substitutions by zirconium, tin and ion complexes (In0.5Nb0.5) and (Fe0.5Nb0.5) have been studied. It has been found that the synthesis is a multi-step process associated with the formation of a number of intermediate phases (depending on the compositions and calcination temperatures). Single-phase solid solutions have been produced at the calcination temperatures in the interval 1000-1100∘C. An increase in the substituting ions concentration leads to a linear increase of the crystal cell size. At the same time, the tolerance factor gets reduced boosting the stability of the antiferroelectric phase as compared to that of the ferroelectric phase.

  4. 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.

  5. Defining critical residues for substrate binding to 1-deoxy-d-xylulose 5-phosphate synthase: Active site substitutions stabilize the pre-decarboxylation intermediate C2α-lactylthiamin diphosphate

    PubMed Central

    Kakalis, Lazaros; Jordan, Frank; Meyers, Caren L. Freel

    2014-01-01

    1-Deoxy-d-xylulose 5-phosphate (DXP) synthase catalyzes formation of DXP from pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) in a thiamin diphosphate (ThDP)-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 uses mutagenesis and a combination of protein fluorescence, circular dichroism and kinetics experiments to investigate the roles of Arg-420, Arg-478 and Tyr-392 in substrate binding and catalysis. The results support a random sequential, preferred order mechanism and predict Arg-420 and Arg-478 are involved in binding of the acceptor substrate, d-GAP. d-Glyceraldehyde, an alternative acceptor substrate lacking the phosphoryl group predicted to interact with Arg-420 and Arg-478, also accelerates decarboxylation of the pre-decarboxylation intermediate C2α-lactylthiamin diphosphate (LThDP) on DXP synthase, indicating this binding interaction is not absolutely required, and the hydroxyaldehyde sufficiently triggers decarboxylation. Unexpectedly, Tyr-392 contributes to d-GAP affinity and is not required for LThDP formation or its d-GAP-promoted decarboxylation. Time-resolved CD spectroscopy and NMR experiments indicate LThDP is significantly stabilized on R420A and Y392F variants compared to wild type DXP synthase in the absence of acceptor substrate, yet these substitutions do not appear to impact the rate of d-GAP-promoted LThDP decarboxylation in the presence of high d-GAP, and LThDP formation remains the rate-limiting step. These results suggest a role of these residues to promote d-GAP binding which in turn facilitates decarboxylation, and further highlight interesting differences between DXP synthase and other ThDP-dependent enzymes. PMID:24767541

  6. Structure-photodynamic activity relationships of substituted zinc trisulfophthalocyanines.

    PubMed

    Cauchon, Nicole; Tian, Hongjian; Langlois, Réjean; La Madeleine, Carole; Martin, Stephane; Ali, Hasrat; Hunting, Darel; van Lier, Johan E

    2005-01-01

    To identify optimal features of metalated sulfophthalocyanine dyes for their use as photosensitizers in the photodynamic therapy of cancer, we synthesized a series of alkynyl-substituted trisulfonated phthalocyanines and compared their amphiphilic properties to a number of parameters related to their photodynamic potency. Varying the length of the substituted alkynyl side-chain modulates the hydrophobic/hydrophilic properties of the dyes providing a linear relationship between their n-octanol/water partition coefficients and retention times on reversed-phase HPLC. Aggregate formation of the dyes in aqueous solution increased with increasing hydrophobicity while monomer formation was favored by the addition of serum proteins or organic solvent. Trisulfonated zinc phthalocyanines bearing hexynyl and nonynyl substituents exhibited high cellular uptake with strong localization at the mitochondrial membranes, which coincided with effective photocytotoxicity toward EMT-6 murine mammary tumor cells. Further increase in the length of the alkynyl chains (dodecynyl, hexadecynyl) did not improve their phototoxicity, likely resulting from extensive aggregation of the dyes in aqueous medium and reduced cell uptake. Aggregation was evident from shifts in the electronic spectra and reduced capacity to generate singlet oxygen. When monomerized through the addition of Cremophor EL all sulfonated zinc phthalocyanines gave similar singlet oxygen yields. Accordingly, differences in the tendency of the dyes to aggregate do not appear to be a determining factor in their photodynamic potency. Our results confirm that the latter in particular relates to their amphiphilic properties, which facilitate cell uptake and intracellular localization at photosensitive sites such as the mitochondria. Combined, these factors play a significant role in the overall photodynamic potency of the dyes.

  7. Substituting activities mediates the effect of cognitive flexibility on physical activity: a daily diary study.

    PubMed

    Kelly, Scout M; Updegraff, John A

    2017-03-02

    Pursuit of physical activity goals often requires modifying plans, but research on these flexible processes is limited. Cognitive flexibility may heighten one's likelihood of using flexible self-regulatory strategies (e.g., substitution), thereby increasing physical activity. This study used daily diary methodology to test the indirect effect of cognitive flexibility on physical activity via activity substitution. A sample of 128 college students (73% female, mean age 19.9) completed baseline measures and cognitive flexibility assessments, then logged physical activity daily for 2 weeks. Activity substitution was defined as adopting an alternate activity on a day another planned activity was unfulfilled. Controlling for baseline activity, intentions, and time, a multilevel mediation model revealed a significant indirect effect of cognitive flexibility on physical activity via activity substitution (b = 81.36, p = .041). Our results indicate that people with greater cognitive flexibility are more likely to use flexible self-regulation, leading to greater physical activity.

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. Relative strengths of axial and equatorial bonds and site preferences for ligand substitution on σ complexes

    NASA Astrophysics Data System (ADS)

    Su, Ming-Der

    An analytical LCAO MO perturbation approach has been developed for treating the electronic structure and some properties of Y-shaped EL3 compounds where E is a main group element A or a transition metal M. Three problems have been considered: (i) the relative strengths of E-L(1,2) (1 and 2 stand for the paired ligands) and E-L(3) (3 stands for the unique ligand) bonds; (ii) the mutual influence of ligands in substituted complexes EL2L'; (iii) the site preference of the stronger donor (or acceptor) of substituents L', L''. Ratios of overlap populations T=NE-L(3)/NE-L(1) were estimated for ns, np and (n-1)d contributions. For 6-10 valence electron AL3 we found T(s)>1, T(p)>1. These contributions all reinforce to make the A-L(3) bond relatively stronger than A-L(1,2) bonds. The ML3 d0-d8 case was also examined and it was found that T(s+p+d)>1, while the T(p) contribution dominates in ML3 (d10) complexes to make M-L(3)>M-L(1,2). The perturbing influence on ligand σ orbital energies δα'=α(L')-α(L), where δα'>0(<0) corresponds to a better donor (acceptor) substituent L', and all changes in overlap populations of different E-L bonds, δN(E-L)/δα', were obtained in terms of the ns, np and (n-1)d contributions. In addition, there are three kinds of substitution type: sub-L(3)-cis-L(1), sub-L(1)-cis-L(2) and sub-L(1)-cis-L(3) effects. It was found that both the nature of E, L', L and steric factors may play important roles in determining the relative bond strengthening of E-L(1,2) and E-L(3) for unsubstituted and substituted EL3 compounds. Y-shaped substituents were examined using first-order perturbation theory, with the finding that the most electronegative atom prefers to substitute at the L(3) site in AL3 and d0-d10 ML3 complexes. The role of π bonding effects is briefly discussed. The results obtained agree with the available experimental and computational data and permit a number of predictions to be made.

  13. 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

  14. 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.

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

    PubMed

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

    2013-01-01

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

  16. Isomorphous substitution and the generation of catalytic activity in VPI-5

    SciTech Connect

    Kraushaar-Czarnetzki, B.; Dogterom, R.J.; Stork, W.H.J.; Emeis, K.A.; Van Braam Houckgeest, J.P. ) )

    1993-05-01

    VPI-5 and substituted derivatives containing silicon or magnesium have been synthesized, analyzed, and tested for their catalytic activity. By means of [sup 29]Si solid-state NMR, it could be shown that SAPO-VPI-5 prepared with polyphosphoric acid as a phosphorus source exhibits both [open quotes]silica patches[close quotes] and isolated silicon atoms incorporated in the framework. Broensted acid sites in SAPO-and MAPO-VPI-5 could be detected by means of infrared spectroscopy in combination with pyridine adsorption. The isomorphously substituted VPI-5 samples were active in the hydroconversion of n-heptane, giving both cracking and isomerization products. The highest activity and the highest yield in iso-heptane, however, were observed with AlPO-VPI-5 as a catalyst. The catalytic activity could be ascribed to the presence of unreacted alumina. Upon treatment with an EDTA solution the Al/P atomic ratio decreased from 1.29 to 1.07, and the purified AlPO-VPI-5 showed considerably reduced catalytic activity. 28 refs., 6 figs., 2 tabs.

  17. 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.

  18. Normal Modes Expose Active Sites in Enzymes

    PubMed Central

    Glantz-Gashai, Yitav; Samson, Abraham O.

    2016-01-01

    Accurate prediction of active sites is an important tool in bioinformatics. Here we present an improved structure based technique to expose active sites that is based on large changes of solvent accessibility accompanying normal mode dynamics. The technique which detects EXPOsure of active SITes through normal modEs is named EXPOSITE. The technique is trained using a small 133 enzyme dataset and tested using a large 845 enzyme dataset, both with known active site residues. EXPOSITE is also tested in a benchmark protein ligand dataset (PLD) comprising 48 proteins with and without bound ligands. EXPOSITE is shown to successfully locate the active site in most instances, and is found to be more accurate than other structure-based techniques. Interestingly, in several instances, the active site does not correspond to the largest pocket. EXPOSITE is advantageous due to its high precision and paves the way for structure based prediction of active site in enzymes. PMID:28002427

  19. 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.

  20. Cd-substituted horse liver alcohol dehydrogenase: catalytic site metal coordination geometry and protein conformation.

    PubMed

    Hemmingsen, L; Bauer, R; Bjerrum, M J; Zeppezauer, M; Adolph, H W; Formicka, G; Cedergren-Zeppezauer, E

    1995-05-30

    The coordination geometry of the catalytic site in Cd-substituted horse liver alcohol dehydrogenase (LADH) has been investigated as a function of pH using the method of perturbed angular correlation of gamma-rays (PAC). LADH in solution fully loaded with cadmium, including radioactive 111mCd in the catalytic site [Cd2(111mCd)Cd2LADH], was studied over the pH range 7.9-11.5. Analysis of the PAC spectra showed the ionization of a group with pKa of 11. This pKa value is about 2 pH units higher than that of native zinc-containing LADH. A pKa of 9.6 was found for the binary complex of Cd2(111mCd)Cd2LADH with NAD+. This value is also about 2 pH units higher than that of the binary complex of native zinc-containing enzyme and NAD+. No pH dependency was detected for the binary complex of Cd2(111mCd)Cd2LADH with NADH within the pH range measured (pH 8.3-11.5). Assuming that metal-coordinated water is the ionizing group [Kvassman, J., & Pettersson, G. (1979) Eur. J. Biochem. 100, 115-123], we conclude that the larger ionic radius of Cd(II) relative to Zn(II) in the catalytic site causes the elevated pKa values of metal-bound water. Interpretation of nuclear quadrupole interaction (NQI) parameters derived from PAC spectra is based on the use of the angular overlap model, using the coordinates for the catalytic zinc site from the 1.8 A resolution crystal structure of the ternary complex between LADH, NADH, and dimethyl sulfoxide as a model.(ABSTRACT TRUNCATED AT 250 WORDS)

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

    PubMed

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

    2007-06-26

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

  2. Different site occupancies in substitution variants of Mo{sub 3}Sb{sub 7}

    SciTech Connect

    Guo, Quansheng; Assoud, Abdeljalil; Kleinke, Holger

    2016-04-15

    A series of samples with the initial formula Mo{sub 3–x}Fe{sub x}Sb{sub 7} and Mo{sub 3–x}Ni{sub x}Sb{sub 7} have been prepared by solid state reaction at 873 K, starting from the elements in the stoichiometric ratios. The materials were compressed via hot-pressing, and their thermoelectric properties determined. Single crystal data show that Ni atoms in part occupy the cubic voids of the Mo{sub 3}Sb{sub 7} structure, as well as replace some Mo atoms, in contrast to the Fe analogues, where we found no evidence for Fe in the cubic voids. Supported by electronic structure calculations, replacing Mo with Fe or Ni increases the valence-electron count, thus decreases the numbers of p-type carriers of Mo{sub 3}Sb{sub 7}. On the other hand, filling the holes with Ni atoms appears to have no such effect. As a consequence, the Ni containing samples exhibit higher electrical conductivity than the Fe analogues, and ultimately lower thermoelectric figure-of-merit, as the p-type carrier concentration remains too high. - Graphical abstract: Trying to substitute Ni atoms for Mo in Mo{sub 3}Sb{sub 7} resulted in partial filling of the cubic voids, in contrast to Mo{sub 3–x}Fe{sub x}Sb{sub 7}. Since the Ni atoms in the Sb{sub 8} cubes do not act as electron donors, the Ni-containing materials exhibit very high carrier concentration, and thus high electric conductivity and low Seebeck coefficient. - Highlights: • Various substitution variants of Mo{sub 3}Sb{sub 7} with Fe and Ni were characterized. • While the Fe atoms replace the Mo atoms, the Ni atoms also fill the cubic voids. • Higher Fe/Ni amount on the Mo sites causes a decrease of the charge carriers. • The Ni atoms on the cubic sites do not decrease the charge carriers. • The lower the carrier concentration, the better the thermoelectric properties.

  3. 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.

  4. 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...

  5. Tuning of the magnetostrictive properties of cobalt ferrite by forced distribution of substituted divalent metal ions at different crystallographic sites

    NASA Astrophysics Data System (ADS)

    Anantharamaiah, P. N.; Joy, P. A.

    2017-03-01

    Comparative studies have been made to understand the role of different crystallographic site preferences of the substituted non-magnetic divalent metal ions in the magnetostrictive properties of cobalt ferrite, by substitution of Zn2+ and/or Mg2+ for Fe3+ in CoMgxFe2-xO4, CoZnxFe2-xO4, and CoMgx/2Znx/2Fe2-xO4 (0.0 ≤ x ≤ 0.2). Detailed Raman spectral and magnetic characterizations are made to extract the information on the tetrahedral/octahedral site preferences of Zn and Mg in the spinel lattice of cobalt ferrite. The structural, microstructural, magnetic, Raman spectral, and magnetostrictive parameters of the studied compositions show distinguishable variations for x < 0.1 and x ≥ 0.1. Co-substitution of a small amount of Mg and Zn for Fe in CoMgx/2Znx/2Fe2-xO4 (x < 0.1) showed relatively larger strain sensitivity, [dλ/dH]max (-2.6 × 10-9 mA-1 for x = 0.05), higher than that for the Mg-substituted samples (-2.05 × 10-9 mA-1 for x = 0.05) and comparable to that for the Zn-substituted samples (-2.47 × 10-9 mA-1 for x = 0.05), without much drop in the maximum value of magnetostriction, λmax (-189 ppm for x = 0.05) compared to that for the unsubstituted counterpart (-221 ppm). The results show that it is possible to obtain high strain sensitivity (at fields <50 kA/m), along with high magnetostriction strain at low magnetic fields (˜250 kA/m), by tuning the distribution of the substituted cations in the tetrahedral and octahedral sites of the cobalt ferrite lattice.

  6. Synthesis of substituted benzimidazolyl curcumin mimics and their anticancer activity.

    PubMed

    Woo, Ho Bum; Eom, Young Woo; Park, Kyu-Sang; Ham, Jungyeob; Ahn, Chan Mug; Lee, Seokjoon

    2012-01-15

    A novel curcumin mimic library (14a-14h and 15a-15h) possessing variously substituted benzimidazole groups was synthesized through the aldol reaction of (E)-4-(4-hydroxy-3-methoxyphenyl)but-3-en-2-one (7) or (E)-4-(3-hydroxy-4-methoxyphenyl)but-3-en-2-one (13) with diversely substituted benzimidazolyl-2-carbaldehyde (12a-12h). The MTT assay of the cancer cells MCF-7, SH-SY5Y, HEP-G2, and H460 showed that compound 14c with IC(50) of 1.0 and 1.9μM has a strong inhibitory effect on the growth of SH-SY5Y and Hep-G2 cells, respectively, and that compound 15h with IC(50) of 1.9μM has a strong inhibitory effect on the growth of MCF-7 cancer cells.

  7. Substitutions of Thr30 provide mechanistic insight into tryptophan-mediated activation of TRAP binding to RNA.

    PubMed

    Payal, Vandana; Gollnick, Paul

    2006-01-01

    TRAP is an 11 subunit RNA binding protein that regulates expression of genes involved in tryptophan biosynthesis and transport in Bacillus subtilis. TRAP is activated to bind RNA by binding up to 11 molecules of l-tryptophan in pockets formed by adjacent subunits. The precise mechanism by which tryptophan binding activates TRAP is not known. Thr30 is in the tryptophan binding pocket. A TRAP mutant in which Thr30 is substituted with Val (T30V) does not bind tryptophan but binds RNA constitutively, suggesting that Thr30 plays a key role in the activation mechanism. We have examined the effects of other substitutions of Thr30. TRAP proteins with small beta-branched aliphatic side chains at residue 30 bind RNA constitutively, whereas those with a small polar side chain show tryptophan-dependent RNA binding. Several mutant proteins exhibited constitutive RNA binding that was enhanced by tryptophan. Although the tryptophan and RNA binding sites on TRAP are distinct and are separated by approximately 7.5 A, several substitutions of residues that interact with the bound RNA restored tryptophan binding to T30V TRAP. These observations support the hypothesis that conformational changes in TRAP relay information between the tryptophan and RNA binding sites of the protein.

  8. 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

  9. 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

    2015-10-20

    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.

  10. Validated ligand mapping of ACE active site

    NASA Astrophysics Data System (ADS)

    Kuster, Daniel J.; Marshall, Garland R.

    2005-08-01

    Crystal structures of angiotensin-converting enzyme (ACE) complexed with three inhibitors (lisinopril, captopril, enalapril) provided experimental data for testing the validity of a prior active site model predicting the bound conformation of the inhibitors. The ACE active site model - predicted over 18 years ago using a series of potent ACE inhibitors of diverse chemical structure - was recreated using published data and commercial software. Comparison between the predicted structures of the three inhibitors bound to the active site of ACE and those determined experimentally yielded root mean square deviation (RMSD) values of 0.43-0.81 Å, among the distances defining the active site map. The bound conformations of the chemically relevant atoms were accurately deduced from the geometry of ligands, applying the assumption that the geometry of the active site groups responsible for binding and catalysis of amide hydrolysis was constrained. The mapping of bound inhibitors at the ACE active site was validated for known experimental compounds, so that the constrained conformational search methodology may be applied with confidence when no experimentally determined structure of the enzyme yet exists, but potent, diverse inhibitors are available.

  11. 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.

  12. Synthesis, antitubercular, antifungal and antibacterial activities of 6-substituted phenyl-2-(3'-substituted phenyl pyridazin-6'-yl)-2,3,4,5-tetrahydropyridazin-3-one.

    PubMed

    Islam, Mojahidul; Siddiqui, Anees A; Rajesh, Ramadoss

    2008-01-01

    A series of 6-substituted phenyl-2-(3'-substituted phenyl pyridazin-6'-yl)-2,3,4,5-tetrahydropyridazin-3-ones has been synthesized. An appropriate aromatic hydrocarbon reacts with succinic anhydride in presence of AlCl3 to yield beta-aroyl propionic acid. The corresponding acid was cyclized with hydrazine hydrate to give 6-(substituted aryl)-2,3,4,5-tetrahydro-3-pyridazinone, which was heated on steam bath with phosphorus(V) oxychloride to yield 3-chloro 6-substituted phenyl pyridazine. This intermediate after reaction with hydrazine hydrate was converted into 3-hydrazino-6-substituted phenyl pyridazine. The resulting product was converted into 6-substituted phenyl-2-(3'-substituted phenyl pyridazin-6'-yl)-2,3,4,5-tetrahydropyridazin-3-one by reacting with substituted aroyl propionic acid. Spectral data (IR, NMR, mass spectra) confirmed the structures of the synthesized compounds. The synthesized compounds were investigated for their in vitro antitubercular, antifungal and antibacterial activities. The results indicated that the synthesized compounds have mild to potent activities with reference to their appropriate reference standards.

  13. A first-principles comparative study of lithium, sodium, and magnesium storage in pure and gallium-doped germanium: Competition between interstitial and substitutional sites

    NASA Astrophysics Data System (ADS)

    Legrain, Fleur; Manzhos, Sergei

    2017-01-01

    Thermodynamics and kinetics of Li, Na, and Mg storage in Ge are studied ab initio. The most stable configurations can consist of tetrahedral, substitutional, or a combination of the two types of sites. In the dilute limit, Li and Na prefer interstitial, while Mg prefers substitutional sites. At higher concentrations of Li, Na, and Mg, there is a combination of interstitial and substitutional sites. This is an important finding, as most previous ab initio studies of alloying type electrode materials ignored substitutional sites. Insertion energies computed at dilute concentration (x = 1/64) show that Na and Mg insertion are not thermodynamically favored in Ge vs. the formation of bulk Na and Mg, as opposed to Li insertion which is favored. We investigate the effect of p-doping of Ge (with Ga) on the thermodynamics and find that it considerably lowers the defect formation energies associated with the insertion of Li/Na/Mg at tetrahedral sites. On the other hand, the energetics associated with Li/Na/Mg insertion at substitutional sites are not significantly affected. In addition, we compute the migration energy barriers for Li/Na/Mg diffusion between two tetrahedral sites (0.38/0.79/0.66 eV), between two substitutional sites (0.77/0.93/1.83 eV), and between two sites of different types (2.15/1.75/0.85 eV).

  14. Specificity of the hepatitis C virus NS3 serine protease: effects of substitutions at the 3/4A, 4A/4B, 4B/5A, and 5A/5B cleavage sites on polyprotein processing.

    PubMed Central

    Kolykhalov, A A; Agapov, E V; Rice, C M

    1994-01-01

    Cleavage at four sites (3/4A, 4A/4B, 4B/5A, and 5A/5B) in the hepatitis C virus polyprotein requires a viral serine protease activity residing in the N-terminal one-third of the NS3 protein. Sequence comparison of the residues flanking these cleavage sites reveals conserved features including an acidic residue (Asp or Glu) at the P6 position, a Cys or Thr residue at the P1 position, and a Ser or Ala residue at the P1' position. In this study, we used site-directed mutagenesis to assess the importance of these and other residues for NS3 protease-dependent cleavages. Substitutions at the P7 to P2' positions of the 4A/4B site had varied effects on cleavage efficiency. Only Arg at the P1 position or Pro at P1' substantially blocked processing at this site. Leu was tolerated at the P1 position, whereas five other substitutions allowed various degrees of cleavage. Substitutions with positively charged or other hydrophilic residues at the P7, P3, P2, and P2' positions did not reduce cleavage efficiency. Five substitutions examined at the P6 position allowed complete cleavage, demonstrating that an acidic residue at this position is not essential. Parallel results were obtained with substrates containing an active NS3 protease domain in cis or when the protease domain was supplied in trans. Selected substitutions blocking or inhibiting cleavage at the 4A/4B site were also examined at the 3/4A, 4B/5A, and 5A/5B sites. For a given substitution, a site-dependent gradient in the degree of inhibition was observed, with a 3/4A site being least sensitive to mutagenesis, followed by the 4A/4B, 4B/5A, and 5A/5B sites. In most cases, mutations abolishing cleavage at one site did not affect processing at the other serine protease-dependent sites. However, mutations at the 3/4A site which inhibited cleavage also interfered with processing at the 4B/5A site. Finally, during the course of these studies an additional NS3 protease-dependent cleavage site has been identified in the NS4B

  15. 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.

  16. 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%.

  17. 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.

  18. Characterization of the active site of ADP-ribosyl cyclase.

    PubMed

    Munshi, C; Thiel, D J; Mathews, I I; Aarhus, R; Walseth, T F; Lee, H C

    1999-10-22

    ADP-ribosyl cyclase synthesizes two Ca(2+) messengers by cyclizing NAD to produce cyclic ADP-ribose and exchanging nicotinic acid with the nicotinamide group of NADP to produce nicotinic acid adenine dinucleotide phosphate. Recombinant Aplysia cyclase was expressed in yeast and co-crystallized with a substrate, nicotinamide. x-ray crystallography showed that the nicotinamide was bound in a pocket formed in part by a conserved segment and was near the central cleft of the cyclase. Glu(98), Asn(107) and Trp(140) were within 3.5 A of the bound nicotinamide and appeared to coordinate it. Substituting Glu(98) with either Gln, Gly, Leu, or Asn reduced the cyclase activity by 16-222-fold, depending on the substitution. The mutant N107G exhibited only a 2-fold decrease in activity, while the activity of W140G was essentially eliminated. The base exchange activity of all mutants followed a similar pattern of reduction, suggesting that both reactions occur at the same active site. In addition to NAD, the wild-type cyclase also cyclizes nicotinamide guanine dinucleotide to cyclic GDP-ribose. All mutant enzymes had at least half of the GDP-ribosyl cyclase activity of the wild type, some even 2-3-fold higher, indicating that the three coordinating amino acids are responsible for positioning of the substrate but not absolutely critical for catalysis. To search for the catalytic residues, other amino acids in the binding pocket were mutagenized. E179G was totally devoid of GDP-ribosyl cyclase activity, and both its ADP-ribosyl cyclase and the base exchange activities were reduced by 10,000- and 18,000-fold, respectively. Substituting Glu(179) with either Asn, Leu, Asp, or Gln produced similar inactive enzymes, and so was the conversion of Trp(77) to Gly. However, both E179G and the double mutant E179G/W77G retained NAD-binding ability as shown by photoaffinity labeling with [(32)P]8-azido-NAD. These results indicate that both Glu(179) and Trp(77) are crucial for catalysis and

  19. An active site mutation increases the polymerase activity of the guinea pig-lethal Marburg virus.

    PubMed

    Koehler, Alexander; Kolesnikova, Larissa; Becker, Stephan

    2016-10-01

    Marburg virus (MARV) causes severe, often fatal, disease in humans and transient illness in rodents. Sequential passaging of MARV in guinea pigs resulted in selection of a lethal virus containing 4 aa changes. A D184N mutation in VP40 (VP40D184N), which leads to a species-specific gain of viral fitness, and three mutations in the active site of viral RNA-dependent RNA polymerase L, which were investigated in the present study for functional significance in human and guinea pig cells. The transcription/replication activity of L mutants was strongly enhanced by a substitution at position 741 (S741C), and inhibited by other substitutions (D758A and A759D) in both species. The polymerase activity of L carrying the S741C substitution was eightfold higher in guinea pig cells than in human cells upon co-expression with VP40D184N, suggesting that the additive effect of the two mutations provides MARV a replicative advantage in the new host.

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

    PubMed Central

    Petrosino, J F; Palzkill, T

    1996-01-01

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

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

    PubMed

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

    2011-07-01

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

  2. Evaluation of Different Oligonucleotide Base Substitutions at CpG Binding sites in Multiplex Bisulfite-PCR sequencing

    PubMed Central

    Lu, Jennifer; Ru, Kelin; Candiloro, Ida; Dobrovic, Alexander; Korbie, Darren; Trau, Matt

    2017-01-01

    Multiplex bisulfite-PCR sequencing is a convenient and scalable method for the quantitative determination of the methylation state of target DNA regions. A challenge of this application is the presence of CpGs in the same region where primers are being placed. A common solution to the presence of CpGs within a primer-binding region is to substitute a base degeneracy at the cytosine position. However, the efficacy of different substitutions and the extent to which bias towards methylated or unmethylated templates may occur has never been evaluated in bisulfite multiplex sequencing applications. In response, we examined the performance of four different primer substitutions at the cytosine position of CpG’s contained within the PCR primers. In this study, deoxyinosine-, 5-nitroindole-, mixed-base primers and primers with an abasic site were evaluated across a series of methylated controls. Primers that contained mixed- or deoxyinosine- base modifications performed most robustly. Mixed-base primers were further selected to determine the conditions that induce bias towards methylated templates. This identified an optimized set of conditions where the methylated state of bisulfite DNA templates can be accurately assessed using mixed-base primers, and expands the scope of bisulfite resequencing assays when working with challenging templates. PMID:28327639

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

    PubMed

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

    2014-06-01

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

  4. Active Site Hydrophobicity and the Convergent Evolution of Paraoxonase Activity in Structurally Divergent Enzymes: The Case of Serum Paraoxonase 1

    PubMed Central

    2016-01-01

    Serum paraoxonase 1 (PON1) is a native lactonase capable of promiscuously hydrolyzing a broad range of substrates, including organophosphates, esters, and carbonates. Structurally, PON1 is a six-bladed β-propeller with a flexible loop (residues 70–81) covering the active site. This loop contains a functionally critical Tyr at position 71. We have performed detailed experimental and computational analyses of the role of selected Y71 variants in the active site stability and catalytic activity in order to probe the role of Y71 in PON1’s lactonase and organophosphatase activities. We demonstrate that the impact of Y71 substitutions on PON1’s lactonase activity is minimal, whereas the kcat for the paraoxonase activity is negatively perturbed by up to 100-fold, suggesting greater mutational robustness of the native activity. Additionally, while these substitutions modulate PON1’s active site shape, volume, and loop flexibility, their largest effect is in altering the solvent accessibility of the active site by expanding the active site volume, allowing additional water molecules to enter. This effect is markedly more pronounced in the organophosphatase activity than the lactonase activity. Finally, a detailed comparison of PON1 to other organophosphatases demonstrates that either a similar “gating loop” or a highly buried solvent-excluding active site is a common feature of these enzymes. We therefore posit that modulating the active site hydrophobicity is a key element in facilitating the evolution of organophosphatase activity. This provides a concrete feature that can be utilized in the rational design of next-generation organophosphate hydrolases that are capable of selecting a specific reaction from a pool of viable substrates. PMID:28026940

  5. Reinforcement value and substitutability of sucrose and wheel running: implications for activity anorexia.

    PubMed

    Belke, Terry W; Pierce, W David; Duncan, Ian D

    2006-09-01

    Choice between sucrose and wheel-running reinforcement was assessed in two experiments. In the first experiment, ten male Wistar rats were exposed to concurrent VI 30 s VI 30 s schedules of wheel-running and sucrose reinforcement. Sucrose concentration varied across concentrations of 2.5, 7.5, and 12.5%. As concentration increased, more behavior was allocated to sucrose and more reinforcements were obtained from that alternative. Allocation of behavior to wheel running decreased, but obtained wheel-running reinforcement did not change. Overall, the results suggested that food-deprived rats were sensitive to qualitative changes in food supply (sucrose concentration) while continuing to defend a level of physical activity (wheel running). In the second study, 15 female Long Evans rats were exposed to concurrent variable ratio schedules of sucrose and wheel-running, wheel-running and wheel-running, and sucrose and sucrose reinforcement. For each pair of reinforcers, substitutability was assessed by the effect of income-compensated price changes on consumption of the two reinforcers. Results showed that, as expected, sucrose substituted for sucrose and wheel running substituted for wheel running. Wheel running, however, did not substitute for sucrose; but sucrose partially substituted for wheel running. We address the implications of the interrelationships of sucrose and wheel running for an understanding of activity anorexia.

  6. New aromatase inhibitors. Synthesis and biological activity of aryl-substituted pyrrolizine and indolizine derivatives.

    PubMed

    Sonnet, P; Dallemagne, P; Guillon, J; Enguehard, C; Stiebing, S; Tanguy, J; Bureau, R; Rault, S; Auvray, P; Moslemi, S; Sourdaine, P; Séralini, G E

    2000-05-01

    We report herein the design and the synthesis of some aryl-substituted pyrrolizine and indolizine derivatives, on the basis of a hypothetical pharmacophore structure designed to fit the catalytic site of the human cytochrome P450 aromatase. The in vitro biological evaluation of these compounds allowed us to point out two new potent non-steroidal aromatase inhibitors, MR 20494 and MR 20492, with IC50 values in the range of 0.1 microM.

  7. Enhancement of Polymerase Activity of the Large Fragment in DNA Polymerase I from Geobacillus stearothermophilus by Site-Directed Mutagenesis at the Active Site

    PubMed Central

    Ma, Yi; Zhang, Beilei; Wang, Meng; Ou, Yanghui

    2016-01-01

    The large fragment of DNA polymerase I from Geobacillus stearothermophilus GIM1.543 (Bst DNA polymerase) with 5′-3′ DNA polymerase activity while in absence of 5′-3′ exonuclease activity possesses high thermal stability and polymerase activity. Bst DNA polymerase was employed in isothermal multiple self-matching initiated amplification (IMSA) which amplified the interest sequence with high selectivity and was widely applied in the rapid detection of human epidemic diseases. However, the detailed information of commercial Bst DNA polymerase is unpublished and well protected by patents, which makes the high price of commercial kits. In this study, wild-type Bst DNA polymerase (WT) and substitution mutations for improving the efficiency of DNA polymerization were expressed and purified in E. coli. Site-directed substitutions of four conserved residues (Gly310, Arg412, Lys416, and Asp540) in the activity site of Bst DNA polymerase influenced efficiency of polymerizing dNTPs. The substitution of residue Gly310 by alanine or leucine and residue Asp540 by glutamic acid increased the efficiency of polymerase activity. All mutants with higher polymerizing efficiency were employed to complete the rapid detection of EV71-associated hand, foot, and mouth disease (HFMD) by IMSA approach with relatively shorter period which is suitable for the primary diagnostics setting in rural and underdeveloped areas. PMID:27981047

  8. 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

  9. Anticancer activity of new coumarin substituted hydrazide-hydrazone derivatives.

    PubMed

    Nasr, Tamer; Bondock, Samir; Youns, Mahmoud

    2014-04-09

    Drug resistance is a major impediment for cancer treatment, to overcome it we designed and synthesized sixteen coumarins bearing hydrazide-hydrazone moiety and evaluated them against human drug-resistant pancreatic carcinoma (Panc-1) cells and drug-sensitive (hepatic carcinoma; Hep-G2 and leukemia; CCRF) cell lines in vitro. The 6-brominated coumarin hydrazide-hydrazone derivatives (BCHHD) 7c, 8c and 10c were more potent than doxorubicin (DOX) against resistant Panc-1 cells. BCHHD 7c showed significant cytotoxicity against all tested cells (IC50: 3.60-6.50 μM) on comparison with all other coumarin hydrazide-hydrazone derivatives (CHHD), whereas BCHHD's 8c and 10c showed significant antiproliferative activity only against resistant Panc-1 cells with IC50 of 2.02 μM and 2.15 μM, respectively. All the investigated BCHHD's were able to activate caspases 3/7 and they could induce apoptosis in resistant Panc-1 cells. Microarray analysis showed that BCHHD 7c induced the expression of apoptotic- and cell cycle arrest (G2/M)- genes in resistant Panc-1 cells. Moreover, BCHHD 7c induced the up-regulation of CDKN1A, DDIT4, GDF-15 and down-regulation of CDC2, CDC20, CDK2 genes. Based on our results, we conclude that 7c could be a potent anticancer drug to overcome drug resistance in cancer and it could be highly beneficial for patients in the clinic.

  10. Active sites of thioredoxin reductases: why selenoproteins?

    PubMed

    Gromer, Stephan; Johansson, Linda; Bauer, Holger; Arscott, L David; Rauch, Susanne; Ballou, David P; Williams, Charles H; Schirmer, R Heiner; Arnér, Elias S J

    2003-10-28

    Selenium, an essential trace element for mammals, is incorporated into a selected class of selenoproteins as selenocysteine. All known isoenzymes of mammalian thioredoxin (Trx) reductases (TrxRs) employ selenium in the C-terminal redox center -Gly-Cys-Sec-Gly-COOH for reduction of Trx and other substrates, whereas the corresponding sequence in Drosophila melanogaster TrxR is -Ser-Cys-Cys-Ser-COOH. Surprisingly, the catalytic competence of these orthologous enzymes is similar, whereas direct Sec-to-Cys substitution of mammalian TrxR, or other selenoenzymes, yields almost inactive enzyme. TrxRs are therefore ideal for studying the biology of selenocysteine by comparative enzymology. Here we show that the serine residues flanking the C-terminal Cys residues of Drosophila TrxRs are responsible for activating the cysteines to match the catalytic efficiency of a selenocysteine-cysteine pair as in mammalian TrxR, obviating the need for selenium. This finding suggests that the occurrence of selenoenzymes, which implies that the organism is selenium-dependent, is not necessarily associated with improved enzyme efficiency. Our data suggest that the selective advantage of selenoenzymes is a broader range of substrates and a broader range of microenvironmental conditions in which enzyme activity is possible.

  11. Rational design of K173A substitution enhances thermostability coupled with catalytic activity of Enterobacter sp. Bn12 lipase.

    PubMed

    Farrokh, Parisa; Yakhchali, Bagher; Karkhane, Ali Asghar

    2014-01-01

    ELBn12 is a lipase isolated from Enterobacter sp. Bn12 with potential application in biotechnology. Homology modeling and rational design were applied to improve thermal stability of the lipase. K173A substitution introduced an AXXXA motif on the lipase model and it may have role in dimerization and thermostability of the protein. Site-directed mutagenesis was performed to construct the lipase variant. The mutated lipase was expressed in Escherichia coli pLysS and partially purified. Thermostability of the mutated lipase after 1 h of incubation at 70°C was twice that of wild-type lipase under the same conditions. Catalytic activity of the variant was about 1.5-fold towards tricaprylin at 60°C and pH 8.0; moreover, the lipase variant preserved its stability within the pH range of 7.0-11.0. Substitution of superficial hydrophilic Lys with hydrophobic Ala residue increased stability of the mutated lipase in the presence of nonionic surfactants, but this substitution caused lower stability towards polar solvents. Analysis of circular dichroism spectroscopy showed that the K173A mutation altered the secondary structure of the lipase into a more helical one. In conclusion, results of this study demonstrate the positive role of generation of a stabilizing protein motif through rational protein engineering that improves the enzyme characteristics.

  12. Synthesis, spectral characterization and antimicrobial activity of substituted thiazolyl derivatives of 2-quinolones.

    PubMed

    Katagi, M S; Bolakatti, G S; Badiger, A M; Satyanarayana, D; Mamledesai, S N; Sujatha, M L

    2013-02-01

    A series of new 3-(2-substituted amino/substituted hydrazino-1,3-thiazol-4-yl)-4-hydroxy-1-methyl/phenylquinolin-2 (1H)-one hydroiodide 3a-3f and 4a-4f derivatives were prepared by heating 3-Acetyl-4-hydroxy-1-methyl/phenyl quinolin-2 (1H)-one 2a-2b with substituted thiourea and substituted thiosemicarbazide in presence of iodine in n-butanol. The title compounds were characterized on the basis of IR, 1H NMR, 13C NMR and Mass spectral (MS) studies. Further title compounds were evaluated for antibacterial and antifungal by Agar diffusion assay method where as antitubercular activity by Micro-plate Alamar Blue Assay (MABA). Among 12 synthesized novel compounds 3a, 3b, 4d exhibited promising antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa. 3a, 3b, 3e, 4d, 4e showed good antifungal activity against Candida albicans and Aspergillus niger. 3a, 3d, 4d showed good antitubercular activity against Mycobacterium tuberculosis H37Rv strain.

  13. Activating Group Recycling in Action: A Rhodium-Catalyzed Carbothiolation Route to Substituted Isoquinolines

    PubMed Central

    2013-01-01

    A new rhodium(I) catalyst allows practical and efficient alkyne carbothiolation reactions to be achieved on synthetically useful ketone-bearing aryl methyl sulfides. The carbothiolation adducts, featuring a ‘recycled methyl sulfide’ activating group, are convenient precursors to highly substituted isoquinolines. PMID:24083625

  14. Isotemporal Substitution Analysis for Physical Activity, Television Watching, and Risk of Depression

    PubMed Central

    Mekary, Rania A.; Lucas, Michel; Pan, An; Okereke, Olivia I.; Willett, Walter C.; Hu, Frank B.; Ding, Eric L.

    2013-01-01

    The isotemporal substitution model (ISM) was previously developed as a methodology to study the time-substitution effects of 1 type of activity for another in a data setting with continuous outcomes. To demonstrate the application of ISM with a dichotomous outcome, we prospectively examined the associations of different activities with various activity displacements with depression risk among 32,900 US women from the Nurses' Health Study who were free from depressive symptoms at baseline (in 1996). During a 10-year follow-up, 5,730 incident depression cases were documented. Results from the ISMs indicated that for each physical activity, differences in magnitude of effects of each activity type were observed, dependent on the activity being displaced/substituted. Notably, an isotemporal substitution gradient was found for television watching, in which its association with depression risk varied by its substitution for slow-, average-, or brisk-paced walking in a gradient toward high depression risk when television watching replaced a faster walking pace (relative risk = 1.18, 95% confidence interval: 1.05, 1.31). Conversely, no association with depression was found for replacement of television watching with 60 minutes/day of slow walking, whereas a lower depression risk (relative risk = 0.85, 95% confidence interval: 0.76, 0.95) was found when 60 minutes/day of brisk walking replaced 60 minutes/day of television watching. Thus, the ISM could offer a more meaningful alternative to the standard nonsubstitution models to support public health recommendations. PMID:23785112

  15. Corrosion Research And Web Site Activities

    NASA Technical Reports Server (NTRS)

    Heidersbach, Robert H.

    2001-01-01

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

  16. Corrosion Research and Web Site Activities

    NASA Technical Reports Server (NTRS)

    Heidersbach, Robert H.

    2002-01-01

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

  17. Substitution of lysine for arginine at position 42 of human transforming growth factor-alpha eliminates biological activity without changing internal disulfide bonds.

    PubMed Central

    Defeo-Jones, D; Tai, J Y; Vuocolo, G A; Wegrzyn, R J; Schofield, T L; Riemen, M W; Oliff, A

    1989-01-01

    Transforming growth factor-alpha (TGF-alpha) is a growth-promoting protein that binds to the epidermal growth factor (EGF) receptor. To identify critical residues that govern TGF-alpha-EGF receptor binding, we prepared site-specific substitution mutants of TGF-alpha. Mutant proteins were tested in receptor-binding and mitogenesis assays. Semiconservative substitutions at positions 4, 12, 18, and 45 decreased biological activity 2.1- to 14-fold. The conservative substitution of lysine for arginine at position 42 completely eliminated biological activity. Amino acid composition analysis of proteolytic fragments from TGF-alpha and the Lys-42 mutant indicated that these proteins contained the same disulfide bonds. These studies suggest that arginine 42 may be a contact point for TGF-alpha-EGF receptor interaction. PMID:2506441

  18. Substituted benzimidazoles with nanomolar activity against respiratory syncytial virus.

    PubMed

    Andries, Koen; Moeremans, Marc; Gevers, Tom; Willebrords, Rudy; Sommen, Cois; Lacrampe, Jean; Janssens, Frans; Wyde, Philip R

    2003-11-01

    A cell-based assay was used to discover compounds inhibiting respiratory syncytial virus (RSV)-induced fusion in HeLa/M cells. A lead compound was identified and subsequent synthesis of >300 analogues led to the identification of JNJ 2408068 (R170591), a low molecular weight (MW 395) benzimidazole derivative with an EC(50) (0.16 nM) against some lab strains almost 100,000 times better than that of ribavirin (15 microM). Antiviral activity was confirmed for subgroup A and B clinical isolates of human RSV and for a bovine RSV isolate. The compound did not inhibit the growth of representative viruses from other Paramyxovirus genera, i.e. HPIV2 and Mumps Virus (genus Rubulavirus), HPIV3 (genus Respirovirus), Measles virus (genus Morbillivirus) and hMPV. Efficacy in cytopathic effect inhibition assays correlated well with efficacy in virus yield reduction assays. A concentration of 10nM reduced RSV production 1000-fold in multi-cycle experiments, irrespective of the multiplicity of infection. Time of addition studies pointed to a dual mode of action: inhibition of virus-cell fusion early in the infection cycle and inhibition of cell-cell fusion at the end of the replication cycle. Two resistant mutants were raised and shown to have single point mutations in the F-gene (S398L and D486N). JNJ 2408068 was also shown to inhibit the release of proinflammatory cytokines IL-6, IL-8 and Rantes from RSV-infected A549 cells.

  19. 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

    2010-08-31

    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.

  20. 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.

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

    USGS Publications Warehouse

    Kendall, William L.; White, Gary C.

    2009-01-01

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

  2. Apoptosis inducing activity of 4-substituted coumarins from Calophyllum brasiliense in human leukaemia HL-60 cells.

    PubMed

    Ito, Chihiro; Murata, Tomiyasu; Itoigawa, Masataka; Nakao, Keisuke; Kaneda, Norio; Furukawa, Hiroshi

    2006-07-01

    With the objective of identifying anti-tumour-promoting agents, we carried out a primary screening of ten 4-substituted coumarins isolated from Calophyllum brasiliense Camb. (Guttiferae), to determine the ability of these compounds to inhibit proliferation of the human leukaemia cell line HL-60. Among the 4-substituted coumarins isolated, calophyllolide (2) and mammea B/BB (3) showed significant cytotoxicity against HL-60 cells. Fluorescence microscopy with Hoechst 33342 staining revealed that the percentage of apoptotic cells with fragmented nuclei and condensed chromatin increased in a time-dependent manner after treatment with calophyllolide (2) or mammea B/BB (3). In addition, the activity of caspase-9 and caspase-3 was also enhanced in a time-dependent manner upon treatment with the 4-substituted coumarins 2 and 3. Caspase-9 and caspase-3 inhibitors suppressed apoptosis induced by 4-substituted coumarins 2 and 3. These results suggest that calophyllolide (2) and mammea B/ BB (3) induced apoptosis in HL-60 cells through activation of the caspase-9/caspase-3 pathway, which is triggered by mitochondrial dysfunction.

  3. 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.

  4. Effects of cation disorder and size on metamagnetism in A-site substituted Pr0.5Ca0.5MnO3 system

    NASA Astrophysics Data System (ADS)

    Mavani, K. R.; Paulose, P. L.

    2005-04-01

    The effects of A-site cation disorder and size on metamagnetism of ABO3 type charge and orbital ordered Pr0.5Ca0.5MnO3 system have been studied by substituting Ba+2 for Ca+2 or La+3 for Pr+3. Substitution of 5% Ba+2 or 5% La+3 drastically reduces the critical magnetic field (Hc) for metamagnetism and induces successive steplike metamagnetic transitions at low temperatures. Interestingly, with further increase in substitution, Hc rises. We find that there is a sharp decrease in electrical resistivity corresponding to the metamagnetic transitions, which is indicative of strongly correlated magnetic and electronic transitions in these manganites.

  5. Synthesis, antimicrobial activity and advances in structure-activity relationships (SARs) of novel tri-substituted thiazole derivatives.

    PubMed

    Reddy, Guda Mallikarjuna; Garcia, Jarem Raul; Reddy, Vemulapati Hanuman; de Andrade, Ageo Meier; Camilo, Alexandre; Pontes Ribeiro, Renan Augusto; de Lazaro, Sergio Ricardo

    2016-11-10

    Trisubstituted thiazoles were synthesized and studied for their antimicrobial activity and supported by theoretical calculations. In addition, MIC, MBC and MFC were also tested. Moreover, the present study was analyzed to scrutinize comprehensive structure-activity relationships. In fact, LUMO orbital energy and orbital orientation was reliable to explain their antibacterial and antifungal assay. Amongst the tested compounds, tri-methyl-substituted thiazole compound showed higher antimicrobial activity and low MIC value due to highest LUMO energy.

  6. Validation of a computational model for predicting the site for electrophilic substitution in aromatic systems.

    PubMed

    Liljenberg, Magnus; Brinck, Tore; Herschend, Björn; Rein, Tobias; Rockwell, Glen; Svensson, Mats

    2010-07-16

    We have investigated the scope and limitations of a method for predicting the regioisomer distribution in electrophilic aromatic substitution reactions that are under kinetic control. This method is based on calculation of the relative stabilities of the sigma-complex intermediates using density functional theory. Predictions from this method can be used quantitatively for halogenations; it agreed to an accuracy of about 1 kcal/mol with experimental observations in 10 of the 11 investigated halogenation reactions. For nitrations, the method gave useful predictions for heterocyclic substrates. The method failed for nitration of monosubstituted benzenes, and we expect that more elaborate model systems, including explicit solvent molecules, will be necessary to obtain quantitatively useful predictions for such cases. For Lewis acid promoted Friedel-Crafts acylations, the method can be expected to give qualitatively correct predictions, that is, to point out the dominating isomer. For substrates where the regioisomeric outcome is highly dependent on the reaction conditions, the method can only be of qualitative use if the concentration of the free Lewis acid is high during the reaction. We have also compared the predictive capacity of the method to that of a modern reactivity index, the average local ionization energy, I(r). The latter method is found to predict the regisolectivity in halogenations and nitrations qualitatively correctly if the positions for the I(r) minima (I(S,min)) are not too sterically hindered but fails for qualitative predictions of F-C reactions. The downscaled I(S,min) values also perform well for the quantitative prediction of regioisomer distributions of halogenations. The accuracy is slightly lower than that for the new method.

  7. 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

  8. Nd substitution in y/ba sites in melt processed YBa2Cu3O7- δ through Nd2O3 additions

    NASA Astrophysics Data System (ADS)

    Varanasi, Chakrapani; Mc Ginn, Paul J.; Blackstead, Howard A.; Pulling, David B.

    1995-12-01

    YBa2Cu3O7- δ (Y123) samples with excess Nd2O3 and Y2O3 additions in the same molar ratios were melt textured in air. In the Nd-doped samples, in addition to Y ion site substitution, partial substitution into the Ba2+ sites is anticipated because of the similar ionic sizes of Nd3+ and Ba2+. The microstructure, Tc, and magnetic properties of Nd-doped samples were analyzed and compared with undoped Y123 and samples with excess Y2O3. The Nd2O3 additions lead to significant magnetization improvements, likely due to both rare earth- and Ba-site substitution by the doped Nd3+ ions, and to increases in Tc. Y2O3 additions resulted in no marked property enhancement.

  9. 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...

  10. 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.

  11. C5-Alkyl-2-methylurea-Substituted Pyridines as a New Class of Glucokinase Activators

    PubMed Central

    2014-01-01

    Glucokinase (GK) activators represent a class of type 2 diabetes therapeutics actively pursued due to the central role that GK plays in regulating glucose homeostasis. Herein we report a novel C5-alkyl-2-methylurea-substituted pyridine series of GK activators derived from our previously reported thiazolylamino pyridine series. Our efforts in optimizing potency, enzyme kinetic properties, and metabolic stability led to the identification of compound 26 (AM-9514). This analogue showed a favorable combination of in vitro potency, enzyme kinetic properties, acceptable pharmacokinetic profiles in preclinical species, and robust efficacy in a rodent PD model. PMID:25516785

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

    PubMed Central

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

    2013-01-01

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

  13. World's First Clinical Case of Gene-Activated Bone Substitute Application

    PubMed Central

    Deev, R. V.; Drobyshev, A. Y.; Isaev, A. A.; Eremin, I. I.

    2016-01-01

    Treatment of patients with large bone defects is a complex clinical problem. We have initiated the first clinical study of a gene-activated bone substitute composed of the collagen-hydroxyapatite scaffold and plasmid DNA encoding vascular endothelial growth factor. The first patient with two nonunions of previously reconstructed mandible was enrolled into the study. Scar tissues were excised; bone defects (5–14 mm) between the mandibular fragments and nonvascularized rib-bone autograft were filled in with the gene-activated bone substitute. No adverse events were observed during 12 months of follow-up. In 3 months, the average density of newly formed tissues within the implantation zone was 402.21 ± 84.40 and 447.68 ± 106.75 HU in the frontal and distal regions, respectively, which correlated with the density of spongy bone. Complete distal bone defect repair with vestibular and lingual cortical plates formation was observed in 6 and 12 months after surgery; thereby the posterior nonunion was successfully eliminated. However, there was partial resorption of the proximal edge of the autograft entailed to relapse of the anterior nonunion. Thus, the first clinical data on the safety and efficacy of the gene-activated bone substitute were obtained. Given a high complexity of the clinical situation the treatment, results might be considered as promising. NCT02293031. PMID:27891264

  14. 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.

  15. [Structural regularities in activated cleavage sites of thrombin receptors].

    PubMed

    Mikhaĭlik, I V; Verevka, S V

    1999-01-01

    Comparison of thrombin receptors activation splitting sites sequences testifies to their similarity both in activation splitting sites of protein precursors and protein proteinase inhibitors reactive sites. In all these sites corresponded to effectory sites P2'-positions are placed by hydrophobic amino-acids only. The regularity defined conforms with previous thesis about the role of effectory S2'-site in regulation of the processes mediated by serine proteinases.

  16. The Effect of Selective D- or Nα-Methyl Arginine Substitution on the Activity of the Proline-Rich Antimicrobial Peptide, Chex1-Arg20

    PubMed Central

    Li, Wenyi; Sun, Zhe; O'Brien-Simpson, Neil M.; Otvos, Laszlo; Reynolds, Eric C.; Hossain, Mohammed A.; Separovic, Frances; Wade, John D.

    2017-01-01

    In vivo pharmacokinetics studies have shown that the proline-rich antimicrobial peptide, A3-APO, which is a discontinuous dimer of the peptide, Chex1-Arg20, undergoes degradation to small fragments at positions Pro6-Arg7 and Val19-Arg20. With the aim of minimizing or abolishing this degradation, a series of Chex1-Arg20 analogs were prepared via Fmoc/tBu solid phase peptide synthesis with D-arginine or, in some cases, peptide backbone Nα-methylated arginine, substitution at these sites. All the peptides were tested for antibacterial activity against the Gram-negative bacterium Klebsiella pneumoniae. The resulting activity of position-7 substitution of Chex1-Arg20 analogs showed that arginine-7 is a crucial residue for maintaining activity against K. pneumoniae. However, arginine-20 substitution had a much less deleterious effect on the antibacterial activity of the peptide. Moreover, none of these peptides displayed any cytotoxicity to HEK and H-4-II-E mammalian cells. These results will aid the development of more effective and stable PrAMPs via judicious amino acid substitutions. PMID:28154813

  17. Inhibition of NADPH oxidase activation in endothelial cells by ortho-methoxy-substituted catechols.

    PubMed

    Johnson, David K; Schillinger, Kurt J; Kwait, David M; Hughes, Chambers V; McNamara, Erin J; Ishmael, Fauod; O'Donnell, Robert W; Chang, Ming-Mei; Hogg, Michael G; Dordick, Jonathan S; Santhanam, Lakshmi; Ziegler, Linda M; Holland, James A

    2002-01-01

    NADPH oxidase is a major enzymatic source of oxygen free radicals in stimulated endothelial cells (ECs). The ortho-methoxy-substituted catechol, apocynin (4-hydroxy-3-methoxyacetophenone), isolated from the traditional medicinal plant Picrorhiza kurroa, inhibits the release of superoxide anion (O2*-) by this enzyme. The compound acts by blocking the assembly of a functional NADPH oxidase complex. The underlying chemistry of this inhibitory activity, and its physiological significance to EC proliferation, have been investigated. A critical event is the reaction of ortho-methoxy-substituted catechols with reactive oxygen species (ROS) and peroxidase. Analysis of this reaction reveals that apocynin is converted to a symmetrical dimer through the formation of a 5,5' carbon-carbon bond. Both reduced glutathione and L-cysteine inhibit this dimerization process. Catechols without the ortho-methoxy-substituted group do not undergo this chemical reaction. Superoxide production by an endothelial cell-free system incubated with apocynin was nearly completely inhibited after a lagtime for inhibition of ca. 2 min. Conversely, O2*- production was nearly completely inhibited, without a lagtime, by incubation with the dimeric form of apocynin. The apocynin dimer undergoes a two-electron transfer reaction with standard redox potentials of -0.75 and -1.34 V as determined by cyclic voltammetry. Inhibition of endothelial NADPH oxidase by apocynin caused a dose-dependent inhibition of cell proliferation. These findings identify a metabolite of an ortho-methoxy-substituted catechol, which may be the active compound formed within stimulated ECs that prevents NADPH oxidase complex assembly and activation.

  18. Structural and thermoelectric properties of A-site substituted (Sr1-x-yCaxNdy)TiO3 perovskites

    NASA Astrophysics Data System (ADS)

    Somaily, Hamoud H.

    Detailed structural results and models are reported for a special class of A-site substituted perovskites, (Sr1-x-yCaxNd y)TiO3, obtained with high resolution NPD data as a function of temperature and Nd composition. Two series with various A-site concentrations were synthesized and investigated. Each series was designed to have a nominally constant tolerance factor. At room temperature (RT), I determine the space groups of the Sr-rich and Sr poor series as being tetragonal I4/mcm and orthorhombic Pbnm, respectively. The RT structures remain unchanged upon increasing the Nd3+ content. However, three different orthorhombic phases, Pbnm, Ibmm, Pbcm, are determined for the Sr-rich series as a function of decreasing temperature; whereas, for the Sr-poor series the orthorhombic Pbnm structure is found to persist throughout the full range of measured temperatures. A phase diagram is constructed and proposed in the temperature range 0-1000 K. Thermoelectric properties of (Sr 1-x-yCaxNdy)TiO3 were also investigated and the best figure of merit ZT=0.07 was obtained with the Sr-rich series.

  19. 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

  20. Design, synthesis and potential 6 Hz psychomotor seizure test activity of some novel 2-(substituted)-3-{[substituted]amino}quinazolin-4(3H)-one.

    PubMed

    Kumar, Praveen; Shrivastava, Birendra; Pandeya, Surendra N; Stables, James P

    2011-04-01

    Thirty new 2-(substituted)-3-{[substituted]amino}quinazolin-4(3H)-one were designed and synthesized keeping in view the structural requirement of pharmacophore and evaluated for anticonvulsant activity and neurotoxicity. The anticonvulsant activity of the titled compounds was assessed using the 6 Hz psychomotor seizure test. The most active compound of the series was 3-({(E)-[3-(4-chloro-3-methylphenoxy)phenyl]methylidene}amino)-2-phenylquinazolin-4(3H)-one PhQZ 7, which showed 100% protection (4/4, 0.5 h) and 75% protection (3/4, 0.25 h) at a dose of 100 mg/kg in mice. A computational study was carried out for calculation of pharmacophore pattern and prediction of pharmacokinetic properties. Titled compounds have also exhibited good binding properties with epilepsy molecular targets such as glutamate, GABA (A) delta and GABA (A) alpha-1 receptors, in Lamarckian genetic algorithm based flexible docking studies.

  1. Synthesis and antileishmanial activity of 6-mono-substituted and 3,6-di-substituted acridines obtained by acylation of proflavine.

    PubMed

    Di Giorgio, Carole; Shimi, Kamal; Boyer, Gérard; Delmas, Florence; Galy, Jean-Pierre

    2007-10-01

    Two new series of diaminoacridinic derivatives obtained from proflavine and N-(6-amino-3-acridinyl)acetamide were synthesised and assessed for their cytotoxic and antileishmanial activities. Two compounds, N-[6-(acetylamino)-3-acridinyl]acetamide and N-[6-(benzoylamino)-3-acridinyl]benzamide demonstrated highly specific antileishmanial properties against the intracellular amastigote form of the parasite. Structure-activity relationships established that the antiproliferative activity against human cells was greatly enhanced by the presence of a benzoylamino group in 6-mono-substituted acridines, while the presence of two acetylamino or benzoylamino groups in 3,6-di-substituted acridines strongly increased the specificity of the molecules for Leishmania parasite, suggesting that symmetric conformations could preferentially interfere with Leishmania metabolism.

  2. 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

  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. [3+3] Cyclocondensation of Disubstituted Biphenyl Dialdehydes: Access to Inherently Luminescent and Optically Active Hexa-substituted C3-Symmetric and Asymmetric Trianglimine Macrocycles.

    PubMed

    Wang, Zhenzhen; Nour, Hany F; Roch, Loïc M; Guo, Minjie; Li, Wenjiao; Baldridge, Kim K; Sue, Andrew C H; Olson, Mark A

    2017-03-03

    A general synthetic route to inherently luminescent and optically active 6-fold substituted C3-symmetric and asymmetric biphenyl-based trianglimines has been developed. The synthesis of these hexa-substituted triangular macrocycles takes advantage of a convenient method for the synthesis of symmetrically and asymmetrically difunctionalized biphenyl dialdehydes through a convergent two-step aromatic nucleophilic substitution-one-pot Suzuki-coupling reaction protocol. A modular [3+3] diamine-dialdehyde cyclocondensation reaction between both the symmetrically and asymmetrically difunctionalized-4,4'-biphenyldialdehydes with enantiomerically pure (1R,2R)-1,2-diaminocyclohexane was employed to construct the hexa-substituted triangular macrocycles. B97-D/6-311G(2d,p) density functional theory determined structures and X-ray crystallographic analysis reveal that the six substituents appended to the biphenyl legs of the trianglimine macrocycles adopt an alternating conformation not unlike the 1,3,5-alternate conformation observed for calix[6]arenes. Reduction of the imine bonds using NaBH4 afforded the corresponding 6-fold substituted trianglamine without the need to alkylate the amine nitrogen atoms which could hinder their later use as metal coordination sites and without having to introduce asymmetric carbons.

  5. 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

  6. Synthesis and κ-opioid receptor activity of furan-substituted salvinorin A analogues.

    PubMed

    Riley, Andrew P; Groer, Chad E; Young, David; Ewald, Amy W; Kivell, Bronwyn M; Prisinzano, Thomas E

    2014-12-26

    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.

  7. 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

  8. 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.

  9. Investigating the spectrum of biological activity of substituted quinoline-2-carboxamides and their isosteres.

    PubMed

    Gonec, Tomas; Bobal, Pavel; Sujan, Josef; Pesko, Matus; Guo, Jiahui; Kralova, Katarina; Pavlacka, Lenka; Vesely, Libor; Kreckova, Eva; Kos, Jiri; Coffey, Aidan; Kollar, Peter; Imramovsky, Ales; Placek, Lukas; Jampilek, Josef

    2012-01-10

    In this study, a series of thirty-five substituted quinoline-2-carboxamides and thirty-three substituted naphthalene-2-carboxamides were prepared and characterized. They were tested for their activity related to the inhibition of photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. Primary in vitro screening of the synthesized compounds was also performed against four mycobacterial species. N-Cycloheptylquinoline-2-carboxamide, N-cyclohexylquinoline-2-carboxamide and N-(2-phenylethyl)quinoline-2-carboxamide showed higher activity against M. tuberculosis than the standards isoniazid or pyrazinamide and 2-(pyrrolidin-1-ylcarbonyl)quinoline and 1-(2-naphthoyl)pyrrolidine expressed higher activity against M. kansasii and M. avium paratuberculosis than the standards isoniazid or pyrazinamide. The most effective antimycobacterial compounds demonstrated insignificant toxicity against the human monocytic leukemia THP-1 cell line. The PET-inhibiting activity expressed by IC(50) value of the most active compound N-benzyl-2-naphthamide was 7.5 μmol/L. For all compounds, the structure-activity relationships are discussed.

  10. Design, synthesis and evaluation of novel 7-aminoalkyl-substituted flavonoid derivatives with improved cholinesterase inhibitory activities.

    PubMed

    Luo, Wen; Chen, Ying; Wang, Ting; Hong, Chen; Chang, Li-Ping; Chang, Cong-Cong; Yang, Ya-Cheng; Xie, Song-Qiang; Wang, Chao-Jie

    2016-02-15

    A novel series of 7-aminoalkyl-substituted flavonoid derivatives 5a-5r were designed, synthesized and evaluated as potential cholinesterase inhibitors. The results showed that most of the synthesized compounds exhibited potent acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities at the micromolar range. Compound 2-(naphthalen-1-yl)-7-(8-(pyrrolidin-1-yl)octyloxy)-4H-chromen-4-one (5q) showed the best inhibitory activity (IC50, 0.64μM for AChE and 0.42μM for BChE) which were better than our previously reported compounds and the commercially available cholinergic agent Rivastigmine. The results from a Lineweaver-Burk plot indicated a mixed-type inhibition for compound 5q with AChE and BChE. Furthermore, molecular modeling study showed that 5q targeted both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. Besides, these compounds (5a-5r) did not affect PC12 and HepG2 cell viability at the concentration of 10μM. Consequently, these flavonoid derivatives should be further investigated as multipotent agents for the treatment of Alzheimer's disease.

  11. Bronchodilator activity of xanthine derivatives substituted with functional groups at the 1- or 7-position.

    PubMed

    Miyamoto, K; Yamamoto, Y; Kurita, M; Sakai, R; Konno, K; Sanae, F; Ohshima, T; Takagi, K; Hasegawa, T; Iwasaki, N

    1993-05-14

    Xanthine derivatives with several functional groups at the 1- or 7-position were synthesized, and their pharmacological activities in guinea pigs were studied. In general, the in vitro tracheal relaxant action and positive chronotropic action of 3-propylxanthines were increased by substitutions with nonpolar functional groups at the 1-position, but decreased by any substitution at the 7-position. On the other hand, because positive chronotropic actions of substituents with allyl, aminoalkyl, alkoxyalkyl, and normal alkyl groups were much less than tracheal muscle became very high with substitutions of 3'-butenyl, (dimethylamino)ethyl, 2'-ethoxyethyl, 3'-methoxypropyl, and n-propyl groups at the 1-position and of 2'-ethoxyethyl, 2'-oxopropyl, and n-propyl groups at the 7-position, compared with theophylline and the corresponding unsubstituted xanthines, 3-propylxanthine and 1-methyl-3-propylxanthine. When compounds were intraduodenally administered to the guinea pig, 1-(2'-ethoxyethyl)-, 1-(3'-methoxypropyl)-, 1-(3'-butenyl)-, and 1-[(dimethylamino)-ethyl]-3-propylxanthines, 1-methyl-7-(2'-oxopropyl)-3-propylxanthine, and denbufylline (1,3-di-n-butyl-7-(2'-oxopropyl)xanthine) effectively inhibited the acetylcholine-induced bronchospasm without heart stimulation or central nervous system-stimulation at the effective dosage range. Particularly, the bronchodilatory effect of 1-(2'-ethoxyethyl)-3-propylxanthine was much stronger and more continuous than those of theophylline and pentoxifylline. On the other hand, there were certain relationships among the in vitro tracheal relaxant activities of these compounds, their affinities for adenosine (A1) receptors in the brain membrane, and their inhibition of cyclic AMP-phosphodiesterase (PDE) in the tracheal muscle. The affinity for A2 receptors of these compounds was very low or negligible. This suggests that both the action on A1 receptors or interaction with adenosine and the cyclic AMP-PDE inhibitory activity contribute

  12. Antimicrobial activity and stability of protonectin with D-amino acid substitutions.

    PubMed

    Qiu, Shuai; Zhu, Ranran; Zhao, Yanyan; An, Xiaoping; Jia, Fengjing; Peng, Jinxiu; Ma, Zelin; Zhu, Yuanyuan; Wang, Jiayi; Su, Jinhuan; Wang, Qingjun; Wang, Hailin; Li, Yuan; Wang, Kairong; Yan, Wenjin; Wang, Rui

    2017-03-16

    The misuse and overuse of antibiotics result in the emergence of resistant bacteria and fungi, which make an urgent need of the new antimicrobial agents. Nowadays, antimicrobial peptides have attracted great attention of researchers. However, the low physiological stability in biological system limits the application of naturally occurring antimicrobial peptides as novel therapeutics. In the present study, we synthesized derivatives of protonectin by substituting all the amino acid residues or the cationic lysine residue with the corresponding D-amino acids. Both the D-enantiomer of protonectin (D-prt) and D-Lys-protonectin (D-Lys-prt) exhibited strong antimicrobial activity against bacteria and fungi. Moreover, D-prt showed strong stability against trypsin, chymotrypsin and the human serum, while D-Lys-prt only showed strong stability against trypsin. Circular dichroism analysis revealed that D-Lys-prt still kept typical α-helical structure in the membrane mimicking environment, while D-prt showed left hand α-helical structure. In addition, propidium iodide uptake assay and bacteria and fungi killing experiments indicated that all D-amino acid substitution or partially D-amino acid substitution analogs could disrupt the integrity of membrane and lead the cell death. In summary, these findings suggested that D-prt and D-Lys-prt might be promising candidate antibiotic agents for therapeutic application against resistant bacteria and fungi infection. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

  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. 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.

  15. [Synthesis and antitumor activity of 5-substituted-2-(pyridyl)benzothiazole compounds].

    PubMed

    Liu, Wen-Hu; Chang, Jin-Xia; Liu, Yi

    2013-01-01

    Fifteen novel 5-substituted-2-(pyridyl)benzothiazole compounds were designed and synthesized by simple hydrolization and condensation reaction of the 2-amino-5-substituent benzothiazole. Activities of these synthesized compounds were evaluated on Bcap-37, HCT-15 and HepG2 tumor cells in vitro by standard MTT assay. 5-Fluorouracil (5-FU) was used as the positive control. The results revealed that most of the new compounds had potent effects on Bcap-37, HCT-15 and HepG2 tumor cells, and had no or less effect on 293T and L02 normal cells. Particularly, compounds 1c and 2e exhibited better activities on HCT-15 and HepG2 cells with IC50 values of 41.59 and 38.65 micromol x L(-1), and 1i showed excellent activities on Bcap-37 and HepG2 cells with IC50 values of 46.63 and 23.51 micromol x L(-1), respectively. The structure-activity relationship of 5-substituted-2-(pyridyl)benzothiazole compounds were also discussed preliminarily.

  16. N-Substituted piperazinyl quinolones as potential cytotoxic agents: structure-activity relationships study.

    PubMed

    Foroumadi, Alireza; Emami, Saeed; Rajabalian, Saeed; Badinloo, Marziyeh; Mohammadhosseini, Negar; Shafiee, Abbas

    2009-03-01

    As part of a continuing search for new potential anticancer candidates in the piperazinyl quinolone series, the cytotoxicity evaluation of new N-substituted piperazinyl quinolones was of our interest. The growth inhibitory activities of 12 new compounds, namely N-[2-(5-chlorothiophen-2-yl)-2-oxoethyl] and N-[2-(5-chlorothiophen-2-yl)-2-oxyiminoethyl] piperazinyl quinolones 1-12 were determined against six cancer cell lines using MTT colorimetric assay. Preliminary screening showed that most of the new N-[2-(5-chlorothiophen-2-yl)ethyl]piperazinyl quinolones 4-12 containing (un)substituted oxime moiety showed significant cytotoxic activity and the modification of functionality on ethyl spacer produced a relatively minor change of activity. Thus, in the piperazinyl quinolone series, cytotoxic activity can be positively modulated through the introduction of 2-(5-chlorothiophen-2-yl)ethyl residue on the piperazine ring. The results revealed that the introduction of 2-(5-chlorothiophen-2-yl)ethyl moiety on the piperazine ring of quinolone antibacterials (ciprofloxacin, norfloxacin and enoxacin) changes the biological profile of piperazinyl quinolones from antibacterials to cytotoxic agents.

  17. Hydroxyproline substitutions stabilize non-glycosylated drosocin against serum proteases without challenging its antibacterial activity.

    PubMed

    Knappe, Daniel; Cassone, Marco; Nollmann, Friederike Inga; Otvos, Laszlo; Hoffmann, Ralf

    2014-04-01

    The increasing incidence of multi- and pan-resistant pathogens demands novel compounds to fight Grampositive and especially Gram-negative bacteria. Among the currently investigated compound classes, antimicrobial peptides (AMPs) inhibiting specific bacterial targets appear especially promising for systemic therapy of infections, although unmodified linear peptides are typically rapidly degraded by serum proteases. Proline-rich AMPs have been heavily investigated in recent years due to their low toxicity and proven in vivo efficacy. Here, we report novel unglycosylated drosocin analogs with extended half-life in mouse serum and improved activity against Gram-negative pathogens Escherichia coli and Klebsiella pneumoniae. Substituting proline (Pro) residues in positions 3, 5, 10, and 14 with trans-4-hydroxy-Lproline ((t)Hyp) improved the antibacterial activity, whereas substitution of Pro-16 reduced the activity. Drosocin analogs with (t)Hyp in positions 3 and 5 were also four to eight times more stable in mouse serum than the unmodified analog. The new compounds were not toxic against human HeLa, HEK293, and HepG2 cell lines and showed no hemolytic activity against human erythrocytes at peptide concentrations of at least 600 µg/mL.

  18. 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.

  19. Substitution scanning identifies a novel, catalytically active ibrutinib-resistant BTK cysteine 481 to threonine (C481T) variant.

    PubMed

    Hamasy, A; Wang, Q; Blomberg, K E M; Mohammad, D K; Yu, L; Vihinen, M; Berglöf, A; Smith, C I E

    2017-01-01

    Irreversible Bruton tyrosine kinase (BTK) inhibitors, ibrutinib and acalabrutinib have demonstrated remarkable clinical responses in multiple B-cell malignancies. Acquired resistance has been identified in a sub-population of patients in which mutations affecting BTK predominantly substitute cysteine 481 in the kinase domain for catalytically active serine, thereby ablating covalent binding of inhibitors. Activating substitutions in the BTK substrate phospholipase Cγ2 (PLCγ2) instead confers resistance independent of BTK. Herein, we generated all six possible amino acid substitutions due to single nucleotide alterations for the cysteine 481 codon, in addition to threonine, requiring two nucleotide substitutions, and performed functional analysis. Replacement by arginine, phenylalanine, tryptophan or tyrosine completely inactivated the catalytic activity, whereas substitution with glycine caused severe impairment. BTK with threonine replacement was catalytically active, similar to substitution with serine. We identify three potential ibrutinib resistance scenarios for cysteine 481 replacement: (1) Serine, being catalytically active and therefore predominating among patients. (2) Threonine, also being catalytically active, but predicted to be scarce, because two nucleotide changes are needed. (3) As BTK variants replaced with other residues are catalytically inactive, they presumably need compensatory mutations, therefore being very scarce. Glycine and tryptophan variants were not yet reported but likely also provide resistance.

  20. Substitution scanning identifies a novel, catalytically active ibrutinib-resistant BTK cysteine 481 to threonine (C481T) variant

    PubMed Central

    Hamasy, A; Wang, Q; Blomberg, K E M; Mohammad, D K; Yu, L; Vihinen, M; Berglöf, A; Smith, C I E

    2017-01-01

    Irreversible Bruton tyrosine kinase (BTK) inhibitors, ibrutinib and acalabrutinib have demonstrated remarkable clinical responses in multiple B-cell malignancies. Acquired resistance has been identified in a sub-population of patients in which mutations affecting BTK predominantly substitute cysteine 481 in the kinase domain for catalytically active serine, thereby ablating covalent binding of inhibitors. Activating substitutions in the BTK substrate phospholipase Cγ2 (PLCγ2) instead confers resistance independent of BTK. Herein, we generated all six possible amino acid substitutions due to single nucleotide alterations for the cysteine 481 codon, in addition to threonine, requiring two nucleotide substitutions, and performed functional analysis. Replacement by arginine, phenylalanine, tryptophan or tyrosine completely inactivated the catalytic activity, whereas substitution with glycine caused severe impairment. BTK with threonine replacement was catalytically active, similar to substitution with serine. We identify three potential ibrutinib resistance scenarios for cysteine 481 replacement: (1) Serine, being catalytically active and therefore predominating among patients. (2) Threonine, also being catalytically active, but predicted to be scarce, because two nucleotide changes are needed. (3) As BTK variants replaced with other residues are catalytically inactive, they presumably need compensatory mutations, therefore being very scarce. Glycine and tryptophan variants were not yet reported but likely also provide resistance. PMID:27282255

  1. MYST protein acetyltransferase activity requires active site lysine autoacetylation.

    PubMed

    Yuan, Hua; Rossetto, Dorine; Mellert, Hestia; Dang, Weiwei; Srinivasan, Madhusudan; Johnson, Jamel; Hodawadekar, Santosh; Ding, Emily C; Speicher, Kaye; Abshiru, Nebiyu; Perry, Rocco; Wu, Jiang; Yang, Chao; Zheng, Y George; Speicher, David W; Thibault, Pierre; Verreault, Alain; Johnson, F Bradley; Berger, Shelley L; Sternglanz, Rolf; McMahon, Steven B; Côté, Jacques; Marmorstein, Ronen

    2012-01-04

    The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases.

  2. MYST protein acetyltransferase activity requires active site lysine autoacetylation

    PubMed Central

    Yuan, Hua; Rossetto, Dorine; Mellert, Hestia; Dang, Weiwei; Srinivasan, Madhusudan; Johnson, Jamel; Hodawadekar, Santosh; Ding, Emily C; Speicher, Kaye; Abshiru, Nebiyu; Perry, Rocco; Wu, Jiang; Yang, Chao; Zheng, Y George; Speicher, David W; Thibault, Pierre; Verreault, Alain; Johnson, F Bradley; Berger, Shelley L; Sternglanz, Rolf; McMahon, Steven B; Côté, Jacques; Marmorstein, Ronen

    2012-01-01

    The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases. PMID:22020126

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

    PubMed

    Hamada, Nagwa Mohamed Mahrous; Abdo, Nadia Yousef Megally

    2015-06-08

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

  4. 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

  5. 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

  6. Using Unnatural Amino Acids to Probe the Energetics of Oxyanion Hole Hydrogen Bonds in the Ketosteroid Isomerase Active Site

    PubMed Central

    2015-01-01

    Hydrogen bonds are ubiquitous in enzyme active sites, providing binding interactions and stabilizing charge rearrangements on substrate groups over the course of a reaction. But understanding the origin and magnitude of their catalytic contributions relative to hydrogen bonds made in aqueous solution remains difficult, in part because of complexities encountered in energetic interpretation of traditional site-directed mutagenesis experiments. It has been proposed for ketosteroid isomerase and other enzymes that active site hydrogen bonding groups provide energetic stabilization via “short, strong” or “low-barrier” hydrogen bonds that are formed due to matching of their pKa or proton affinity to that of the transition state. It has also been proposed that the ketosteroid isomerase and other enzyme active sites provide electrostatic environments that result in larger energetic responses (i.e., greater “sensitivity”) to ground-state to transition-state charge rearrangement, relative to aqueous solution, thereby providing catalysis relative to the corresponding reaction in water. To test these models, we substituted tyrosine with fluorotyrosines (F-Tyr’s) in the ketosteroid isomerase (KSI) oxyanion hole to systematically vary the proton affinity of an active site hydrogen bond donor while minimizing steric or structural effects. We found that a 40-fold increase in intrinsic F-Tyr acidity caused no significant change in activity for reactions with three different substrates. F-Tyr substitution did not change the solvent or primary kinetic isotope effect for proton abstraction, consistent with no change in mechanism arising from these substitutions. The observed shallow dependence of activity on the pKa of the substituted Tyr residues suggests that the KSI oxyanion hole does not provide catalysis by forming an energetically exceptional pKa-matched hydrogen bond. In addition, the shallow dependence provides no indication of an active site electrostatic

  7. Synthesis and monoamine oxidase inhibitory activities of some 3-(4-fluorophenyl)-5-aryl-n-substituted-4,5-dihydro-(1H)-pyrazole-1-carbothioamide derivatives.

    PubMed

    Koç, G Ş; Tan, O U; Uçar, G; Yildirim, E; Erol, K; Palaska, E

    2014-11-01

    28 new 3-(4-fluorophenyl)-5-aryl-N-substituted-4,5-dihydro-1H-pyrazole-1-carbothioamide derivatives were synthesized and evaluated in vitro for their monoamine oxidase (MAO) A and B inhibitory activity and selectivity. The derivatives substituted by halogen on the fifth position of pyrazole ring, inhibited MAO-A enzyme with a high selectivity index. On the other hand, compounds substituted with 2-naphthyl inhibited MAO-B enzyme with a moderate selectivity index. Docking studies were done to highlight the interactions of the most active derivative with the active site of MAO-A. In addition, in vivo antidepressant and anxiolytic activities of the compounds having selective MAO-A inhibitory effects, were investigated by using Porsolt forced swimming and elevated plus-maze tests respectively. 3-(4-Fluorophenyl)-5-(4-chloro-phenyl)-N-allyl-4,5-dihydro-1H-pyrazole-1-carbothio-amide has antidepressant, 3-(4-fluorophenyl)-5-(4-chlorophenyl)-N-methyl-4,5-dihydro-1H-pyrazole-1-carbothioamide and 3-(4-fluoro-phenyl)-5-(4-bromophenyl)-N-ethyl-4,5-dihydro-1H-pyrazole-1-carbothioamide have anxiolytic activity.

  8. Conformational plasticity surrounding the active site of NADH oxidase from Thermus thermophilus

    PubMed Central

    Miletti, Teresa; Di Trani, Justin; Jr Levros, Louis-Charles; Mittermaier, Anthony

    2015-01-01

    Biotechnological applications of enzymes can involve the use of these molecules under nonphysiological conditions. Thus, it is of interest to understand how environmental variables affect protein structure and dynamics and how this ultimately modulates enzyme function. NADH oxidase (NOX) from Thermus thermophilus exemplifies how enzyme activity can be tuned by reaction conditions, such as temperature, cofactor substitution, and the addition of cosolutes. This enzyme catalyzes the oxidation of reduced NAD(P)H to NAD(P)+ with the concurrent reduction of O2 to H2O2, with relevance to biosensing applications. It is thermophilic, with an optimum temperature of approximately 65°C and sevenfold lower activity at 25°C. Moderate concentrations (≈1M) of urea and other chaotropes increase NOX activity by up to a factor of 2.5 at room temperature. Furthermore, it is a flavoprotein that accepts either FMN or the much larger FAD as cofactor. We have used nuclear magnetic resonance (NMR) titration and 15N spin relaxation experiments together with isothermal titration calorimetry to study how NOX structure and dynamics are affected by changes in temperature, the addition of urea and the substitution of the FMN cofactor with FAD. The majority of signals from NOX are quite insensitive to changes in temperature, cosolute addition, and cofactor substitution. However, a small cluster of residues surrounding the active site shows significant changes. These residues are implicated in coupling changes in the solution conditions of the enzyme to changes in catalytic activity. PMID:25970557

  9. In vivo reshaping the catalytic site of nucleoside 2'-deoxyribosyltransferase for dideoxy- and didehydronucleosides via a single amino acid substitution.

    PubMed

    Kaminski, Pierre Alexandre; Dacher, Priscilla; Dugué, Laurence; Pochet, Sylvie

    2008-07-18

    Nucleoside 2'-deoxyribosyltransferases catalyze the transfer of 2-deoxyribose between bases and have been widely used as biocatalysts to synthesize a variety of nucleoside analogs. The genes encoding nucleoside 2'-deoxyribosyltransferase (ndt) from Lactobacillus leichmannii and Lactobacillus fermentum underwent random mutagenesis to select variants specialized for the synthesis of 2',3'-dideoxynucleosides. An Escherichia coli strain, auxotrophic for uracil and unable to use 2',3'-dideoxyuridine, cytosine, and 2',3'-dideoxycytidine as a source of uracil was constructed. Randomly mutated lactobacilli ndt libraries from two species, L. leichmannii and L. fermentum, were screened for the production of uracil with 2',3'-dideoxyuridine as a source of uracil. Several mutants suitable for the synthesis of 2',3'-dideoxynucleosides were isolated. The nucleotide sequence of the corresponding genes revealed a single mutation (G --> A transition) leading to the substitution of a small aliphatic amino acid by a nucleophilic one, A15T (L. fermentum) or G9S (L. leichmannii), respectively. We concluded that the "adaptation" of the nucleoside 2'-deoxyribosyltransferase activity to 2,3-dideoxyribosyl transfer requires an additional hydroxyl group on a key amino acid side chain of the protein to overcome the absence of such a group in the corresponding substrate. The evolved proteins also display significantly improved nucleoside 2',3'-didehydro-2',3'-dideoxyribosyltransferase activity.

  10. Synthesis and structure-activity relationships of novel amino/nitro substituted 3-arylcoumarins as antibacterial agents.

    PubMed

    Matos, Maria J; Vazquez-Rodriguez, Saleta; Santana, Lourdes; Uriarte, Eugenio; Fuentes-Edfuf, Cristina; Santos, Ysabel; Muñoz-Crego, Angeles

    2013-01-24

    A new series of amino/nitro-substituted 3-arylcoumarins were synthesized and their antibacterial activity against clinical isolates of Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative) was evaluated. Some of these molecules exhibited antibacterial activity against S. aureus comparable to the standards used (oxolinic acid and ampicillin). The preliminary structure-activity relationship (SAR) study showed that the antibacterial activity against S. aureus depends on the position of the 3-arylcoumarin substitution pattern. With the aim of finding the structural features for the antibacterial activity and selectivity, in the present manuscript different positions of nitro, methyl, methoxy, amino and bromo substituents on the 3-arylcoumarin scaffold were reported.

  11. [Effect of the structural integrity of Na,K-ATPase preparations on the ability of lithium to substitute for sodium in enzyme activation].

    PubMed

    Skul'skiĭ, I A; Krestinskaia, T V; Pisareva, L N; Baklanova, S M; Solius, A A

    1982-01-01

    The ability of Li+ to substitute for Na+ in activating ouabain-sensitive ATPase of the rat kidney was studied on three kinds of the enzyme preparation: a) crude membrane fraction, b) purified enzyme, c) tissue slices. The preparations were free of endogenous Na+ and K+. The ATPase activity of slices was estimated both biochemically and cytochemically. In slices, the extent of Li+--Na+-isomorphism was about 10-fold higher than that in the purified enzyme. It is concluded that the biochemical procedures involved in the enzyme isolation procedure cause the damage of membranes, thus decreasing Li+-affinity to the Na+-dependent site.

  12. QSAR modeling for anti-human African trypanosomiasis activity of substituted 2-Phenylimidazopyridines

    NASA Astrophysics Data System (ADS)

    Masand, Vijay H.; El-Sayed, Nahed N. E.; Mahajan, Devidas T.; Mercader, Andrew G.; Alafeefy, Ahmed M.; Shibi, I. G.

    2017-02-01

    In the present work, sixty substituted 2-Phenylimidazopyridines previously reported with potent anti-human African trypanosomiasis (HAT) activity were selected to build genetic algorithm (GA) based QSAR models to determine the structural features that have significant correlation with the activity. Multiple QSAR models were built using easily interpretable descriptors that are directly associated with the presence or the absence of a structural scaffold, or a specific atom. All the QSAR models have been thoroughly validated according to the OECD principles. All the QSAR models are statistically very robust (R2 = 0.80-0.87) with high external predictive ability (CCCex = 0.81-0.92). The QSAR analysis reveals that the HAT activity has good correlation with the presence of five membered rings in the molecule.

  13. 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.

  14. Unraveling the effect of La A-site substitution on oxygen ion diffusion and oxygen catalysis in perovskite BaFeO3 by data-mining molecular dynamics and density functional theory.

    PubMed

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

    2015-10-07

    BaFeO3 (BFO) is a promising parent material for high-temperature oxygen catalysis. The effects of La substitution on the oxygen ion diffusion and oxygen catalysis in A-site La-substituted BFO are studied by combining data-driven molecular dynamics (MD) simulations and density functional theory (DFT) calculations. The data-driven MD simulations are capable of providing atomic level information regarding oxygen jumps at different sites, bridging the resolution gap of analysis between MD and DFT. The simulations identify several effects due to the introduction of La. First, according to simple electroneutrality considerations and DFT calculations, La tends to decrease the concentration of oxygen vacancies in BFO. Second, La substitution lowers the activation energy of local oxygen migration, providing faster paths for oxygen diffusion. The MD analysis predicts a higher hopping rate through La-containing bottlenecks as well as easier oxygen jumps from the La-rich cages and lower dwell times of oxygen in those cages. DFT calculations confirm a lower migration energy through La-containing bottlenecks. Third, the electrocatalytic activity of the material decreases with La, as indicated by a lower O p-band center and higher oxygen vacancy formation energies.

  15. Identification of active sites in amidase: Evolutionary relationship between amide bond- and peptide bond-cleaving enzymes

    PubMed Central

    Kobayashi, Michihiko; Fujiwara, Yoshie; Goda, Masahiko; Komeda, Hidenobu; Shimizu, Sakayu

    1997-01-01

    Mainly based on various inhibitor studies previously performed, amidases came to be regarded as sulfhydryl enzymes. Not completely satisfied with this generally accepted interpretation, we performed a series of site-directed mutagenesis studies on one particular amidase of Rhodococcus rhodochrous J1 that was involved in its nitrile metabolism. For these experiments, the recombinant amidase was produced as the inclusion body in Escherichia coli to greatly facilitate its recovery and subsequent purification. With regard to the presumptive active site residue Cys203, a Cys203 → Ala mutant enzyme still retained 11.5% of the original specific activity. In sharp contrast, substitutions in certain other positions in the neighborhood of Cys203 had a far more dramatic effect on the amidase. Glutamic acid substitution of Asp191 reduced the specific activity of the mutant enzyme to 1.33% of the wild-type activity. Furthermore, Asp191 → Asn substitution as well as Ser195 → Ala substitution completely abolished the specific activity. It would thus appear that, among various conserved residues residing within the so-called signature sequence common to all amidases, the real active site residues are Asp191 and Ser195 rather than Cys203. Inasmuch as an amide bond (CO-NH2) in the amide substrate is not too far structurally removed from a peptide bond (CO-NH-), the signature sequences of various amidases were compared with the active site sequences of various types of proteases. It was found that aspartic acid and serine residues corresponding to Asp191 and Ser195 of the Rhodococcus amidase are present within the active site sequences of aspartic proteinases, thus suggesting the evolutionary relationship between the two. PMID:9342349

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

    SciTech Connect

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

    1990-04-17

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

  17. 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.

  18. 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.

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

    NASA Astrophysics Data System (ADS)

    Olson, Mark A.; Scovill, John P.; Hack, Dallas 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.

  20. 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.

  1. Novel derivatives of nitro-substituted salicylic acids: Synthesis, antimicrobial activity and cytotoxicity.

    PubMed

    Paraskevopoulos, Georgios; Krátký, Martin; Mandíková, Jana; Trejtnar, František; Stolaříková, Jiřina; Pávek, Petr; Besra, Gurdyal; Vinšová, Jarmila

    2015-11-15

    Inspired by the high antituberculous activity of novel nitro-substituted derivatives and based on promising predicted ADMET properties we have synthesized a series of 33 salicylanilides containing nitro-group in their salicylic part and evaluated them for their in vitro antimycobacterial, antimicrobial and antifungal activities. The presence of nitro-group in position 4 of the salicylic acid was found to be beneficial and the resulting molecules exhibited minimum inhibitory concentrations (MICs) ranging from 2 to 32 μM against Mycobacterium tuberculosis. The best activity was found for 2-hydroxy-4-nitro-N-[4-(trifluoromethyl)phenyl]benzamide (MIC=2 μM). 4-Nitrosalicylanilides were also found to be active against all Staphylococcus species tested while for MRSA strain 2-hydroxy-4-nitro-N-[4-(trifluoromethyl)phenyl]benzamide's MIC was 0.98 μM. None of the nitrosalicylanilides was active against Enterococcus sp. J 14365/08 and no considerable activity was found against Gram-negative bacteria or fungi. The hepatotoxicity of all nitrosalicylanilides was found to be in the range of their MICs for HepG2 cells.

  2. 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

  3. 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

  4. Exotoxin A of Pseudomonas aeruginosa: substitution of glutamic acid 553 with aspartic acid drastically reduces toxicity and enzymatic activity.

    PubMed Central

    Douglas, C M; Collier, R J

    1987-01-01

    Glutamic acid 553 of Pseudomonas aeruginosa exotoxin A (ETA) has been identified by photoaffinity labeling as a residue within the NAD binding site (S.F. Carroll and R.J. Collier, J. Biol. Chem. 262:8707-8711, 1987). To explore the function of Glu-553 we used oligonucleotide-directed mutagenesis to replace this residue with Asp in cloned ETA and expressed the mutant gene in Escherichia coli K-12. ADP-ribosylation activity of Asp-553 ETA in cell extracts was about 1,800-fold lower and toxicity for mouse L-M929 fibroblasts was at least 10,000-fold lower than that of the wild-type toxin. Extracts containing Asp-553 ETA inhibited the cytotoxicity of authentic ETA on L-M929 fibroblasts, suggesting that the mutant toxin competes for ETA receptors. The results indicate that Glu-553 is crucial for ADP-ribosylation activity and, consequently, cytotoxicity of ETA. Substitution or deletion of this residue may be a route to new ETA vaccines. Images PMID:2889718

  5. Di-substituted cyclohexyl derivatives bind to two identical sites with positive cooperativity on the voltage-gated potassium channel, K(v)1.3.

    PubMed

    Schmalhofer, William A; Slaughter, Robert S; Matyskiela, Mary; Felix, John P; Tang, Yui S; Rupprecht, Kathleen; Kaczorowski, Gregory J; Garcia, Maria L

    2003-04-29

    Di-substituted cyclohexyl (DSC) derivatives inhibit the voltage-gated potassium channel, K(v)1.3, and have immunosuppressant activity (Schmalhofer et al. (2002) Biochemistry 41, 7781-7794). This class of inhibitors displays Hill coefficients of near 2 in functional assays, and trans DSC analogues appear to selectively interact with K(v)1.3 channel conformations related to C-type inactivation. To further understand the details of the DSC inhibitor interaction with potassium channels, trans-1-(N-n-propylcarbamoyloxy)-4-phenyl-4-(3-(2-methoxyphenyl)-3-oxo-2-azaprop-1-yl)cyclo-hexane (trans-NPCO-DSC) was radiolabeled with tritium, and its binding characteristics to K(v)1.3 channels were determined. Specific binding of [(3)H]-trans-NPCO-DSC to K(v)1.3 channels is a saturable, time-dependent, and fully reversible process. Saturation binding isotherms and competition binding experiments are consistent with the presence of two receptor sites for DSC derivatives on the K(v)1.3 channel that display positive allosteric cooperativity. The high affinity interaction of [(3)H]-trans-NPCO-DSC with K(v)1.3 channels appears to correlate with the rates of C-type inactivation of the channel. These data, taken together, mark the first demonstration of the existence of multiple binding sites for an inhibitor of an ion channel and suggest that the high affinity interaction of trans-NPCO-DSC and similar inhibitors with K(v)1.3 channels could be exploited for the development of selective molecules that target this protein.

  6. Control of Substrate Specificity by Active-Site Residues in Nitrobenzene Dioxygenase

    PubMed Central

    Ju, Kou-San; Parales, Rebecca E.

    2006-01-01

    Nitrobenzene 1,2-dioxygenase from Comamonas sp. strain JS765 catalyzes the initial reaction in nitrobenzene degradation, forming catechol and nitrite. The enzyme also oxidizes the aromatic rings of mono- and dinitrotoluenes at the nitro-substituted carbon, but the basis for this specificity is not understood. In this study, site-directed mutagenesis was used to modify the active site of nitrobenzene dioxygenase, and the contribution of specific residues in controlling substrate specificity and enzyme performance was evaluated. The activities of six mutant enzymes indicated that the residues at positions 258, 293, and 350 in the α subunit are important for determining regiospecificity with nitroarene substrates and enantiospecificity with naphthalene. The results provide an explanation for the characteristic specificity with nitroarene substrates. Based on the structure of nitrobenzene dioxygenase, substitution of valine for the asparagine at position 258 should eliminate a hydrogen bond between the substrate nitro group and the amino group of asparagine. Up to 99% of the mononitrotoluene oxidation products formed by the N258V mutant were nitrobenzyl alcohols rather than catechols, supporting the importance of this hydrogen bond in positioning substrates in the active site for ring oxidation. Similar results were obtained with an I350F mutant, where the formation of the hydrogen bond appeared to be prevented by steric interference. The specificity of enzymes with substitutions at position 293 varied depending on the residue present. Compared to the wild type, the F293Q mutant was 2.5 times faster at oxidizing 2,6-dinitrotoluene while retaining a similar Km for the substrate based on product formation rates and whole-cell kinetics. PMID:16517627

  7. 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.

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

    PubMed Central

    2015-01-01

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

  9. 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.

  10. Syntheses and antibacterial activity of soluble 9-bromo substituted indolizinoquinoline-5,12-dione derivatives.

    PubMed

    Yang, Hui; Wang, Hao-Wen; Zhu, Teng-Wei; Yu, Le-Mao; Chen, Jian-Wen; Wang, Lu-Xia; Shi, Lei; Li, Ding; Gu, Lian-Quan; Huang, Zhi-Shu; An, Lin-Kun

    2017-02-15

    In our previous research, 9-bromo indolizinoquinoline-5,12-dione 1 has been found to be a good anti-MRSA agent. However, it had very low bioavailability in vivo possibly due to its low solubility in water. In order to obtain the derivatives with higher anti-MRSA activity and good water solubility, twenty eight bromo-substituted indolizinoquinoline-5,12-dione derivatives were synthesized in the present study. The antibacterial activity of the synthesized compounds was evaluated against one gram-negative and some gram-positive bacterial strains including 100 clinical MRSA strains. The UV assays were carried out to determine the solubility of six active compounds 16, 21, 23 and 27-29. The most potent compound 28 exhibited strong activity against clinical MRSA strains with both MIC50 and MIC90 values lower than 7.8 ng/mL. Compound 27 had good water solubility of 1.98 mg/mL and strong activity against clinical MRSA strains with MIC50 value of 63 ng/mL and MIC90 value of 125 ng/mL, 16-fold higher than that of Vancomycin.

  11. 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)

  12. 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

  13. The effect of N-substituted alkyl groups on the anticonvulsant activities ofN-Cbz-alpha-amino-N-alkylsuccinimides.

    PubMed

    Lee, J; Son, K; Jung, K; Choi, J; Park, M

    1997-02-01

    For the purpose of defining the effects of theN-substituted alkyl groups on the anticonvulsant activities ofN-Cbz-alpha-aminosuccinimides, various (R)-and(S)-N-alkyl substitutedN-Cbz-alpha-aminosuccinimides (1 and2) were prepared from the corresponding (R)-and(S)-N-Cbz-aspartic acid by using known reaction and were evaluated the anticonvulsant activies in the MES and PTZ tests, including their neurotoxicities. The most active compound in the MES test was(R)-N-Cbz-alpha-amino-N-methylsuccinimide (1b) (ED(50)=52.5 mg/kg, Pl-3.2). And in case of the PTZ test,(R)-N-Cbz-alpha-amino-N-ethylsuccinimide (1c) was the most active compound (ED(50)=32.5 mg/kg, Pl=3.1). The order of anticonvulsant activities of these compounds against the MES test, as judged from the ED(50) values for theR series (1), wasN-methyl >N-isobutyl > non-substituted >N-ethyl,N-allyl >N-benzyl compound; for theS series (2)N-methyl >N-allyl > non-substituted >N-isobutyl >N-ethyl >N-benzyl compound. The anticonvulsant activities in the PTZ tests of these compounds exhibited somewhat different pattern; for theR series (1)N-ethyl >N-methyl >N-isobutyl> non-substituted >N-allyl >N-benzyl compound in order of decreasing activity; forS series (2)N-ethyl >N-allyl, non-substituted >N-isobutyl >N-methyl >N-benzyl compound in order of decreasing activity.

  14. 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.

  15. The protein conformation of Cd-substituted horse liver alcohol dehydrogenase and its metal-site coordination geometry in binary and ternary inhibitor complexes.

    PubMed

    Hemmingsen, L; Bauer, R; Bjerrum, M J; Adolph, H W; Zeppezauer, M; Cedergren-Zeppezauer, E

    1996-10-15

    The coordination geometry of the metal at the active site in Cd-substituted horse liver alcohol dehydrogenase (LADH) has been investigated for the binary complexes of LADH with imidazole, isobutyramide, decanoic acid and Cl-, and for the ternary complexes of LADH with NADH and imidazole, NADH and isobutyramide, NAD+ and decanoic acid and NAD+ and Cl-, by using the method of perturbed angular correlation of gamma-rays (PAC). The spectral results are consistent with a flexible structure around the metal for the binary complexes with inhibitors. For ternary complexes, however, a rigid structure is observed. An exception is the ternary complex between LADH, NADH and imidazole, in which the metal site is still flexible. Comparing with available structures determined by X-ray crystallography, we found a correlation between open structures and flexible metal sites, and between closed structures and rigid metal sites. This indicates that the PAC technique can be applied to distinguish the two conformations in solution. The spectral parameters, omega(o) and eta, of the experiments, except for the complexes with imidazole, fall into two groups: one with low omega(o) and one with high omega(o) (eta is relatively constant in all experiments). In this work it is clarified that the low omega(o) values are connected with the presence of a negatively charged solvent ligand. Using an angular-overlap approach to interpret the results, the low omega(o) values are found to be compatible with a coordination geometry where the S-Cd-S (Cys174 and Cys46 coordinate to the metal) angle is about 110 degrees as suggested in [Hemmingsen, L., Bauer, R., Danielsen, E., Bjerrum. M. J., Zeppezauer, M., Adolph, H. W., Formicka, G. & Cedergren-Zeppezauer, E. (1995) Biochemistry 34, 7145-7153], whereas high omega(o) values are compatible with an S-Cd-S angle of 130 degrees. The presence of a negatively charged metal ligand, therefore, might trigger the movement of the sulfur of Cys174. As it is

  16. Activity of Cu{sup 2+} ions on the tetrahedral and octahedral sites of spinel oxide catalysts for CO oxidation

    SciTech Connect

    Ghose, J.; Murthy, K.S.R.C.

    1996-09-01

    In studies of CO oxidation on substituted copper chromite spinel oxide catalyst decreases as the Cu{sup 2+} content of the catalyst decreases, either by substitution with a divalent ion, i.e., Cu{sub 1-x} Mg{sub x} [Cr{sub 2}]O{sub 4}, or by reduction of Cu{sup 2+} to Cu{sup 1+}. Crystallographic studies have shown that Cu[Cr{sub 2}]O{sub 4} changes from normal to partially inverse when Cr{sup 3+} is replaced by Al{sup 3+}. Thus, in aluminum-substituted copper chromite catalysts, copper is present on both tetrahedral and octahedral sites of the spinel lattice, i.e., Cu{sub 1-x}Al{sub x} [Cu{sub x}Cr{sub 2-(x+y)}Al{sub y}]O{sub 4}. ESCA studies have shown that upon Al substitution some of the tetrahedral Cu{sup 2+} ions are reduced to Cu{sup 1+} and this causes a reduction in the catalytic activity of the catalysts. The present work was taken up to compare the activity of Cu{sup 2+} on tetrahedral sites with that on octahedral sites of the spinel oxide catalysts. For this, CO oxidation studies were carried out on the inverse spinel CuFe{sub 2}O{sub 4} and on the normal spinel CuRh{sub 2}O{sub 4} catalysts. 7 refs., 1 fig.

  17. 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.

  18. 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.

  19. 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.

  20. Activation of Graphenic Carbon Due to Substitutional Doping by Nitrogen: Mechanistic Understanding from First Principles.

    PubMed

    Bhattacharjee, Joydeep

    2015-05-07

    Nitrogen-doped graphene and carbon nanotubes are popularly in focus as metal-free electrocatalysts for oxygen reduction reactions (ORR) central to fuel cells. N-doped CNTs have been also reported to chemisorb mutually, promising a route to their robust predetermined assembly into devices and mechanical reinforcements. We propose from first principles a common mechanistic understanding of these two aspects pointing further to a generic chemical activation of carbon atoms due to substitution by nitrogen in experimentally observed configurations. Wannier-function based orbital resolved study of mechanisms suggests increase in C-N bond-orders in attempt to retain π-conjugation among carbon atoms, causing mechanical stress and loss of charge neutrality of nitrogen and carbon atoms, which remedially facilitate chemical activation of N-coordinated C atoms, enhancing sharply with increasing coordination to N and proximity to zigzag edges. Activated C atoms facilitate covalent adsorption of radicals in general, diradicals like O2 relevant to ORR, and also other similarly activated C atoms, leading to self-assembly of graphenic nanostructures while remaining inert to ordinary graphenic C atoms.

  1. Controlled Orientation of Active Sites in a Nanostructured Multienzyme Complex

    PubMed Central

    Lim, Sung In; Yang, Byungseop; Jung, Younghan; Cha, Jaehyun; Cho, Jinhwan; Choi, Eun-Sil; Kim, Yong Hwan; Kwon, Inchan

    2016-01-01

    Multistep cascade reactions in nature maximize reaction efficiency by co-assembling related enzymes. Such organization facilitates the processing of intermediates by downstream enzymes. Previously, the studies on multienzyme nanocomplexes assembled on DNA scaffolds demonstrated that closer interenzyme distance enhances the overall reaction efficiency. However, it remains unknown how the active site orientation controlled at nanoscale can have an effect on multienzyme reaction. Here, we show that controlled alignment of active sites promotes the multienzyme reaction efficiency. By genetic incorporation of a non-natural amino acid and two compatible bioorthogonal chemistries, we conjugated mannitol dehydrogenase to formate dehydrogenase with the defined active site arrangement with the residue-level accuracy. The study revealed that the multienzyme complex with the active sites directed towards each other exhibits four-fold higher relative efficiency enhancement in the cascade reaction and produces 60% more D-mannitol than the other complex with active sites directed away from each other. PMID:28004799

  2. 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.

  3. In vitro activity of ciprofloxacin compared with those of other new fluorinated piperazinyl-substituted quinoline derivatives.

    PubMed Central

    Van Caekenberghe, D L; Pattyn, S R

    1984-01-01

    The in vitro antimicrobial activity of ciprofloxacin (Bay o 9867) was compared with those of the other new fluorated piperazinyl-substituted quinoline derivatives: norfloxacin, ofloxacin, and pefloxacin, as well as with those of the earlier analogs, nalidixic acid and oxolinic acid. Virtually no resistance against the new fluorated piperazinyl-substituted quinoline derivatives was observed. As a rule, ciprofloxacin was about four times more active in vitro than the other new fluorated piperazinyl-substituted quinoline derivatives. The antibacterial spectrum of the new fluorated piperazinyl-substituted quinoline derivatives included, among other susceptible species, Pseudomonas aeruginosa, Streptococcus faecalis, Staphylococcus spp., and members of the family Enterobacteriaceae. In the case of ciprofloxacin, ofloxacin, and perfloxacin, anaerobes were included in the antibacterial spectrum as well. There was no cross resistance between the earlier analogs and the new fluorated piperazinyl-substituted quinoline derivatives. Regression analysis of MIC pairs for individual strains illustrated almost mathematical correlation between the antimicrobial activities of the quinoline derivatives. PMID:6732221

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

    PubMed

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

    2012-06-01

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

  5. A phosphorylation site in the ftz homeodomain is required for activity.

    PubMed Central

    Dong, J; Hung, L H; Strome, R; Krause, H M

    1998-01-01

    The Drosophila homeodomain-containing protein Fushi tarazu (Ftz) is expressed sequentially in the embryo, first in alternate segments, then in specific neuroblasts and neurons in the central nervous system, and finally in parts of the gut. During these different developmental stages, the protein is heavily phosphorylated on different subsets of Ser and Thr residues. This stage-specific phosphorylation suggests possible roles for signal transduction pathways in directing tissue-specific Ftz activities. Here we show that one of the Ftz phosphorylation sites, T263 in the N-terminus of the Ftz homeodomain, is phosphorylated in vitro by Drosophila embryo extracts and protein kinase A. In the embryo, mutagenesis of this site to the non-phosphorylatable residue Ala resulted in loss of ftz-dependent segments. Conversely, substitution of T263 with Asp, which is also non-phosphorylatable, but which successfully mimics phosphorylated residues in a number of proteins, rescued the mutant phenotype. This suggests that T263 is in the phosphorylated state when functioning normally in vivo. We also demonstrate that the T263 substitutions of Ala and Asp do not affect Ftz DNA-binding activity in vitro, nor do they affect stability or transcriptional activity in transfected S2 cells. This suggests that T263 phosphorylation is most likely required for a homeodomain-mediated interaction with an embryonically expressed protein. PMID:9545243

  6. 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.

  7. 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

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

    PubMed Central

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

    2015-01-01

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

  9. Synthesis and anti-cancer activities of new sulfonamides 4-substituted-triazolyl nucleosides.

    PubMed

    Alaoui, Soukaina; Dufies, Maeva; Driowya, Mohsine; Demange, Luc; Bougrin, Khalid; Robert, Guillaume; Auberger, Patrick; Pagès, Gilles; Benhida, Rachid

    2017-03-09

    Nucleoside analogues are among the most known drugs commonly used in antiviral and anticancer chemotherapies. Among them, those featuring a five-membered ring nucleobase are of utmost interest such as the anti-cancer agent AICAR or the anti-viral drug ribavirin. Despite its low activity in vitro in different cell lines, AICAR is under clinical development for several pathologies, thanks to its original mode of action. Indeed, AICAR induced autophagy cell death and is able, following this mechanism, to circumvent resistance to apoptotic drugs including kinase inhibitors currently on the market. To improve the activity of AICAR, we report herein an efficient synthesis of new series of sulfonamide-4-substituted-1,2,3-triazolyl nucleosides using a Cu-catalyzed 1,3-dipolar cycloaddition. All these molecules have been fully characterized and evaluated against two aggressive tumor cell lines, RCC4 and MDA-MB-231. Among them, nucleoside analogue 5i belonging to the ribose series was found to be 19 to 66-fold more active than AICAR. Western blot analyses on RCC4 cells showed that 5i displayed an interesting mode of action by inducing both apoptosis and autophagy cell death, making therefore this class of molecules highly promising for further hit-to-lead optimization.

  10. The influence of metakaolin substitution by slag in alkali-activated inorganic binders for civil engineering

    NASA Astrophysics Data System (ADS)

    Kadlec, J.; Rieger, D.; Kovářík, T.; Novotný, P.; Franče, P.; Pola, M.

    2017-02-01

    In this study the effect of metakaolin replacement by milled blast furnace slag in alkali-activated geopolymeric binder was investigated in accordance to their rheological and mechanical properties. It was demonstrated that slag addition into the metakaolin binder can improve mechanical properties of final products. Our investigation was focused on broad interval of metakaolin substitution in the range from 100 to 40 volume per cents of metakaolin so that the volume content of solids in final binder was maintained constant. Prepared binders were activated by alkaline solution of potassium silicate with silicate module of 1.61. The particle size analyses were performed for determination of particle size distribution. The rheological properties were determined in accordance to flow properties by measurements on Ford viscosity cup and by oscillatory measurements of hardening process. For the investigation of hardening process, the strain controlled small amplitude oscillatory rheometry was used in plane-plate geometry. For determination of applied mechanical properties were binders filled by ceramic grog in the granularity range 0-1 mm. The filling was maintained constant at 275 volume per cents in accordance to ratio of solids in dry binder. The mechanical properties were investigated after 1, 7 and 28 days and microstructure was documented by scanning electron microscopy. The results indicate that slag addition have beneficial effect not only on mechanical properties of hardened binder but also on flow properties of fresh geopolymer paste and subsequent hardening kinetics of alkali-activated binders.

  11. Synthesis and evaluation of 7-substituted 4-aminoquinoline analogs for antimalarial activity

    PubMed Central

    Hwang, Jong Yeon; Kawasuji, Takashi; Lowes, David J.; Clark, Julie A.; Connelly, Michele C.; Zhu, Fangyi; Guiguemde, W. Armand; Sigal, Martina S.; Wilson, Emily B.; DeRisi, Joseph L.; Guy, R. Kiplin

    2013-01-01

    We previously reported that substituted 4-aminoquinolines with a phenylether substituent at the 7-position of the quinoline ring and the capability of intramolecular hydrogen bonding between the protonated amine on the side chain and a hydrogen bond acceptor on the amine’s alkyl substituents exhibited potent antimalarial activity against the multi-drug resistant strain P. falciparum W2. We employed a parallel synthetic method to generate diaryl ether, biaryl, and alkylaryl 4-aminoquinoline analogs, in the background of a limited number of side chain variations that had previously afforded potent 4-aminoquinolines. All subsets were evaluated for their antimalarial activity against the chloroquine-sensitive strain 3D7 and the chloroquine-resistant K1 and cytotoxicity mammalian cell lines. While all three arrays showed good antimalarial activity, only the biaryl-containing subset showed consistently good potency against the drug-resistant K1strain good selectivity with regard to mammalian cytotoxicity. Overall, our data indicate that the biaryl-containing series contains promising candidates for further study. PMID:21910466

  12. 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.

  13. Design, synthesis and structure-activity relationships of substituted oxazole-benzamide antibacterial inhibitors of FtsZ.

    PubMed

    Stokes, Neil R; Baker, Nicola; Bennett, James M; Chauhan, Pramod K; Collins, Ian; Davies, David T; Gavade, Maruti; Kumar, Dushyant; Lancett, Paul; Macdonald, Rebecca; Macleod, Leanne; Mahajan, Anu; Mitchell, Jeffrey P; Nayal, Narendra; Nayal, Yashodanand Nandan; Pitt, Gary R W; Singh, Mahipal; Yadav, Anju; Srivastava, Anil; Czaplewski, Lloyd G; Haydon, David J

    2014-01-01

    The design, synthesis and structure-activity relationships of a series of oxazole-benzamide inhibitors of the essential bacterial cell division protein FtsZ are described. Compounds had potent anti-staphylococcal activity and inhibited the cytokinesis of the clinically-significant bacterial pathogen Staphylococcus aureus. Selected analogues possessing a 5-halo oxazole also inhibited a strain of S. aureus harbouring the glycine-to-alanine amino acid substitution at residue 196 of FtsZ which conferred resistance to previously reported inhibitors in the series. Substitutions to the pseudo-benzylic carbon of the scaffold improved the pharmacokinetic properties by increasing metabolic stability and provided a mechanism for creating pro-drugs. Combining multiple substitutions based on the findings reported in this study has provided small-molecule inhibitors of FtsZ with enhanced in vitro and in vivo antibacterial efficacy.

  14. Site-directed mutagenesis of the toxin from the Chinese scorpion Buthus martensii Karsch (BmKAS): insight into sites related to analgesic activity.

    PubMed

    Cui, Yong; Song, Yong-Bo; Ma, Lin; Liu, Yan-Feng; Li, Guo-Dong; Wu, Chun-Fu; Zhang, Jing-Hai

    2010-10-01

    This study utilized the E. coli expression system to investigate the role of amino acid residues in toxin from the Chinese scorpion--Buthus martensii Karsch (BmKAS). To evaluate the extent to which residues of the toxin core contribute to its analgesic activity, ten mutants of BmKAS were obtained by PCR. Using site-directed mutagenesis, all of these residues were substituted with different amino acids. This study represents a thorough mapping and elucidation of the epitopes that form the molecular basis of the toxin's analgesic activity. Our results showed large mutant-dependent differences that emphasize the important roles of the studied residues.

  15. Structure-activity relationships of 2alpha-substituted androstenedione analogs as aromatase inhibitors and their aromatization reactions.

    PubMed

    Numazawa, Mitsuteru; Handa, Wakako; Hasegawa, Chie; Takahashi, Madoka

    2005-12-01

    Aromatase catalyzes the conversion of androstenedione (1a, AD) to estrone through three sequential oxygenations of the 19-methyl group. To gain insight into the spatial nature of the AD binding (active) site of aromatase in relation to the catalytic function of the enzyme, we tested for the ability of 2alpha-substituted (halogeno, alkyl, hydroxy, and alkoxy) ADs (1b-1i) to inhibit aromatase in human placental microsomes as well as their ability to serve as a substrate for the enzyme. All of the steroids inhibited the enzyme in a competitive manner with the apparent K(i)'s ranging from 45 to 1150 nM. 2alpha-Halogeno (F, Cl, and Br) and 2alpha-alkyl (CH3 and CH2CH3) steroids 1b-1f were powerful to good inhibitors (Ki=45-171 nM) whereas steroids 1g-1i, having an oxygen function (hydroxy or alkoxy) at C-2alpha, were poor inhibitors (Ki=670-1150 nM). Aromatization of some of the steroids with placental microsomes was analyzed by gas chromatography-mass spectrometry, indicating that the aromatization rate of the bromide 1d was about two-fold that of the natural substrate AD and that of 2alpha-methoxide 1h was similar to that of AD. Kinetic analysis of the aromatization of androgens revealed that a good substrate was not essentially a good inhibitor for aromatase.

  16. Perspective: On the active site model in computational catalyst screening

    NASA Astrophysics Data System (ADS)

    Reuter, Karsten; Plaisance, Craig P.; Oberhofer, Harald; Andersen, Mie

    2017-01-01

    First-principles screening approaches exploiting energy trends in surface adsorption represent an unparalleled success story in recent computational catalysis research. Here we argue that our still limited understanding of the structure of active sites is one of the major bottlenecks towards an ever extended and reliable use of such computational screening for catalyst discovery. For low-index transition metal surfaces, the prevalently chosen high-symmetry (terrace and step) sites offered by the nominal bulk-truncated crystal lattice might be justified. For more complex surfaces and composite catalyst materials, computational screening studies will need to actively embrace a considerable uncertainty with respect to what truly are the active sites. By systematically exploring the space of possible active site motifs, such studies might eventually contribute towards a targeted design of optimized sites in future catalysts.

  17. Diffusional correlations among multiple active sites in a single enzyme.

    PubMed

    Echeverria, Carlos; Kapral, Raymond

    2014-04-07

    Simulations of the enzymatic dynamics of a model enzyme containing multiple substrate binding sites indicate the existence of diffusional correlations in the chemical reactivity of the active sites. A coarse-grain, particle-based, mesoscopic description of the system, comprising the enzyme, the substrate, the product and solvent, is constructed to study these effects. The reactive and non-reactive dynamics is followed using a hybrid scheme that combines molecular dynamics for the enzyme, substrate and product molecules with multiparticle collision dynamics for the solvent. It is found that the reactivity of an individual active site in the multiple-active-site enzyme is reduced substantially, and this effect is analyzed and attributed to diffusive competition for the substrate among the different active sites in the enzyme.

  18. Crystal structure of native and Cd/Cd-substituted Dioclea guianensis seed lectin. A novel manganese-binding site and structural basis of dimer-tetramer association.

    PubMed

    Wah, D A; Romero, A; Gallego del Sol, F; Cavada, B S; Ramos, M V; Grangeiro, T B; Sampaio, A H; Calvete, J J

    2001-07-20

    Diocleinae legume lectins are a group of oligomeric proteins whose subunits display a high degree of primary structure and tertiary fold conservation but exhibit considerable diversity in their oligomerisation modes. To elucidate the structural determinants underlaying Diocleinae lectin oligomerisation, we have determined the crystal structures of native and cadmium-substituted Dioclea guianensis (Dguia) seed lectin. These structures have been solved by molecular replacement using concanavalin (ConA) coordinates as the starting model, and refined against data to 2.0 A resolution. In the native (Mn/Ca-Dguia) crystal form (P4(3)2(1)2), the asymmetric unit contains two monomers arranged into a canonical legume lectin dimer, and the tetramer is formed with a symmetry-related dimer. In the Cd/Cd-substituted form (I4(1)22), the asymmetric unit is occupied by a monomer. In both crystal forms, the tetrameric association is achieved by the corresponding symmetry operators. Like other legume lectins, native D. guianensis lectin contains manganese and calcium ions bound in the vicinity of the saccharide-combining site. The architecture of these metal-binding sites (S1 and S2) changed only slightly in the cadmium/cadmium-substituted form. A highly ordered calcium (native lectin) or cadmium (Cd/Cd-substituted lectin) ion is coordinated at the interface between dimers that are not tetrameric partners in a similar manner as the previously identified Cd(2+) in site S3 of a Cd/Ca-ConA. An additional Mn(2+) coordination site (called S5), whose presence has not been reported in crystal structures of any other homologous lectin, is present in both, the Mn/Ca and the Cd/Cd-substituted D. guianensis lectin forms. On the other hand, comparison of the primary and quaternary crystal structures of seed lectins from D. guianensis and Dioclea grandiflora (1DGL) indicates that the loop comprising residues 117-123 is ordered to make interdimer contacts in the D. grandiflora lectin structure

  19. Robotics at Savannah River site: activity report

    SciTech Connect

    Byrd, J.S.

    1984-09-01

    The objectives of the Robotics Technology Group at the Savannah River Laboratory are to employ modern industrial robots and to develop unique automation and robotic systems to enhance process operations at the Savannah River site (SRP and SRL). The incentives are to improve safety, reduce personnel radiation exposure, improve product quality and productivity, and to reduce operating costs. During the past year robotic systems have been installed to fill chemical dilution vials in a SRP laboratory at 772-F and remove radioactive waste materials in the SRL Californium Production Facility at 773-A. A robotic system to lubricate an extrusion press has been developed and demonstrated in the SRL robotics laboratory and is scheduled for installation at the 321-M fuel fabrication area. A mobile robot was employed by SRP for a radiation monitoring task at a waste tank top in H-Area. Several other robots are installed in the SRL robotics laboratories and application development programs are underway. The status of these applications is presented in this report.

  20. A single amino acid substitution confers enhanced methylation activity of mammalian Dnmt3b on chromatin DNA.

    PubMed

    Shen, Li; Gao, Ge; Zhang, Ying; Zhang, He; Ye, Zhiqiang; Huang, Shichao; Huang, Jinyan; Kang, Jiuhong

    2010-10-01

    Dnmt3a and Dnmt3b are paralogous enzymes responsible for de novo DNA methylation but with distinguished biological functions. In mice, disruption of Dnmt3b but not Dnmt3a causes global DNA hypomethylation, especially in repetitive sequences, which comprise the large majority of methylated DNA in the genome. By measuring DNA methylation activity of Dnmt3a and Dnmt3b homologues from five species, we found that mammalian Dnmt3b possessed significantly higher methylation activity on chromatin DNA than Dnmt3a and non-mammalian Dnmt3b. Sequence comparison and mutagenesis experiments identified a single amino acid substitution (I662N) in mammalian Dnmt3b as being crucial for its high chromatin DNA methylation activity. Further mechanistic studies demonstrated this substitution markedly enhanced the binding of Dnmt3b to nucleosomes and hence increased the chromatin DNA methylation activity. Moreover, this substitution was crucial for Dnmt3b to efficiently methylate repetitive sequences, which increased dramatically in mammalian genomes. Consistent with our observation that Dnmt3b evolved more rapidly than Dnmt3a during the emergence of mammals, these results demonstrated that the I662N substitution in mammalian Dnmt3b conferred enhanced chromatin DNA methylation activity and contributed to functional adaptation in the epigenetic system.

  1. Novel 5-substituted benzyloxy-2-arylbenzofuran-3-carboxylic acids as calcium activated chloride channel inhibitors

    PubMed Central

    Kumar, Satish; Namkung, Wan; Verkman, A. S.; Sharma, Pawan K.

    2013-01-01

    Transmembrane protein 16A (TMEM16A) channels are recently discovered membrane proteins that functions as a calcium activated chloride channel (CaCC). CaCCs are major regulators of various physiological processes, such as sensory transduction, epithelial secretion, smooth muscle contraction and oocyte fertilization. Thirty novel 5-substituted benzyloxy-2-arylbenzofuran-3-carboxylic acids (B01–B30) were synthesized and evaluated for their TMEM16A inhibitory activity by using short circuit current measurements in Fischer rat thyroid (FRT) cells expressing human TMEM16A. IC50 values were calculated using YFP fluorescence plate reader assay. Final compounds, having free carboxylic group displayed significant inhibition. Eight of the novel compounds B02, B13, B21, B23, B25, B27, B28, B29 exhibit excellent CaCCs inhibition with IC50 value <6 μM, with compound B25 exhibiting the lowest IC50 value of 2.8 ± 1.3 μM. None of the tested ester analogs of final benzofuran derivatives displayed TMEM16A/CaCCs inhibition. PMID:22739085

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

    PubMed

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

    2016-08-08

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

  3. 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.

  4. 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

  5. Enaminones as building blocks for the synthesis of substituted pyrazoles with antitumor and antimicrobial activities.

    PubMed

    Riyadh, Sayed M

    2011-02-22

    Novel N-arylpyrazole-containing enaminones 2a,b were synthesized as key intermediates. Reactions of 2a,b with active methylene compounds in acetic acid in the presence of ammonium acetate afforded substituted pyridine derivatives 5a-d. Enaminones 2a,b also reacted with aliphatic amines such as hydrazine hydrate and hydroxylamine hydrochloride to give bipyrazoles 8a,b and pyrazolylisoxazoles 9a,b, respectively. On the other hand, treatment of 2a,b with a heterocyclic amine and its diazonium salt yielded the respective [1,2,4]triazolo[4,3-a]pyrimidines 12a,b and pyrazolylcarbonyl[1,2,4]triazolo-[3,4-c][1,2,4]triazines 14a,b. Moreover, 2-thioxo-2,3-dihydro-1H-pyrido[2,3-d]pyrimidin-4-one (17) was prepared via reaction of enaminone 2a with aminothiouracil (15). Cyclocondensation of 17 with the appropriate hydrazonoyl chlorides 18a-c gave the corresponding pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-5-ones 21a-c. The cytotoxic effects of compounds 2b, 14a and 17 against human breast cell line (MCF-7) and liver carcinoma cell line (HEPG2) were screened and in both lines they showed inhibition effects comparable to those of 5-fluorouracil, used as a standard. The antimicrobial activity of some products chosen as representative examples was also evaluated.

  6. 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.

  7. Site Directed Mutagenesis of Schizosaccharomyces pombe Glutathione Synthetase Produces an Enzyme with Homoglutathione Synthetase Activity

    PubMed Central

    Dworeck, Tamara; Zimmermann, Martin

    2012-01-01

    Three different His-tagged, mutant forms of the fission yeast glutathione synthetase (GSH2) were derived by site-directed mutagenesis. The mutant and wild-type enzymes were expressed in E. coli DH5α and affinity purified in a two-step procedure. Analysis of enzyme activity showed that it was possible to shift the substrate specificity of GSH2 from Gly (km 0,19; wild-type) to β-Ala or Ser. One mutation (substitution of Ile471, Cy472 to Met and Val and Ala 485 and Thr486 to Leu and Pro) increased the affinity of GSH2 for β-Ala (km 0,07) and lowered the affinity for Gly (km 0,83), which is a characteristic of the enzyme homoglutathione synthetase found in plants. Substitution of Ala485 and Thr486 to Leu and Pro only, increased instead the affinity of GSH2 for Ser (km 0,23) as a substrate, while affinity to Gly was preserved (km 0,12). This provides a new biosynthetic pathway for hydroxymethyl glutathione, which is known to be synthesized from glutathione and Ser in a reaction catalysed by carboxypeptidase Y. The reported findings provide further insight into how specific amino acids positioned in the GSH2 active site facilitate the recognition of different amino acid substrates, furthermore they support the evolutionary theory that homoglutathione synthetase evolved from glutathione synthetase by a single gene duplication event. PMID:23091597

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

    PubMed

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

    1998-03-01

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

  9. Effect of Ni-site substitutions in superconducting La3Ni2B2N3-δ

    NASA Astrophysics Data System (ADS)

    Michor, H.; Hilscher, G.; Krendelsberger, R.; Rogl, P.; Bourée, F.

    1998-12-01

    We investigated the effect of Ni/Co and Ni/Cu substitutions upon the superconducting and normal state properties of La3(Ni1-xMx)2B2N3-δ (M=Co, Cu). X-ray and neutron diffraction studies reveal a limited solid solubility for Cu: (0substitution only the initial depression rate, dTc/dx~=-0.23 K/(mol % Cu), could be determined. The results clearly show that the suppression of superconductivity is primarily caused by the reduction of N(Ef) concomitant with a fall of the coupling strength λ, while there are no hints for other effects like magnetic pair breaking. The comparison with the results reported for Y(Ni1-xCox)2B2C shows that the electronic changes induced by Ni/Co substitution are very similar in both systems.

  10. Comparison of catalytic activity of bismuth substituted cobalt ferrite nanoparticles synthesized by combustion and co-precipitation method

    NASA Astrophysics Data System (ADS)

    Kiran, Venkat Savunthari; Sumathi, Shanmugam

    2017-01-01

    In this study, cobalt ferrite and bismuth substituted cobalt ferrite (CoFe2-xBixO4x=0, 0.1) nanoparticles were synthesized by two different methods viz combustion and co-precipitation. The nanoparticles were characterized by powder X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), diffuse reflectance spectroscopy (DRS), scanning electron microscopy-energy dispersive X-ray analyzer (SEM-EDX) and vibrating sample magnetometer (VSM). The results of powder XRD pattern showed an increase in lattice parameter and decrease in particle size of cobalt ferrite by the substitution of bismuth. Catalytic activity of cobalt ferrite and bismuth substituted cobalt ferrite nanoparticles synthesized by two different methods were compared for the reduction of 4-nitrophenol to 4-aminophenol using NaBH4 as a reducing agent.

  11. Community Update on Site Activities, July 19, 2013

    EPA Pesticide Factsheets

    In an effort to engage and inform community members interested in the New Bedford Harbor Superfund Site cleanup, EPA will be issuing periodic topic-based fact sheets that will provide background information and updates about ongoing activities.

  12. Mutational analysis of the lac regulatory region: second-site changes that activate mutant promoters.

    PubMed Central

    Rothmel, R K; LeClerc, J E

    1989-01-01

    Second-site mutations that restored activity to severe lacP1 down-promoter mutants were isolated. This was accomplished by using a bacteriophage f1 vector containing a fusion of the mutant E. coli lac promoters with the structural gene for chloramphenicol acetyltransferase (CAT), so that a system was provided for selecting phage revertants (or pseudorevertants) that conferred resistance of phage-infected cells to chloramphenicol. Among the second-site changes that relieved defects in mutant lac promoters, the only one that restored lacP1 activity was a T----G substitution at position -14, a weakly conserved site in E. coli promoters. Three other sequence changes, G----A at -2, A----T at +1, and C----A at +10, activated nascent promoters in the lac regulatory region. The nascent promoters conformed to the consensus rule, that activity is gained by sequence changes toward homology with consensus sequences at the -35 and -10 regions of the promoter. However, the relative activities of some promoters cannot be explained solely by consideration of their conserved sequence elements. Images PMID:2660105

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

    SciTech Connect

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

    2006-03-01

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

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. Substituted 2-Aminopyrimidines Selective for α7-Nicotinic Acetylcholine Receptor Activation and Association with Acetylcholine Binding Proteins.

    PubMed

    Kaczanowska, Katarzyna; Camacho Hernandez, Gisela Andrea; Bendiks, Larissa; Kohs, Larissa; Cornejo-Bravo, Jose Manuel; Harel, Michal; Finn, M G; Taylor, Palmer

    2017-03-15

    Through studies with ligand binding to the acetylcholine binding protein (AChBP), we previously identified a series of 4,6-substituted 2-aminopyrimidines that associate with this soluble surrogate of the nicotinic acetylcholine receptor (nAChR) in a cooperative fashion, not seen for classical nicotinic agonists and antagonists. To examine receptor interactions of this structural family on ligand-gated ion channels, we employed HEK cells transfected with cDNAs encoding three requisite receptor subtypes: α7-nAChR, α4β2-nAChR, and a serotonin receptor (5-HT3AR), along with a fluorescent reporter. Initial screening of a series of over 50 newly characterized 2-aminopyrimidines with affinity for AChBP showed only two to be agonists on the α7-nAChR below 10 μM concentration. Their unique structural features were incorporated into design of a second subset of 2-aminopyrimidines yielding several congeners that elicited α7 activation with EC50 values of 70 nM and Kd values for AChBP in a similar range. Several compounds within this series exhibit specificity for the α7-nAChR, showing no activation or antagonism of α4β2-nAChR or 5-HT3AR at concentrations up to 10 μM, while others were weaker antagonists (or partial agonists) on these receptors. Analysis following cocrystallization of four ligand complexes with AChBP show binding at the subunit interface, but with an orientation or binding pose that differs from classical nicotinic agonists and antagonists and from the previously analyzed set of 2-aminopyrimidines that displayed distinct cooperative interactions with AChBP. Orientations of aromatic side chains of these complexes are distinctive, suggesting new modes of binding at the agonist-antagonist site and perhaps an allosteric action for heteromeric nAChRs.

  19. 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.

  20. 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.

  1. An Aberrant Splice Acceptor Site Due to a Novel Intronic Nucleotide Substitution in MSX1 Gene Is the Cause of Congenital Tooth Agenesis in a Japanese Family

    PubMed Central

    Tatematsu, Tadashi; Kimura, Masashi; Nakashima, Mitsuko; Machida, Junichiro; Yamaguchi, Seishi; Shibata, Akio; Goto, Hiroki; Nakayama, Atsuo; Higashi, Yujiro; Miyachi, Hitoshi; Shimozato, Kazuo; Matsumoto, Naomichi; Tokita, Yoshihito

    2015-01-01

    Congenital tooth agenesis is caused by mutations in the MSX1, PAX9, WNT10A, or AXIN2 genes. Here, we report a Japanese family with nonsyndromic tooth agenesis caused by a novel nucleotide substitution in the intronic region between exons 1 and 2 of the MSX1 gene. Because the mutation is located 9 bp before exon 2 (c.452-9G>A), we speculated that the nucleotide substitution would generate an abnormal splice site. Using cDNA analysis of an immortalized patient blood cell, we confirmed that an additional 7-nucleotide sequence was inserted at the splice junction between exons 1 and 2 (c.451_452insCCCTCAG). The consequent frameshift generated a homeodomain-truncated MSX1 (p.R151fsX20). We then studied the subcellular localization of truncated MSX1 protein in COS cells, and observed that it had a whole cell distribution more than a nuclear localization, compared to that of wild-type protein. This result suggests a deletion of the nuclear localization signal, which is mapped to the MSX1 homeodomain. These results indicate that this novel intronic nucleotide substitution is the cause of tooth agenesis in this family. To date, most MSX1 variants isolated from patients with tooth agenesis involve single amino acid substitutions in the highly conserved homeodomain or deletion mutants caused by frameshift or nonsense mutations. We here report a rare case of an intronic mutation of the MSX1 gene responsible for human tooth agenesis. In addition, the missing tooth patterns were slightly but significantly different between an affected monozygotic twin pair of this family, showing that epigenetic or environmental factors also affect the phenotypic variations of missing teeth among patients with nonsyndromic tooth agenesis caused by an MSX1 haploinsufficiency. PMID:26030286

  2. Structural role of the active-site metal in the conformation of Trypanosoma brucei phosphoglycerate mutase.

    PubMed

    Mercaldi, Gustavo F; Pereira, Humberto M; Cordeiro, Artur T; Michels, Paul A M; Thiemann, Otavio H

    2012-06-01

    Phosphoglycerate mutases (PGAMs) participate in both the glycolytic and the gluconeogenic pathways in reversible isomerization of 3-phosphoglycerate and 2-phosphoglycerate. PGAMs are members of two distinct protein families: enzymes that are dependent on or independent of the 2,3-bisphosphoglycerate cofactor. We determined the X-ray structure of the monomeric Trypanosoma brucei independent PGAM (TbiPGAM) in its apoenzyme form, and confirmed this observation by small angle X-ray scattering data. Comparing the TbiPGAM structure with the Leishmania mexicana independent PGAM structure, previously reported with a phosphoglycerate molecule bound to the active site, revealed the domain movement resulting from active site occupation. The structure reported here shows the interaction between Asp319 and the metal bound to the active site, and its contribution to the domain movement. Substitution of the metal-binding residue Asp319 by Ala resulted in complete loss of independent PGAM activity, and showed for the first time its involvement in the enzyme's function. As TbiPGAM is an attractive molecular target for drug development, the apoenzyme conformation described here provides opportunities for its use in structure-based drug design approaches. Database Structural data for the Trypanosoma brucei 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGAM) has been deposited with the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank under code 3NVL.

  3. Unusual transformation of substituted-3-formylchromones to pyrimidine analogues: synthesis and antimicrobial activities of 5-(o-hydroxyaroyl)pyrimidines.

    PubMed

    Raj, Tilak; Singh, Narinder; Ishar, M P S

    2013-11-15

    Substituted-3-formylchromones (4a-e) on reaction with 1,3-bis-dimethylaminomethylene-thiourea (5) in refluxing toluene solution give novel substituted 5-(o-hydroxyaroyl)pyrimidines (6a-e) in high yields. A mechanistic rationalization of the formation of products (6a-e) is proffered. Antimicrobial activities of all the synthesized compounds (6a-e) were evaluated against various fungal and bacterial strains. Compound 6d display significant antifungal activity (MIC 15) against Geotrichum candidum in comparison fluconazole used as positive control. Some of the compounds also display good antibacterial activity. Cytotoxic profile of compound 6d against HeLa cells indicates that at concentration (20 μM) no significant cell death (~2%) was observed.

  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. Identification of putative active site residues of ACAT enzymes.

    PubMed

    Das, Akash; Davis, Matthew A; Rudel, Lawrence L

    2008-08-01

    In this report, we sought to determine the putative active site residues of ACAT enzymes. For experimental purposes, a particular region of the C-terminal end of the ACAT protein was selected as the putative active site domain due to its high degree of sequence conservation from yeast to humans. Because ACAT enzymes have an intrinsic thioesterase activity, we hypothesized that by analogy with the thioesterase domain of fatty acid synthase, the active site of ACAT enzymes may comprise a catalytic triad of ser-his-asp (S-H-D) amino acid residues. Mutagenesis studies revealed that in ACAT1, S456, H460, and D400 were essential for activity. In ACAT2, H438 was required for enzymatic activity. However, mutation of D378 destabilized the enzyme. Surprisingly, we were unable to identify any S mutations of ACAT2 that abolished catalytic activity. Moreover, ACAT2 was insensitive to serine-modifying reagents, whereas ACAT1 was not. Further studies indicated that tyrosine residues may be important for ACAT activity. Mutational analysis showed that the tyrosine residue of the highly conserved FYXDWWN motif was important for ACAT activity. Furthermore, Y518 was necessary for ACAT1 activity, whereas the analogous residue in ACAT2, Y496, was not. The available data suggest that the amino acid requirement for ACAT activity may be different for the two ACAT isozymes.

  6. 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

  7. 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.

  8. 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.

  9. Activity-composition relationship in Tschermak's substituted Fe biotites at 700°C, 2 kbar

    NASA Astrophysics Data System (ADS)

    Benisek, Artur; Dachs, E.; Redhammer, Günther; Tippelt, Gerold; Amthauer, Georg

    1996-09-01

    The reaction-displacement technique was applied to the end-member reaction annite = sanidine + magnetite + H2 in order to determine the activity of the annite component ( a Ann) in iron biotites with variable degrees of the Tschermak's substitution ([6]Fe + [4]Si = [6]Al + [4]Al). Based on the simplified relation a Ann = f H 2/ foH2 ( foH2 = hydrogen fugacity of the end-member reaction at P, T), two types of experiments were performed at 700°C / 2 kbar: Type I used Fe-Al biotites of known starting composition together with sanidine + magnetite + H2O. This assemblage was exposed to various f H 2 conditions ( f H 2 < foH2) produced in the pressure vessel either by using different ratios of water/oil as pressure medium ( f H 2 in this case was measured by the hydrogen sensor technique), or by the Ni'NiO buffer. The composition of the Fe-Al biotites changed through incorporation or release of the annite component in response to the externally imposed f H 2. By using opposite biotite starting compositions, the equilibrium composition as a function of f H2 was bracketed. For type II, f H 2 in equilibrium with a specific combination of fine-grained Fe-Al biotite (+ sanidine + magnetite + H2O) was measured internally by application of the hydrogen sensor technique. Both type I and type II experiments yield consistent results demonstrating that a fine-grained assemblage of Fe-Al biotite (+ sanidine + magnetite + H2O) is able to act as a sliding-scale buffer. The final chemical composition of the Fe-Al biotite after the experiments was determined by electron microprobe and Mössbauer spectroscopy. The [4]Al and [6]Al in the biotites are coupled according to the Tschermak's substitution. In the tetrahedral sheet 0.1 Al-atoms per formula unit are present in excess to the amount required to balance [6]Al, and all Fe-Al biotites contain 8 10% Fe3+. Therefore, they are not members of the pure annite - siderophyllite join, but have an almost constant amount (15 Mol%) of two

  10. Exploring the role of putative active site amino acids and pro-region motif of recombinant falcipain-2: a principal hemoglobinase of Plasmodium falciparum.

    PubMed

    Kumar, Amit; Dasaradhi, P V N; Chauhan, Virander S; Malhotra, Pawan

    2004-04-23

    Falcipain-2 is one of the principal hemoglobinases of Plasmodium falciparum, a human malaria parasite. It has a typical papain family cysteine protease structural organization, a large pro-domain, a mature domain with conserved active site amino acids. Pro-domain of falcipain-2 also contains two important conserved motifs, "GNFD" and "ERFNIN." The "GNFD" motif has been shown to be responsible for correct folding and stability in case of many papain family proteases. In the present study, we carried out site-directed mutagenesis to assess the roles of active site residues and pro-domain residues for the activity of falcipain-2. Our results showed that substitutions of putative active site residues; Q36, C42, H174, and N204 resulted in complete loss of falcipain-2 activity, while W206 and D155 mutants retained partial/complete activity in comparison to the wild type falcipain-2. Homology modeling data also corroborate the results of mutagenesis; Q36, C42, H174, N204, and W206 residues form the active site loop of the enzyme and D155 lie outside the active pocket. Substitutions in the pro-region did not affect the activity of falcipain-2. This implies that falcipain-2 shares active site residues with other members of papain family, however pro-region of falcipain-2 does not play any role in the activity of enzyme.

  11. 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.

  12. Photodynamic activity of substituted zinc trisulfophthalocyanines: role of plasma membrane damage.

    PubMed

    Cauchon, Nicole; Nader, Moni; Bkaily, Ghassan; van Lier, Johan E; Hunting, Darel

    2006-01-01

    We recently reported that variations in cellular phototoxicity among a series of alkynyl-substituted zinc trisulfophthalocyanines (ZnPcS3Cn) correlates with their hydrophobicity, with the most amphiphilic derivatives showing the highest cell uptake and phototoxicity. In this study we address the role of the plasma membrane in the photodynamic response as it relates to the overall hydrophobicity of the photosensitizer. The membrane tracker dye 1-[4(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), which is incorporated into plasma membranes by endocytosis, was used to establish plasma membrane uptake by EMT-6 cells of the ZnPcS3C, by colocalization, and TMA-DPH membrane uptake rates after photodynamic therapy were used to quantify membrane damage. TMA-DPH colocalization patterns show plasma membrane uptake of the photosensitizers after short 1 h incubation periods. TMA-DPH plasma membrane uptake rates after illumination of the photosensitizer-treated cells show a parabolic relationship with photosensitizer hydrophobicity that correlates well with the phototoxicity of the ZnPcS3C,. After a 1 h incubation period, overall phototoxicity correlates closely with the postillumination rate of TMA-DPH incorporation into the cell membrane, suggesting a major role of plasma membrane damage in the overall PDT effect. In contrast, after a 24 h incubation, phototoxicity shows a stronger but imperfect correlation with total cellular photosensitizer uptake rather than TMA-DPH membrane uptake, suggesting a partial shift in the cellular damage responsible for photosensitization from the plasma membrane to intracellular targets. We conclude that plasma membrane localization of the amphiphilic ZnPcS3C6-C9 is a major factor in their overall photodynamic activity.

  13. Functional properties and active-site topographies of factor X Gla- and prothrombin Gla-domain chimeras of activated protein C.

    PubMed

    Qureshi, Shabir H; Yang, Likui; Manithody, Chandrashekhara; Bae, Jong-Sup; Rezaie, Alireza R

    2008-09-01

    Substitution of the Gla-domain of activated protein C (APC) with the Gla-domain of prothrombin (APC-PTGla) improves the anticoagulant activity of APC independent of protein S. Previous FRET studies showed that this substitution alters the active-site topography of this mutant, rendering it identical to the active site of the APC-protein S complex. In this study, we characterized the functional properties and the active-site topography of another APC chimera containing the Gla-domain of factor X (APC-FXGla). We discovered that the anticoagulant activity of this mutant was similarly improved independent of protein S. The average distance of the closest approach (L) between the donor dye fluorescein attached to the active site of APC derivatives and the acceptor dye octadecylrhodamine incorporated into PC/PS vesicles was determined to be 99 A for APC and 84-86 A for both APC-PTGla and APC-FXGla. Protein S minimally influenced the L values of the APC chimeras, however, it lowered this value to 87 A for wild-type APC. Further studies revealed that neither chimera elicits a protective signaling response in the TNF-alpha-activated endothelial cells. These results suggest that unique structural features within the Gla-domain of APC enable the protease to interact with endothelial protein C receptor in the antiinflammatory pathway, while the same features also cause an inherently lower specific activity for APC in the anticoagulant pathway. This adaptation has made APC a cofactor-dependent protease, requiring the cofactor function of protein S for its optimal anticoagulant function, which appears to involve the alteration of the active-site topography of APC above the membrane surface.

  14. Conformational transition of the lid helix covering the protease active site is essential for the ATP-dependent protease activity of FtsH.

    PubMed

    Suno, Ryoji; Shimoyama, Masakazu; Abe, Akiko; Shimamura, Tatsuro; Shimodate, Natsuka; Watanabe, Yo-hei; Akiyama, Yoshinori; Yoshida, Masasuke

    2012-09-21

    When bound to ADP, ATP-dependent protease FtsH subunits adopt either an "open" or "closed" conformation. In the open state, the protease catalytic site is located in a narrow space covered by a lidlike helix. This space disappears in the closed form because the lid helix bends at Gly448. Here, we replaced Gly448 with various residues that stabilize helices. Most mutants retained low ATPase activity and bound to the substrate protein, but lost protease activity. However, a mutant proline substitution lost both activities. Our study shows that the conformational transition of the lid helix is essential for the function of FtsH.

  15. 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

    2015-03-16

    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.

  16. α-(Substituted-phenoxyacetoxy)-α-heterocyclylmethylphosphonates: synthesis, herbicidal activity, inhibition on pyruvate dehydrogenase complex (PDHc), and application as postemergent herbicide against broadleaf weeds.

    PubMed

    He, Hong-Wu; Peng, Hao; Wang, Tao; Wang, Chubei; Yuan, Jun-Lin; Chen, Ting; He, Junbo; Tan, Xiaosong

    2013-03-13

    Pyruvate dehydrogenase complex (PDHc) is the site of action of a new class of herbicides. On the basis of the previous work for O,O'-dimethyl α-(substituted-phenoxyacetoxy)alkylphosphonates (I), further synthetic modifications were made by introducing a fural and a thienyl group to structure I. A series of α-(substituted-phenoxyacetoxy)-α-heterocyclylmethylphosphonate derivatives (II) were synthesized as potential inhibitors of PDHc. The postemergent activity of the title compounds II was evaluated in greenhouse experiments. The in vitro efficacy of II against PDHc was also examined. Compounds II with fural as R(3) and 2,4-dichloro as X and Y showed significant herbicidal activity and effective inhibition against PDHc from plants. O,O'-Dimethyl α-(2,4-dichlorophenoxyacetoxy)-α-(furan-2-yl)methylphosphonate II-17 had higher inhibitory potency against PDHc from Pisum sativum than against PDHc from Oryza sativa in vitro and was most effective against broadleaf weeds at 50 and 300 ai g/ha. II-17 was safe for maize and rice even at the dose of 900-1200 ai g/ha. Field trials at different regions in China showed that II-17 (HWS) could control a broad spectrum of broad-leaved and sedge weeds at the rate of 225-375 ai g/ha for postemergent applications in maize fields. II-17 (HWS) displayed potential utility as a selective herbicide.

  17. Promoter-proximal polyadenylation sites reduce transcription activity

    PubMed Central

    Andersen, Pia K.; Lykke-Andersen, Søren; Jensen, Torben Heick

    2012-01-01

    Gene expression relies on the functional communication between mRNA processing and transcription. We previously described the negative impact of a point-mutated splice donor (SD) site on transcription. Here we demonstrate that this mutation activates an upstream cryptic polyadenylation (CpA) site, which in turn causes reduced transcription. Functional depletion of U1 snRNP in the context of the wild-type SD triggers the same CpA event accompanied by decreased RNA levels. Thus, in accordance with recent findings, U1 snRNP can shield premature pA sites. The negative impact of unshielded pA sites on transcription requires promoter proximity, as demonstrated using artificial constructs and supported by a genome-wide data set. Importantly, transcription down-regulation can be recapitulated in a gene context devoid of splice sites by placing a functional bona fide pA site/transcription terminator within ∼500 base pairs of the promoter. In contrast, promoter-proximal positioning of a pA site-independent histone gene terminator supports high transcription levels. We propose that optimal communication between a pA site-dependent gene terminator and its promoter critically depends on gene length and that short RNA polymerase II-transcribed genes use specialized termination mechanisms to maintain high transcription levels. PMID:23028143

  18. 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.

  19. 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.

  20. Activity of ceftazidime/avibactam against isogenic strains of Escherichia coli containing KPC and SHV β-lactamases with single amino acid substitutions in the Ω-loop

    PubMed Central

    Winkler, Marisa L.; Papp-Wallace, Krisztina M.; Bonomo, Robert A.

    2015-01-01

    Objectives The objective of this study was to explore the activity of ceftazidime and ceftazidime/avibactam against a collection of isogenic strains of Escherichia coli DH10B possessing SHV and KPC β-lactamases containing single amino acid substitutions in the Ω-loop (residues 164–179). Methods Ceftazidime and ceftazidime/avibactam MICs were determined by the agar dilution method for a panel of isogenic E. coli strains expressing SHV-1 and KPC-2 with amino acid substitutions at positions 164, 167, 169 or 179. Two KPC-2 β-lactamase variants that possessed elevated MICs of ceftazidime/avibactam were selected for further biochemical analyses. Results Avibactam restored susceptibility to ceftazidime for all Ω-loop variants of SHV-1 with MICs <8 mg/L. In contrast, several of the Arg164 and Asp179 variants of KPC-2 demonstrated MICs of ceftazidime/avibactam >8 mg/L. β-Lactamase kinetics showed that the Asp179Asn variant of KPC-2 demonstrated enhanced kinetic properties against ceftazidime. The Ki app, k2/K and koff of the Arg164Ala and Asp179Asn variant KPC-2 β-lactamases indicated that avibactam effectively inhibited these enzymes. Conclusions Several KPC-2 variants demonstrating ceftazidime resistance as a result of single amino acid substitutions in the Ω-loop were not susceptible to ceftazidime/avibactam (MICs >8 mg/L). We hypothesize that this observation is due to the stabilizing interactions (e.g. hydrogen bonds) of ceftazidime within the active site of variant β-lactamases that prevent avibactam from binding to and inhibiting the β-lactamase. As ceftazidime/avibactam is introduced into the clinic, monitoring for new KPC-2 variants that may exhibit increased ceftazidime kinetics as well as resistance to this novel antibiotic combination will be important. PMID:25957381

  1. Functional consequences of single amino acid substitutions in calmodulin-activated adenylate cyclase of Bordetella pertussis.

    PubMed Central

    Glaser, P; Munier, H; Gilles, A M; Krin, E; Porumb, T; Bârzu, O; Sarfati, R; Pellecuer, C; Danchin, A

    1991-01-01

    Calmodulin-activated adenylate cyclase of Bordetella pertussis and Bacillus anthracis are two cognate bacterial toxins. Three short regions of 13-24 amino acid residues in these proteins exhibit between 66 and 80% identity. Site-directed mutagenesis of four residues in B. pertussis adenylate cyclase situated in the second (Asp188, Asp190) and third (His298, Glu301) segments of identity were accompanied by important decrease, or total loss, of enzyme activity. The calmodulin-binding properties of mutated proteins showed no important differences when compared to the wild-type enzyme. Apart from the loss of enzymatic activity, the most important change accompanying replacement of Asp188 by other amino acids was a dramatic decrease in binding of 3'-anthraniloyl-2'-deoxyadenosine 5'-triphosphate, a fluorescent analogue of ATP. From these results we concluded that the two neighbouring aspartic acid residues in B. pertussis adenylate cyclase, conserved in many other ATP-utilizing enzymes, are essential for binding the Mg(2+)-nucleotide complex, and for subsequent catalysis. Replacement of His298 and Glu301 by other amino acid residues affected the nucleotide-binding properties of adenylate cyclase to a lesser degree suggesting that they might be important in the mechanism of enzyme activation by calmodulin, rather than being involved directly in catalysis. PMID:2050107

  2. New aromatase inhibitors. Synthesis and biological activity of pyridyl-substituted tetralone derivatives.

    PubMed

    Bayer, H; Batzl, C; Hartmann, R W; Mannschreck, A

    1991-09-01

    The (E)-2-(4-pyridylmethylene)-1-tetralones 1-7 (1, H; 2, 5-OCH3; 3, 6-OCH3; 4, 7-OCH3; 5, 5-OH; 6, 6-OH; 7, 7-OH) were obtained by aldol condensation of the corresponding 1-tetralones with 4-pyridinecarboxaldehyde, and in the case of the OH compounds 5 and 7 subsequent ether cleavage of the OCH3-substituted 2-(4-pyridylmethylene)-1-tetralones. Catalytic hydrogenation of 1-4 gave the 2-(4-pyridylmethyl)-1-tetralones 8-11 (8, H; 9, 5-OCH3; 10, 6-OCH3; 11, 7-OCH3). Subsequent ether cleavage of 9-11 led to the corresponding OH compounds 12-14 (12, 5-OH; 13, 6-OH; 14, 7-OH). The enantiomers of 11 and 12 were separated semipreparatively by HPLC on triacetylcellulose. All compounds (1-14) showed an inhibition of human placental aromatase exhibiting relative potencies from 2.2 to 213 [compounds 6 and (+)-12, respectively; aromatase inhibitory potency of aminoglutethimide (AG) = 1]. The compounds exhibited no or only a weak inhibition of desmolase [cholesterol side chain cleavage enzyme; maximum activity shown by 12, 23% inhibition (25 microM); AG, 53% inhibition (25 microM)]. In vivo, however, the compounds were not superior to AG as far as the reduction of the plasma estradiol concentration and the mammary carcinoma (MC) inhibiting properties are concerned (PMSG-primed SD rats as well as DMBA-induced MC of the SD rat, pre- and postmenopausal experiments, and the transplantable MXT-MC of the BD2F1 mouse). This is due to a fast decrease of the plasma E2 concentration inhibiting effect as could be shown by a kinetic experiment. In addition, select compounds inhibited rat ovarian aromatase much less than human placental aromatase (12, factor of 10). Estrogenic effects as a cause for the poor in vivo activity of the test compounds could be excluded, since they did not show affinity for the estrogen receptor.

  3. Copper (II) complexes possessing alkyl-substituted polypyridyl ligands: Structural characterization and in vitro antitumor activity.

    PubMed

    Angel, Noah R; Khatib, Raneen M; Jenkins, Julia; Smith, Michelle; Rubalcava, Justin M; Le, Brian Khoa; Lussier, Daniel; Chen, Zhuo Georgia; Tham, Fook S; Wilson, Emma H; Eichler, Jack F

    2017-01-01

    In an effort to find alternatives to the antitumor drug cisplatin, a series of copper (II) complexes possessing alkyl-substituted polypyridyl ligands have been synthesized. Eight new complexes are reported herein: μ-dichloro-bis{2,9-di-sec-butyl-1,10-phenanthrolinechlorocopper(II)} {[((di-sec-butyl)phen)ClCu(μ-Cl)2CuCl((di-sec-butyl)phen)]}(1), 2-sec-butyl-1,10-phenanthrolinedichlorocopper(II) {([mono-sec-butyl)phen) CuCl2} (2), 2,9-di-n-butyl-1,10-phenanthrolinedichlorocopper(II) {[(di-n-butyl)phen) CuCl2}(3), 2-n-butyl-1,10-phenanthrolinedichlorocopper(II) {[(mono-n-butyl)phen) CuCl2} (4), 2,9-di-methyl-1,10-phenanthrolineaquadichlorocopper(II) {[(di-methyl)phen) Cu(H2O)Cl2}(5), μ-dichloro-bis{6-sec-butyl-2,2'-bipyridinedichlorocopper(II)} {((mono-sec-butyl)bipy) ClCu(μ-Cl)2CuCl((mono-sec-butyl)bipy)} (6), 6,6'-di-methyl-2,2'-bipyridinedichlorocopper(II) {(6,6'-di-methyl)bipy) CuCl2} (7), and 4,4'-dimethyl-2,2'-bipyridinedichlorocopper(II) {(4,4'-di-methyl)bipy) CuCl2} (8). These complexes have been characterized via elemental analysis, UV-vis spectroscopy, and mass spectrometry. Single crystal X-ray diffraction experiments revealed the complexes synthesized with the (di-sec-butyl)phen ligand (1) and (mono-sec-butyl)bipy ligand (6) crystallized as dimers in which two copper(II) centers are bridged by two chloride ligands. Conversely, complexes 2, 7, and 8 were isolated as monomeric species possessing distorted tetrahedral geometries, and the [((di-methyl)phen)Cu(H2O)Cl2] (5) complex was isolated as a distorted square pyramidal monomer possessing a coordinating aqua ligand. Compounds 1-8 were evaluated for their in vitro antitumor efficacy. Compounds 1, 5, and 7 in particular were found to exhibit remarkable activity against human derived lung cancer cells, yet this class of copper(II) compounds had minimal cytotoxic effect on non-cancerous cells. In vitro control experiments indicate the activity of the copper(II) complexes most likely does not arise from the

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

    SciTech Connect

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

    2012-02-28

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

  5. Active and regulatory sites of cytosolic 5'-nucleotidase.

    PubMed

    Pesi, Rossana; Allegrini, Simone; Careddu, Maria Giovanna; Filoni, Daniela Nicole; Camici, Marcella; Tozzi, Maria Grazia

    2010-12-01

    Cytosolic 5'-nucleotidase (cN-II), which acts preferentially on 6-hydroxypurine nucleotides, is essential for the survival of several cell types. cN-II catalyses both the hydrolysis of nucleotides and transfer of their phosphate moiety to a nucleoside acceptor through formation of a covalent phospho-intermediate. Both activities are regulated by a number of phosphorylated compounds, such as diadenosine tetraphosphate (Ap₄A), ADP, ATP, 2,3-bisphosphoglycerate (BPG) and phosphate. On the basis of a partial crystal structure of cN-II, we mutated two residues located in the active site, Y55 and T56. We ascertained that the ability to catalyse the transfer of phosphate depends on the presence of a bulky residue in the active site very close to the aspartate residue that forms the covalent phospho-intermediate. The molecular model indicates two possible sites at which adenylic compounds may interact. We mutated three residues that mediate interaction in the first activation site (R144, N154, I152) and three in the second (F127, M436 and H428), and found that Ap₄A and ADP interact with the same site, but the sites for ATP and BPG remain uncertain. The structural model indicates that cN-II is a homotetrameric protein that results from interaction through a specific interface B of two identical dimers that have arisen from interaction of two identical subunits through interface A. Point mutations in the two interfaces and gel-filtration experiments indicated that the dimer is the smallest active oligomerization state. Finally, gel-filtration and light-scattering experiments demonstrated that the native enzyme exists as a tetramer, and no further oligomerization is required for enzyme activation.

  6. Novel N-substituted indole Schiff bases as dual inhibitors of cyclooxygenase-2 and 5-lipoxygenase enzymes: Synthesis, biological activities in vitro and docking study.

    PubMed

    Lamie, Phoebe F; Ali, Waleed A M; Bazgier, Vaclav; Rárová, Lucie

    2016-11-10

    Two new series of N-substituted indole derivatives 4a-l and 5a-h were synthesized. Their chemical structures were confirmed using spectroscopic tools including IR, (1)H NMR, (13)C NMR mass spectroscopy and elemental analyses. The results showed no significant cytotoxic activity on either cancer or normal human cells. Anti-inflammatory activity for all target compounds was evaluated in vitro. Compounds 5a-h were found to have better anti-inflammatory activity than 4a-l. The inhibitory activity of COX-2 and 5-LOX were tested for 5a-h. Three compounds, 5c, 5d and 5f showed excellent COX-2 inhibitory activity with IC50 ranging from 0.98 to 1.23 μM compared to the reference celecoxib (1.54 μM). These compounds had a reasonable selectivity index between 7.03 and 8.05. Additionally, p-methylbenzoyl derivative 5g (IC50 = 5.78 μM) had superior 5-LOX inhibitory activity, higher than quercetin. 5e was close to quercetin in its LOX inhibitory activity. Compounds 5a-h were docked inside the active site of COX-2 and 5-LOX enzymes.

  7. Beta zero thalassemia caused by a base substitution that creates an alternative splice acceptor site in an intron.

    PubMed Central

    Metherall, J E; Collins, F S; Pan, J; Weissman, S M; Forget, B G

    1986-01-01

    A thalassemic beta-globin gene cloned from a haplotype I chromosome contains a T to G transversion at position 116 of IVS1 which results in the generation of an abnormal alternative acceptor splice site. Transient expression studies revealed a 4-fold decrease in the amount of RNA produced with greater than 99% of it being abnormally spliced despite preservation of the normal acceptor splice site at position 130. These results suggest that the mutation at IVS1 position 116 results in beta zero thalassemia. A closely related mutation at position 110 of IVS1 also generates a novel acceptor site and results in a similar decrease in total mRNA produced, but approximately 20% of the mRNA produced is normally spliced and thus the phenotype is that of beta + thalassemia. These observations suggest that short range position effects may play a dramatic role in the choice of potential splice acceptor sites. We demonstrate the presence of abnormally spliced mRNA in reticulocytes of affected individuals and show the mutation at IVS1 position 116 segregating from the mutation at IVS1 position 110 in a three generation pedigree. The mutation results in the creation of a MaeI restriction site, as do a number of other thalassemic mutations, and we demonstrate some difficulties that may arise in the differential diagnosis of these mutations. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:3780671

  8. Detoxification of alkyl methylphosphonofluoridates by an oxime-substituted β-cyclodextrin--an in vitro structure-activity study.

    PubMed

    Bierwisch, Anne; Zengerle, Michael; Thiermann, Horst; Kubik, Stefan; Worek, Franz

    2014-01-13

    Detoxification rates of a series of alkyl methylphosphonofluoridates by an oxime-substituted β-cyclodextrin (β-CD) were assessed quantitatively by using an AChE inhibition assay. The cyclodextrin (CD) derivative was identified in previous work as a highly active cyclosarin scavenger. Here, a structure-activity relationship was established by investigating the effect of this CD on the detoxification of sarin derivatives differing in the structure of the alkoxy residue. The results show that detoxification rates correlate with the steric bulk and chain length of the alkoxy group in the organophosphonate (OP). OPs with larger, more bulky residues are detoxified more rapidly, with the exception of soman, which is bearing a pinacolyloxy side chain. In addition, the substituted CD was in every case more active than unsubstituted, native β-CD with up to a 400-fold difference. Comparing the kinetic results obtained with the known thermodynamic stabilities of related β-CD complexes indicate that detoxification rates generally increase when the alkoxy residue on the OP is exchanged by a residue, which forms a more stable complex with β-CD. This correlation lends support to the proposed mode of action of the substituted CD, involving initial complexation of the OP followed by reaction between the CD and the OP. The moderate to high efficacy on the detoxification of sarin derivatives suggests the potential applicability of this CD as a small molecule scavenger for G-type nerve agents.

  9. Chiral monolithic absorbent constructed by optically active helical-substituted polyacetylene and graphene oxide: preparation and chiral absorption capacity.

    PubMed

    Li, Weifei; Wang, Bo; Yang, Wantai; Deng, Jianping

    2015-02-01

    Chiral monolithic absorbent is successfully constructed for the first time by using optically active helical-substituted polyacetylene and graphene oxide (GO). The preparative strategy is facile and straightforward, in which chiral-substituted acetylene monomer (Ma), cross-linker (Mb), and alkynylated GO (Mc) undergo copolymerization to form the desired monolithic absorbent in quantitative yield. The resulting monoliths are characterized by circular dichroism, UV-vis absorption, scanning electron microscopy (SEM), FT-IR, Raman, energy-dispersive spectrometer (EDS), X-ray diffraction (XRD), Brunauer-Emmett-Teller (BET), XPS, and thermogravimetric analysis (TGA) techniques. The polymer chains derived from Ma form chiral helical structures and thus provide optical activity to the monoliths, while GO sheets contribute to the formation of porous structures. The porous structure enables the monolithic absorbents to demonstrate a large swelling ratio in organic solvents, and more remarkably, the helical polymer chains provide optical activity and further enantio-differentiating absorption ability. The present study establishes an efficient and versatile methodology for preparing novel functional materials, in particular monolithic chiral materials based on substituted polyacetylene and GO.

  10. Effects of 7-O Substitutions on Estrogenic and Antiestrogenic Activities of Daidzein Analogues in MCF-7 Breast Cancer Cells

    PubMed Central

    Jiang, Quan; Payton-Stewart, Florastina; Elliott, Steven; Driver, Jennifer; Rhodes, Lyndsay V.; Zhang, Qiang; Zheng, Shilong; Bhatnagar, Deepak; Boue, Stephen M.; Collins-Burow, Bridgette M.; Sridhar, Jayalakshmi; Stevens, Cheryl; McLachlan, John A.; Wiese, Thomas E.; Burow, Matthew E.; Wang, Guangdi

    2010-01-01

    Daidzein (1) is a natural estrogenic isoflavone. We report here that 1 can be transformed into antiestrogenic ligands by simple alkyl substitutions of the 7-hydroxyl hydrogen. To test the effect of such structural modifications on the hormonal activities of the resulting compounds, a series of daidzein analogues have been designed and synthesized. When MCF-7 cells were treated with the analogues, those resulting from hydrogen substitution by isopropyl (3d), isobutyl (3f), cyclopentyl (3g), and pyrano- (2), inhibited cell proliferation, estrogen-induced transcriptional activity, and estrogen receptor (ER) regulated progesterone receptor (PgR) gene expression. However, methyl (3a) and ethyl (3b) substitutions of the hydroxyl proton only led to moderate reduction of the estrogenic activities. These results demonstrated the structural requirements for the transformation of daidzein from an ER agonist to an antagonist. The most effective analogue, 2 was found to reduce in vivo estrogen stimulated MCF-7 cell tumorigenesis using a xenograft mouse model. PMID:20669983

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

    PubMed Central

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

    1990-01-01

    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 (ester vs. amide). 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. Images PMID:2395880

  12. Design and synthesis of 4-substituted quinazolines as potent EGFR inhibitors with anti-breast cancer activity.

    PubMed

    Ahmed, Marwa; Magdy, Naja

    2016-09-23

    Cancer is a major health problem to human beings around the world. Many quinazoline derivatives were reported to have potent cytotoxic activity. Our aim in this work is the discovery of potent epidermal growth factor receptor (EGFR) inhibitors with anti-breast cancer activity containing 4-substituted quinazoline pharmacophore. Novel series of 4-substituted 6,8-dibromo-2-(4-chlorophenyl)-quinazoline derivatives have been designed and synthesized. New derivatives were tested against MCF-7 (human breast carcinoma cell line) and screened for their inhibition activity against epidermal growth factor receptor tyrosine kinase (EGFR-TK). Most of the tested compounds show potent antiproliferative activity and EGFR-TK inhibitory activity. Compounds VIIIc and VIIIb exerted powerful cytotoxic activity (IC50 3.1 and 6.3 µM) with potent inhibitory percent (91.1 and 88.4%) against EGFR-TK. Compounds IX, VIIa, X, VIIb, VIc, V, IV, VIa and VIb showed promising cytotoxic effects with IC50 range (12-79 µM) with good activity against EGFR-TK with the inhibitory percent (85.4-60.8%). On the other hand, compounds VIIc, VIIIa exerted low cytotoxic effects as revealed from their IC50 value (124 and 144 µM) with low activity against EGFR-TK with inhibitory percent 30.6 and 29.1% respectively.

  13. BAX Activation is Initiated at a Novel Interaction Site

    PubMed Central

    Gavathiotis, Evripidis; Suzuki, Motoshi; Davis, Marguerite L.; Pitter, Kenneth; Bird, Gregory H.; Katz, Samuel G.; Tu, Ho-Chou; Kim, Hyungjin; Cheng, Emily H.-Y.; Tjandra, Nico; Walensky, Loren D.

    2008-01-01

    BAX is a pro-apoptotic protein of the BCL-2 family stationed in the cytosol until activated by a diversity of stress stimuli to induce cell death. Anti-apoptotic proteins such as BCL-2 counteract BAX-mediated cell death. Although an interaction site that confers survival functionality has been defined for anti-apoptotic proteins, an activation site has not been identified for BAX, rendering its explicit trigger mechanism unknown. We previously developed Stabilized Alpha-Helix of BCL-2 domains (SAHBs) that directly initiate BAX-mediated mitochondrial apoptosis. Here we demonstrate by NMR analysis that BIM SAHB binds BAX at an interaction site that is distinct from the canonical binding groove characterized for anti-apoptotic proteins. The specificity of the BIM SAHB-BAX interaction is highlighted by point mutagenesis that abrogates functional activity, confirming that BAX activation is initiated at this novel structural location. Thus, we have now defined a BAX interaction site for direct activation, establishing a new target for therapeutic modulation of apoptosis. PMID:18948948

  14. Tricyclic covalent inhibitors selectively target Jak3 through an active site thiol.

    PubMed

    Goedken, Eric R; Argiriadi, Maria A; Banach, David L; Fiamengo, Bryan A; Foley, Sage E; Frank, Kristine E; George, Jonathan S; Harris, Christopher M; Hobson, Adrian D; Ihle, David C; Marcotte, Douglas; Merta, Philip J; Michalak, Mark E; Murdock, Sara E; Tomlinson, Medha J; Voss, Jeffrey W

    2015-02-20

    The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. We have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). We found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC50 < 100 nm) inhibit Jak3 activity in cell-based assays. These results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases.

  15. 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

  16. Involvement of novel autophosphorylation sites in ATM activation.

    PubMed

    Kozlov, Sergei V; Graham, Mark E; Peng, Cheng; Chen, Philip; Robinson, Phillip J; Lavin, Martin F

    2006-08-09

    ATM kinase plays a central role in signaling DNA double-strand breaks to cell cycle checkpoints and to the DNA repair machinery. Although the exact mechanism of ATM activation remains unknown, efficient activation requires the Mre11 complex, autophosphorylation on S1981 and the involvement of protein phosphatases and acetylases. We report here the identification of several additional phosphorylation sites on ATM in response to DNA damage, including autophosphorylation on pS367 and pS1893. ATM autophosphorylates all these sites in vitro in response to DNA damage. Antibodies against phosphoserine 1893 revealed rapid and persistent phosphorylation at this site after in vivo activation of ATM kinase by ionizing radiation, paralleling that observed for S1981 phosphorylation. Phosphorylation was dependent on functional ATM and on the Mre11 complex. All three autophosphorylation sites are physiologically important parts of the DNA damage response, as phosphorylation site mutants (S367A, S1893A and S1981A) were each defective in ATM signaling in vivo and each failed to correct radiosensitivity, genome instability and cell cycle checkpoint defects in ataxia-telangiectasia cells. We conclude that there are at least three functionally important radiation-induced autophosphorylation events in ATM.

  17. 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.

  18. Effects of carbohydrate polymers applicable in saliva substitutes on the anti-Candida activity of a histatin-derived peptide.

    PubMed

    Ruissen, Anita L A; Groenink, Jasper; Lommerse, Cock H; Van 't Hof, Wim; Veerman, Enno C I; Nieuw Amerongen, Arie V

    2002-11-01

    The effects of polymers applicable in saliva substitutes on the anti-Candida activity of the cationic antimicrobial peptide dhvar1 were investigated. Dhvar1 is a derivative of the 14 C-terminal amino acids of histatin 5. The effects of the following polymers were tested: uncharged hydroxyethylcellulose (HEC), negatively charged xanthan (XG) and three types of negatively charged carboxymethylcellulose (CMC) of identical mass but different degrees of carboxylic acid-group substitution (DS). The effects were tested at pH 4.0, 7.0 and 8.5 in a killing assay. HEC had no effect at any pH tested; XG and the three types of CMC caused a decrease in activity at increasing concentrations. Within the CMC group, inhibition increased slightly with increasing DS. These results suggest that the reduction in activity associated with these polymers is the result of electrostatic interaction between the positively charged peptides and the negatively charged polymers. In the absence of polymers, no effect of pH was found on the activity of dhvar1. In the presence of the charged polymers XG and CMC, lowering the pH from 7.0 to 4.0 resulted in a decrease of dhvar1 activity. It was concluded that, with respect to the retention of activity, HEC is the most appropriate polymer for use in combination with dhvar1. However, for use in saliva substitutes XG seems more suitable because of its rheological properties. If XG or CMC are to be used, their reductive effect on the anti-Candida activity of dhvar1 should be compensated for by increasing the peptide dose.

  19. In Vitro Antiviral Activity of 6-Substituted 9-β-d-Ribofuranosylpurine 3′,5′-Cyclic Phosphates

    PubMed Central

    Sidwell, Robert W.; Huffman, John H.; Allen, Lois B.; Meyer, Rich B.; Shuman, Dennis A.; Simon, Lionel N.; Robins, Roland K.

    1974-01-01

    A series of twelve recently synthesized 6-substituted derivatives of 9-β-d-ribofuranosylpurine 3′,5′-cyclic phosphate (RPcMP) were evaluated for in vitro antiviral activity, using inhibition of viral cytopathogenic effect as the primary parameter for evaluation. Inhibition of the development of intra- and extracellular virus titer was used as a secondary criterion with certain viruses. Five derivatives were considered to have significant antiviral activity. 6-Hydroxylamino-RPcMP was active against type 1 herpes simplex, cytomegalo-, and vaccinia viruses. 6-Thio-RPcMP was inhibitory to types 1 and 2 herpes simplex, cytomegalo-, vaccinia, and type 3 parainfluenza viruses. The 6-methylthio derivative was active against types 1 and 2 herpes simplex, cytomegalo-, and vaccinia viruses, and types 1A, 2, 8, and 13 rhinoviruses; alteration of this 6-substitution to 6-ethylthio or to 6-benzylthio weakened the herpes- and vaccinia virus activity of the compound, but each continued to have significant antirhinovirus activity. The effect of time of addition of 6-methylthio-RPcMP to type 1 herpes simplex virus-infected cells was determined; the compound was most active when added prior to the virus. Early removal of the compound from the infected cells markedly reduced its antiviral effectiveness. PMID:15825420

  20. Multi-enhanced-phonon scattering modes in Ln-Me-A sites co-substituted LnMeA11O19 ceramics.

    PubMed

    Lu, Haoran; Wang, Chang-An; Huang, Yong; Xie, Huimin

    2014-10-29

    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 La(3+), Mg(2+) and Al(3+) ions with large ionic radius Ba(2+), Zn(2+) and Ti(4+), 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.

  1. 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

  2. Multi-Enhanced-Phonon Scattering Modes in Ln-Me-A Sites co-substituted LnMeA11O19 Ceramics

    NASA Astrophysics Data System (ADS)

    Lu, Haoran; Wang, Chang-An; Huang, Yong; Xie, Huimin

    2014-10-01

    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.

  3. 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.

  4. 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…

  5. Spectroscopic studies of the active site of galactose oxidase

    SciTech Connect

    Knowles, P.F.; Brown, R.D. III; Koenig, S.H.

    1995-07-19

    X-ray absorption and EPR spectroscopy have been used to probe the copper site structure in galactose oxidase at pH 4.5 and 7.0. the results suggest that there are no major differences in the structure of the tetragonal Cu(II) site at these pH values. Analysis of the extended X-ray absorption fine structure (EXAFS) indicates that four N,O scatterers are present at approximately 2 {Angstrom}; these are presumably the equatorial ligands. In addition, the EXAFS data establish that oxidative activation to produce the active-site tyrosine radical does not cause major changes in the copper coordination environment. Therefore results obtained on the one-electron reduced enzyme, containing Cu(II) but not the tyrosine radical, probably also apply to the catalytically active Cu(II)/tyrosine radical state. Solvent water exchange, inhibitor binding, and substrate binding have been probed via nuclear magnetic relaxation dispersion (NMRD) measurements. The NMRD profile of galactose oxidase is quantitatively consistent with the rapid exchange of a single, equatorial water ligand with a Cu(II)-O separation of about 2.4 {Angstrom}. Azide and cyanide displace this coordinated water. The binding of azide and the substrate dihydroxyacetone produce very similar effects on the NMRD profile of galactose oxidase, indicating that substrates also bind to the active site Cu(II) in an equatorial position.

  6. 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.

  7. 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.

  8. Etude des magnétites dopéesa`la chaux: Substitution du fer par le calcium en site tétraédrique

    NASA Astrophysics Data System (ADS)

    G´rardin, R.; Bonazebi, A.; Millon, E.; Brice, J. F.; Evrard, O.; Sanchez, J. P.

    1989-01-01

    A 1200°C, par réaction entre solides, il est possible de dissoudre partiellement le ferrite monocalcique CaFe 2O 4 dans la magnétite Fe 3O 4. Le taux maximum de substitution du cation Fe 2+ par le cation Ca 2+ est de 20% atomique. Dans la solution solide de type spinelle, les ions Ca 2+ se placent presque exclusivement en site tétraédrique. Cette distribution cationique est confirmée par l'analyse des propriétés radiocristallographiques, spectrométrie Mössbauer du 57Fe et des propriétés magnétiques.

  9. Structure-activity relationship of daptomycin analogues with substitution at (2S, 3R) 3-methyl glutamic acid position.

    PubMed

    Lin, Du'an; Lam, Hiu Yung; Han, Wenbo; Cotroneo, Nicole; Pandya, Bhaumik A; Li, Xuechen

    2017-02-01

    Daptomycin is a highly effective lipopeptide antibiotic against Gram-positive pathogens. The presence of (2S, 3R) 3-methyl glutamic acid (mGlu) in daptomycin has been found to be important to the antibacterial activity. However the role of (2S, 3R) mGlu is yet to be revealed. Herein, we reported the syntheses of three daptomycin analogues with (2S, 3R) mGlu substituted by (2S, 3R) methyl glutamine (mGln), dimethyl glutamic acid and (2S, 3R) ethyl glutamic acid (eGlu), respectively, and their antibacterial activities. The detailed synthesis of dimethyl glutamic acid was also reported.

  10. 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.

  11. Amino acid substitutions in the poliovirus maturation cleavage site affect assembly and result in accumulation of provirions.

    PubMed Central

    Ansardi, D C; Morrow, C D

    1995-01-01

    The assembly of infectious poliovirus virions requires a proteolytic cleavage between an asparagine-serine amino acid pair (the maturation cleavage site) in VP0 after encapsidation of the genomic RNA. In this study, we have investigated the effects that mutations in the maturation cleavage site have on P1 polyprotein processing, assembly of subviral intermediates, and encapsidation of the viral genomic RNA. We have made mutations in the maturation cleavage site which change the asparagine-serine amino acid pair to either glutamine-glycine or threonine-serine. The mutations were created by site-directed mutagenesis of P1 cDNAs which were recombined into wild-type vaccinia virus to generate recombinant vaccinia viruses. The P1 polyproteins expressed from the recombinant vaccinia viruses were analyzed for proteolytic processing and assembly defects in cells coinfected with a recombinant vaccinia virus (VV-P3) that expresses the poliovirus 3CD protease. A trans complementation system using a defective poliovirus genome was utilized to assess the capacity of the mutant P1 proteins to encapsidate genomic RNA (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 67:3684-3690, 1993). The mutant P1 proteins containing the glutamine-glycine amino acid pair (VP4-QG) and the threonine-serine pair (VP4-TS) were processed by 3CD provided in trans from VV-P3. The processed capsid proteins VP0, VP3, and VP1 derived from the mutant precursor VP4-QG were unstable and failed to assemble into subviral structures in cells coinfected with VV-P3. However, the capsid proteins derived from VP4-QG did assemble into empty-capsid-like structures in the presence of the defective poliovirus genome. In contrast, the capsid proteins derived from processing of the VP4-TS mutant assembled into subviral intermediates both in the presence and in the absence of the defective genome RNA. By a sedimentation analysis, we determined that the capsid proteins derived from the VP4-TS precursor

  12. 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

  13. 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.

  14. Identification of Arg-12 in the active site of Escherichia coli K1 CMP-sialic acid synthetase.

    PubMed Central

    Stoughton, D M; Zapata, G; Picone, R; Vann, W F

    1999-01-01

    Escherichia coli K1 CMP-sialic acid synthetase catalyses the synthesis of CMP-sialic acid from CTP and sialic acid. The active site of the 418 amino acid E. coli enzyme was localized to its N-terminal half. The bacterial CMP-sialic acid synthetase enzymes have a conserved motif, IAIIPARXXSKGLXXKN, at their N-termini. Several basic residues have been identified at or near the active site of the E. coli enzyme by chemical modification and site-directed mutagenesis. Only one of the lysines in the N-terminal motif, Lys-21, appears to be essential for activity. Mutation of Lys-21 in the N-terminal motif results in an inactive enzyme. Furthermore, Arg-12 of the N-terminal motif appears to be an active-site residue, based on the following evidence. Substituting Arg-12 with glycine or alanine resulted in inactive enzymes, indicating that this residue is required for enzymic activity. The Arg-12-->Lys mutant was partially active, demonstrating that a positive charge is required at this site. Steady-state kinetic analysis reveals changes in k(cat), K(m) and K(s) for CTP, which implicates Arg-12 in catalysis and substrate binding. PMID:10510306

  15. 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

    2016-09-06

    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.

  16. Changes in active site histidine hydrogen bonding trigger cryptochrome activation

    PubMed Central

    Ganguly, Abir; Manahan, Craig C.; Top, Deniz; Yee, Estella F.; Lin, Changfan; Young, Michael W.; Thiel, Walter; Crane, Brian R.

    2016-01-01

    Cryptochrome (CRY) is the principal light sensor of the insect circadian clock. Photoreduction of the Drosophila CRY (dCRY) flavin cofactor to the anionic semiquinone (ASQ) restructures a C-terminal tail helix (CTT) that otherwise inhibits interactions with targets that include the clock protein Timeless (TIM). All-atom molecular dynamics (MD) simulations indicate that flavin reduction destabilizes the CTT, which undergoes large-scale conformational changes (the CTT release) on short (25 ns) timescales. The CTT release correlates with the conformation and protonation state of conserved His378, which resides between the CTT and the flavin cofactor. Poisson-Boltzmann calculations indicate that flavin reduction substantially increases the His378 pKa. Consistent with coupling between ASQ formation and His378 protonation, dCRY displays reduced photoreduction rates with increasing pH; however, His378Asn/Arg variants show no such pH dependence. Replica-exchange MD simulations also support CTT release mediated by changes in His378 hydrogen bonding and verify other responsive regions of the protein previously identified by proteolytic sensitivity assays. His378 dCRY variants show varying abilities to light-activate TIM and undergo self-degradation in cellular assays. Surprisingly, His378Arg/Lys variants do not degrade in light despite maintaining reactivity toward TIM, thereby implicating different conformational responses in these two functions. Thus, the dCRY photosensory mechanism involves flavin photoreduction coupled to protonation of His378, whose perturbed hydrogen-bonding pattern alters the CTT and surrounding regions. PMID:27551082

  17. Beta-thiomaltosides as active site probes for alpha-amylase.

    PubMed

    Stankiewicz, P J; Cascio, D; McPherson, A

    1983-12-01

    A series of substituted 1-thio-beta-D-maltopyranosides was synthesized and confirmed by elemental analysis, optical rotation, NMR, and liquid chromatography. These compounds were shown by several biochemical techniques to bind to the active site of alpha-amylase. Steady-state kinetic studies showed the compounds to be competitive inhibitors, with affinities lying within the range of the natural ligands, maltose and maltotriose. Affinity chromatography employing p-aminophenyl-1-thio-beta-D-maltopyranoside linked to Sepharose provides a relatively simple procedure for alpha-amylase purification. The binding of p-bromphenyl-1-thio-beta-D-maltoside was observed in crystals of alpha-amylase using X-ray crystallography, and through the use of difference Fourier analysis its interaction at 5.0-A resolution with the active site of the enzyme has been visualized. The inhibitor binds in a long, deep cleft that divides the two major domains of the enzyme. These studies are believed to provide a first step toward the rational design of ligands for the physiological regulation of starch breakdown and utilization through modulation of alpha-amylase activity.

  18. The active site structure and mechanism of phosphoenolpyruvate utilizing enzymes

    SciTech Connect

    Cheng, K.C.

    1989-01-01

    Arginine specific reagents showed irreversible inhibition of avian liver mitochondrial phosphoenolpyruvate carboxykinase. Potent protection against modification was elicited by CO{sub 2} or CO{sub 2} in the presence of other substrates. Labeling of enzyme with (7-{sup 14}C) phenylglyoxal showed that 1 or 2 arginines are involved in CO{sub 2} binding and activation. Peptide map studies showed this active site arginine residues is located at position 289. Histidine specific reagents showed pseudo first order inhibition of avian mitochondrial phosphoenolpyruvate carboxykinase activity. The best protection against modification was elicited by IDP or IDP and Mn{sup +2}. One histidine residue is at or near the phosphoenolpyruvate binding site as demonstrated in the increased absorbance at 240 nm and proton relaxation rate studies. Circular dichroism studies reveal that enzyme structure was perturbed by diethylpyrocarbonate modification. Metal binding studies suggest that this enzyme has only one metal binding site. The putative binding sites from several GTP and phosphoenolpyruvate utilizing enzymes are observed in P-enolpyruvate carboxykinase from different species.

  19. Synthesis and appetite suppressant activity of 1-aryloxy-2-substituted aminomethyltetrahydronaphthalenes as conformationally rigid analogues of fluoxetine.

    PubMed

    Bhandari, Kalpana; Srivastava, Shipra; Shankar, Girija; Nath, Chandishwar

    2006-04-15

    Several 1-aryloxy-2-substituted aminomethyltetrahydronaphthalenes (7-21) as conformationally rigid analogues of fluoxetine were synthesized and evaluated for their anorexigenic and antidepressant activities. For SAR studies the related acyclic analogues (22-27) were also prepared. Out of the 21 synthesized compounds, 10 compounds (9, 10, 11, 15, 16, 18, 21, 22, 23 and 27) exhibited significant anorexigenic activity (at 75 micromol/kg). Interestingly, all the compounds (7-20, 22-26) were devoid of antidepressant effect, except for compounds 21 and 27 in which the antidepressant activity was retained. Compound 16 emerged as the most active compound of the series with better anorexigenic activity (97.92%) compared to fluoxetine (76.25%) and even with a clinically used drug sibutramine, thus providing a new structural lead for appetite suppressants.

  20. Investigating the activity of 2-substituted alkyl-6-(2,5-dioxopyrrolidin-1-yl)hexanoates as skin penetration enhancers.

    PubMed

    Brychtova, Katerina; Opatrilova, Radka; Raich, Ivan; Kalinowski, Danuta S; Dvorakova, Lenka; Placek, Lukas; Csollei, Jozef; Richardson, Des R; Jampilek, Josef

    2010-12-15

    Skin penetration enhancers are used in the formulation of transdermal delivery systems for drugs that are otherwise not sufficiently skin-permeable. We generated two series of esters by multi-step synthesis with substituted 6-aminohexanoic acid as potential transdermal penetration enhancers by multi-step synthesis. The synthesis of all newly prepared compounds is presented here. Structure confirmation of all generated compounds was accomplished by (1)H NMR, (13)C NMR, IR and MS spectroscopy. All the prepared compounds were analyzed using RP-HPLC and their lipophilicity (logk) was determined. The hydrophobicity (logP/ClogP) of the studied compounds was also calculated using two commercially available programs and 3D structures of the selected compounds were investigated by means of ab initio calculations of geometry and molecular dynamic simulations. All the synthesized esters were tested for their in vitro transdermal penetration-enhancing activity and showed higher enhancement ratios than oleic acid. The highest enhancement ratios were exhibited by compound 5f (C((2)) substituted with piperidine-2-one, C(11) ester chain) and 5a (C((2)) substituted with piperidine-2-one, C(6) ester chain). The series with a ω-lactam ring (piperidin-2-one; 5a-g), showed slightly higher activities than those with morpholine (6a-6g). All of the agents showed minimal anti-proliferative activity (IC(50) >6.25μM), indicating they would have low cytotoxicity when administered as chemical penetration enhancers. The relationships between the lipophilicity and the chemical structure of the studied compounds, as well as the correlation between their chemical structure and transdermal penetration-enhancing activity, are discussed.

  1. 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

  2. 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

  3. Synthesis and antirhinovirus activity of 8-substituted analogues of 6-(dimethylamino)-9-(4-methylbenzyl)-2-(trifluoromethyl)-9H-purine.

    PubMed

    Kelley, J L; Linn, J A; Selway, J W

    1991-01-01

    Several 8-substituted analogues of 6-(dimethylamino) -9-(4-methylbenzyl)-2-(trifluoromethyl)-9H-purine (1) were synthesized and tested for activity against rhinovirus type 1B. Among 16 8-substituted analogues, the 8-amino (3) and 8-bromo (2) analogues were most active with IC50s of 0.36 and 1.4 microM, respectively, under conditions where 1 had an IC50 of 0.03 microM.

  4. Structural features and activity of Brazzein and its mutants upon substitution of a surfaced exposed alanine.

    PubMed

    Ghanavatian, Parisa; Khalifeh, Khosrow; Jafarian, Vahab

    2016-12-01

    Brazzein (Brz) is a member of sweet-tasting protein containing four disulfide bonds. It was reported as a compact and heat-resistant protein. Here, we have used site-directed mutagenesis and replaced a surface-exposed alanine with aspartic acid (A19D mutant), lysine (A19K mutant) and glycine (A19G mutant). Activity comparisons of wild-type (WT) and mutants using taste panel test procedure showed that A19G variant has the same activity as WT protein. However, introduction of a positive charge in A19K mutant led to significant increase in Brz's sweetness, while A19D has reduced sweetness compared to WT protein. Docking studies showed that mutation at position 19 results in slight chain mobility of protein at the binding surface and changing the patterns of interactions toward more effective binding of E9K variant in the concave surface of sweet taste receptor. Far-UV CD data spectra have a characteristic shape of beta structure for all variants, however different magnitudes of spectra suggest that beta-sheet structure in WT and A19G is more stable than that of A19D and A19K. Equilibrium unfolding studies with fluorescence spectroscopy and using urea and dithiothritol (DTT) as chemical denaturants indicates that A19G mutant gains more stability against urea denaturation; while conformational stability of A19D and A19K decreases when compared with WT and A19G variants. We concluded that the positive charge at the surface of protein is important factor responsible for the interaction of protein with the human sweet receptor and Ala(19) can be considered as a key region for investigating the mechanism of the interaction of Brz with corresponding receptor.

  5. A camel-derived MERS-CoV with a variant spike protein cleavage site and distinct fusion activation properties

    PubMed Central

    Millet, Jean Kaoru; Goldstein, Monty E; Labitt, Rachael N; Hsu, Hung-Lun; Daniel, Susan; Whittaker, Gary R

    2016-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) continues to circulate in both humans and camels, and the origin and evolution of the virus remain unclear. Here we characterize the spike protein of a camel-derived MERS-CoV (NRCE-HKU205) identified in 2013, early in the MERS outbreak. NRCE-HKU205 spike protein has a variant cleavage motif with regard to the S2′ fusion activation site—notably, a novel substitution of isoleucine for the otherwise invariant serine at the critical P1′ cleavage site position. The substitutions resulted in a loss of furin-mediated cleavage, as shown by fluorogenic peptide cleavage and western blot assays. Cell–cell fusion and pseudotyped virus infectivity assays demonstrated that the S2′ substitutions decreased spike-mediated fusion and viral entry. However, cathepsin and trypsin-like protease activation were retained, albeit with much reduced efficiency compared with the prototypical EMC/2012 human strain. We show that NRCE-HKU205 has more limited fusion activation properties possibly resulting in more restricted viral tropism and may represent an intermediate in the complex pattern of MERS-CoV ecology and evolution. PMID:27999426

  6. Identification of Ice Nucleation Active Sites on Silicate Dust Particles

    NASA Astrophysics Data System (ADS)

    Zolles, Tobias; Burkart, Julia; Häusler, Thomas; Pummer, Bernhard; Hitzenberger, Regina; Grothe, Hinrich

    2015-04-01

    Mineral dusts originating from Earth's crust are known to be important atmospheric ice nuclei. In agreement with earlier studies, feldspar was found as the most active of the tested natural mineral dusts [1-3]. Nevertheless, among those structures K-feldspar showed by far the highest ice nucleation activity. In this study, the reasons for its activity and the difference in the activity of the different feldspars were investigated in closer details. Conclusions are drawn from scanning electron microscopy, X-ray powder diffraction, infrared spectroscopy, and oil-immersion freezing experiments. We give a potential explanation of the increased ice nucleation activity of K-feldspar. The ice nucleating sites are very much dependent on the alkali ion present by altering the water structure and the feldspar surface. The higher activity of K-feldspar can be attributed to the presence of potassium ions on the surface and surface bilayer. The alkali-ions have different hydration shells and thus an influence on the ice nucleation activity of feldspar. Chaotropic behavior of Calcium and Sodium ions are lowering the ice nucleation potential of the other feldspars, while kosmotropic Potassium has a neutral or even positive effect. Furthermore we investigated the influence of milling onto the ice nucleation of quartz particles. The ice nucleation activity can be increased by mechanical milling, by introducing more molecular, nucleation active defects to the particle surface. This effect is larger than expected by plane surface increase. [1] Atkinson et al. The Importance of Feldspar for Ice Nucleation by Mineral Dust in Mixed-Phase Clouds. Nature 2013, 498, 355-358. [2] Yakobi-Hancock et al.. Feldspar Minerals as Efficient Deposition Ice Nuclei. Atmos. Chem. Phys. 2013, 13, 11175-11185. [3] Zolles et al. Identification of Ice Nucleation Active Sites on Feldspar Dust Particles. J. Phys. Chem. A 2015 accepted.

  7. Face the Edges: Catalytic Active Sites of Nanomaterials

    PubMed Central

    Ni, Bing

    2015-01-01

    Edges are special sites in nanomaterials. The atoms residing on the edges have different environments compared to those in other parts of a nanomaterial and, therefore, they may have different properties. Here, recent progress in nanomaterial fields is summarized from the viewpoint of the edges. Typically, edge sites in MoS2 or metals, other than surface atoms, can perform as active centers for catalytic reactions, so the method to enhance performance lies in the optimization of the edge structures. The edges of multicomponent interfaces present even more possibilities to enhance the activities of nanomaterials. Nanoframes and ultrathin nanowires have similarities to conventional edges of nanoparticles, the application of which as catalysts can help to reduce the use of costly materials. Looking beyond this, the edge structures of graphene are also essential for their properties. In short, the edge structure can influence many properties of materials. PMID:27980960

  8. Synthesis and structure-activity relationship study of substituted caffeate esters as antinociceptive agents modulating the TREK-1 channel.

    PubMed

    Rodrigues, Nuno; Bennis, Khalil; Vivier, Delphine; Pereira, Vanessa; C Chatelain, Franck; Chapuy, Eric; Deokar, Hemantkumar; Busserolles, Jérôme; Lesage, Florian; Eschalier, Alain; Ducki, Sylvie

    2014-03-21

    The TWIK-related K(+) channel, TREK-1, has recently emerged as an attractive therapeutic target for the development of a novel class of analgesic drugs. It has been reported that TREK-1 -/- mice were more sensitive than wild-type mice to painful stimuli, suggesting that activation of TREK-1 could result in pain inhibition. Here we report the synthesis of a series of substituted caffeate esters (12a-u) based on the hit compound CDC 2 (cinnamyl 3,4-dihydroxyl-α-cyanocinnamate). These analogs were evaluated for their ability to modulate TREK-1 channel by electrophysiology and for their in vivo antinociceptive activity (acetic acid induced-writhing assay) leading to the identification a series of novel molecules able to activate TREK-1 and displaying potent analgesic activity in vivo.

  9. Active sites in char gasification: Final technical report

    SciTech Connect

    Wojtowicz, M.; Lilly, W.D.; Perkins, M.T.; Hradil, G.; Calo, J.M.; Suuberg, E.M.

    1987-09-01

    Among the key variables in the design of gasifiers and combustors is the reactivity of the chars which must be gasified or combusted. Significant loss of unburned char is unacceptable in virtually any process; the provision of sufficient residence time for complete conversion is essential. A very wide range of reactivities are observed, depending upon the nature of the char in a process. The current work focuses on furthering the understanding of gasification reactivities of chars. It has been well established that the reactivity of char to gasification generally depends upon three principal factors: (1) the concentration of ''active sites'' in the char; (2) mass transfer within the char; and (3) the type and concentration of catalytic impurities in the char. The present study primarily addresses the first factor. The subject of this research is the origin, nature, and fate of active sites in chars derived from parent hydrocarbons with coal-like structure. The nature and number of the active sites and their reactivity towards oxygen are examined in ''model'' chars derived from phenol-formaldehyde type resins. How the active sites are lost by the process of thermal annealing during heat treatment of chars are studied, and actual rate for the annealing process is derived. Since intrinsic char reactivities are of primary interest in the present study, a fair amount of attention was given to the model char synthesis and handling so that the effect of catalytic impurities and oxygen-containing functional groups in the chemical structure of the material were minimized, if not completely eliminated. The project would not be considered complete without comparing characteristic features of synthetic chars with kinetic behavior exhibited by natural chars, including coal chars.

  10. Tuning Metal-Organic Frameworks with Open-Metal Sites and Its Origin for Enhancing CO2 Affinity by Metal Substitution.

    PubMed

    Park, Joonho; Kim, Heejin; Han, Sang Soo; Jung, Yousung

    2012-04-05

    Reducing anthropogenic carbon emission is a problem that requires immediate attention. Metal-organic frameworks (MOFs) have emerged as a promising new materials platform for carbon capture, of which Mg-MOF-74 offers chemospecific affinity toward CO2 because of the open Mg sites. Here we tune the binding affinity of CO2 for M-MOF-74 by metal substitution (M = Mg, Ca, and the first transition metal elements) and show that Ti- and V-MOF-74 can have an enhanced affinity compared to Mg-MOF-74 by 6-9 kJ/mol. Electronic structure calculations suggest that the origin of the major affinity trend is the local electric field effect of the open metal site that stabilizes CO2, but forward donation from the lone-pair electrons of CO2 to the empty d-levels of transition metals as in a weak coordination bond makes Ti and V have an even higher binding strength than Mg, Ca, and Sc.

  11. 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.

  12. 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

  13. [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).

  14. Active-site-mutagenesis study of rat liver betaine-homocysteine S-methyltransferase.

    PubMed

    González, Beatriz; Campillo, Nuria; Garrido, Francisco; Gasset, María; Sanz-Aparicio, Juliana; Pajares, María A

    2003-03-15

    A site-directed-mutagenesis study of putative active-site residues in rat liver betaine-homocysteine S-methyltransferase has been carried out. Identification of these amino acids was based on data derived from a structural model of the enzyme. No alterations in the CD spectra or the gel-filtration chromatography elution pattern were observed with the mutants, thus suggesting no modification in the secondary structure content or in the association state of the proteins. All the mutants obtained showed a reduction of the enzyme activity, the most dramatic effect being that of Glu(159), followed by Tyr(77) and Asp(26). Changes in affinity for either of the substrates, homocysteine or betaine, were detected when substitutions were performed of Glu(21), Asp(26), Phe(74) and Cys(186). Interestingly, Asp(26), postulated to be involved in homocysteine binding, has a strong effect on affinity for betaine. The relevance of these results is discussed in the light of very recent structural data obtained for the human enzyme.

  15. 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.

  16. Nest predation increases with parental activity: separating nest site and parental activity effects.

    PubMed Central

    Martin, T E; Scott, J; Menge, C

    2000-01-01

    Alexander Skutch hypothesized that increased parental activity can increase the risk of nest predation. We tested this hypothesis using ten open-nesting bird species in Arizona, USA. Parental activity was greater during the nestling than incubation stage because parents visited the nest frequently to feed their young during the nestling stage. However, nest predation did not generally increase with parental activity between nesting stages across the ten study species. Previous investigators have found similar results. We tested whether nest site effects might yield higher predation during incubation because the most obvious sites are depredated most rapidly. We conducted experiments using nest sites from the previous year to remove parental activity. Our results showed that nest sites have highly repeatable effects on nest predation risk; poor nest sites incurred rapid predation and caused predation rates to be greater during the incubation than nestling stage. This pattern also was exhibited in a bird species with similar (i.e. controlled) parental activity between nesting stages. Once nest site effects are taken into account, nest predation shows a strong proximate increase with parental activity during the nestling stage within and across species. Parental activity and nest sites exert antagonistic influences on current estimates of nest predation between nesting stages and both must be considered in order to understand current patterns of nest predation, which is an important source of natural selection. PMID:11413645

  17. Active site amino acid sequence of human factor D.

    PubMed

    Davis, A E

    1980-08-01

    Factor D was isolated from human plasma by chromatography on CM-Sephadex C50, Sephadex G-75, and hydroxylapatite. Digestion of reduced, S-carboxymethylated factor D with cyanogen bromide resulted in three peptides which were isolated by chromatography on Sephadex G-75 (superfine) equilibrated in 20% formic acid. NH2-Terminal sequences were determined by automated Edman degradation with a Beckman 890C sequencer using a 0.1 M Quadrol program. The smallest peptide (CNBr III) consisted of the NH2-terminal 14 amino acids. The other two peptides had molecular weights of 17,000 (CNBr I) and 7000 (CNBr II). Overlap of the NH2-terminal sequence of factor D with the NH2-terminal sequence of CNBr I established the order of the peptides. The NH2-terminal 53 residues of factor D are somewhat more homologous with the group-specific protease of rat intestine than with other serine proteases. The NH2-terminal sequence of CNBr II revealed the active site serine of factor D. The typical serine protease active site sequence (Gly-Asp-Ser-Gly-Gly-Pro was found at residues 12-17. The region surrounding the active site serine does not appear to be more highly homologous with any one of the other serine proteases. The structural data obtained point out the similarities between factor D and the other proteases. However, complete definition of the degree of relationship between factor D and other proteases will require determination of the remainder of the primary structure.

  18. 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.

  19. [Mechanism of arginine deiminase activity by site-directed mutagenesis].

    PubMed

    Li, Lifeng; Ni, Ye; Sun, Zhihao

    2012-04-01

    Arginine deiminase (ADI) has been studied as a potential anti-cancer agent for inhibiting arginine-auxotrophic tumors (such as melanomas and hepatocellular carcinomas) in phase III clinical trials. In this work, we studied the molecular mechanism of arginine deiminase activity by site-directed mutagenesis. Three mutation sites, A128, H404 and 1410, were introduced into wild-type ADI gene by QuikChange site-directed mutagenesis method, and four ADI mutants M1 (A128T), M2 (H404R), M3 (I410L), and M4 (A128T, H404R) were obtained. The ADI mutants were individually expressed in Escherichia coli BL21 (DE3), and the enzymatic properties of the purified mutant proteins were determined. The results show that both A128T and H404R had enhanced optimum pH, higher activity and stability of ADI under physiological condition (pH 7.4), as well as reduced K(m) value. This study provides an insight into the molecular mechanism of the ADI activity, and also the experimental evidence for the rational protein evolution in the future.

  20. Potential sites of CFTR activation by tyrosine kinases

    PubMed Central

    Billet, Arnaud; Jia, Yanlin; Jensen, Timothy J.; Hou, Yue-Xian; Chang, Xiu-Bao; Riordan, John R.; Hanrahan, John W.

    2016-01-01

    ABSTRACT The CFTR chloride channel is tightly regulated by phosphorylation at multiple serine residues. Recently it has been proposed that its activity is also regulated by tyrosine kinases, however the tyrosine phosphorylation sites remain to be identified. In this study we examined 2 candidate tyrosine residues near the boundary between the first nucleotide binding domain and the R domain, a region which is important for channel function but devoid of PKA consensus sequences. Mutating tyrosines at positions 625 and 627 dramatically reduced responses to Src or Pyk2 without altering the activation by PKA, suggesting they may contribute to CFTR regulation. PMID:26645934

  1. 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.

  2. The T210M Substitution in the HLA-a*02:01 gp100 Epitope Strongly Affects Overall Proteasomal Cleavage Site Usage and Antigen Processing*

    PubMed Central

    Textoris-Taube, Kathrin; Keller, Christin; Liepe, Juliane; Henklein, Petra; Sidney, John; Sette, Alessandro; Kloetzel, Peter M.; Mishto, Michele

    2015-01-01

    MHC class I-restricted epitopes, which carry a tumor-specific mutation resulting in improved MHC binding affinity, are preferred T cell receptor targets in innovative adoptive T cell therapies. However, T cell therapy requires efficient generation of the selected epitope. How such mutations may affect proteasome-mediated antigen processing has so far not been studied. Therefore, we analyzed by in vitro experiments the effect on antigen processing and recognition of a T210M exchange, which previously had been introduced into the melanoma gp100209–217tumor epitope to improve the HLA-A*02:01 binding and its immunogenicity. A quantitative analysis of the main steps of antigen processing shows that the T210M exchange affects proteasomal cleavage site usage within the mutgp100201–230 polypeptide, leading to the generation of an unique set of cleavage products. The T210M substitution qualitatively affects the proteasome-catalyzed generation of spliced and non-spliced peptides predicted to bind HLA-A or -B complexes. The T210M substitution also induces an enhanced production of the mutgp100209–217 epitope and its N-terminally extended peptides. The T210M exchange revealed no effect on ERAP1-mediated N-terminal trimming of the precursor peptides. However, mutant N-terminally extended peptides exhibited significantly increased HLA-A*02:01 binding affinity and elicited CD8+ T cell stimulation in vitro similar to the wtgp100209–217 epitope. Thus, our experiments demonstrate that amino acid exchanges within an epitope can result in the generation of an altered peptide pool with new antigenic peptides and in a wider CD8+ T cell response also towards N-terminally extended versions of the minimal epitope. PMID:26507656

  3. Structure-Activity Relationship Studies of Amino Acid Substitutions in Radiolabeled Neurotensin Conjugates.

    PubMed

    Mascarin, Alba; Valverde, Ibai E; Mindt, Thomas L

    2016-01-05

    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.

  4. Activity of 2-substituted lysophosphatidic acid (LPA) analogs at LPA receptors: discovery of a LPA1/LPA3 receptor antagonist.

    PubMed

    Heise, C E; Santos, W L; Schreihofer, A M; Heasley, B H; Mukhin, Y V; Macdonald, T L; Lynch, K R

    2001-12-01

    The physiological implications of lysophosphatidic acid occupancy of individual receptors are largely unknown because selective agonists/antagonists are unavailable currently. The molecular cloning of three high-affinity lysophosphatidic acid receptors, LPA1, LPA2, and LPA3, provides a platform for developing receptor type-selective ligands. Starting with an N-acyl ethanolamide phosphate LPA analog, we made a series of substitutions at the second carbon to generate compounds with varying spatial, stereochemical, and electronic characteristics. Analysis of this series at each recombinant LPA receptor using a guanosine 5'-O-(3-[35S]thio)triphosphate (GTP[gamma35S]) binding assay revealed sharp differences in activity. Our results suggest that these receptors have one spatially restrictive binding pocket that interacts with the 2-substituted moieties and prefers small hydrophobic groups and hydrogen bonding functionalities. The agonist activity predicted by the GTP[gamma35S] binding assay was reflected in the activity of a subset of compounds in increasing arterial pressure in anesthetized rats. One compound with a bulky hydrophobic group (VPC12249) was a dual LPA1/LPA3 competitive antagonist. Several compounds that had smaller side chains were found to be LPA1-selective agonists.

  5. MSK1 activity is controlled by multiple phosphorylation sites

    PubMed Central

    McCOY, Claire E.; Campbell, David G.; Deak, Maria; Bloomberg, Graham B.; Arthur, J. Simon C.

    2004-01-01

    MSK1 (mitogen- and stress-activated protein kinase) is a kinase activated in cells downstream of both the ERK1/2 (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) cascades. In the present study, we show that, in addition to being phosphorylated on Thr-581 and Ser-360 by ERK1/2 or p38, MSK1 can autophosphorylate on at least six sites: Ser-212, Ser-376, Ser-381, Ser-750, Ser-752 and Ser-758. Of these sites, the N-terminal T-loop residue Ser-212 and the ‘hydrophobic motif’ Ser-376 are phosphorylated by the C-terminal kinase domain of MSK1, and their phosphorylation is essential for the catalytic activity of the N-terminal kinase domain of MSK1 and therefore for the phosphorylation of MSK1 substrates in vitro. Ser-381 is also phosphorylated by the C-terminal kinase domain, and mutation of Ser-381 decreases MSK1 activity, probably through the inhibition of Ser-376 phosphorylation. Ser-750, Ser-752 and Ser-758 are phosphorylated by the N-terminal kinase domain; however, their function is not known. The activation of MSK1 in cells therefore requires the activation of the ERK1/2 or p38 MAPK cascades and does not appear to require additional signalling inputs. This is in contrast with the closely related RSK (p90 ribosomal S6 kinase) proteins, whose activity requires phosphorylation by PDK1 (3-phosphoinositide-dependent protein kinase 1) in addition to phosphorylation by ERK1/2. PMID:15568999

  6. Synthesis and characterization of a new fluorogenic active-site titrant of serine proteases.

    PubMed

    Livingston, D C; Brocklehurst, J R; Cannon, J F; Leytus, S P; Wehrly, J A; Peltz, S W; Peltz, G A; Mangel, W F

    1981-07-21

    The molecule 3',6'-bis(4-guanidinobenzoyloxy)-5-[N'-(4-carboxyphenyl)thioureido[spirop]isobenzofuran-1-(3H),9'-[9H]xanthen]-3-one, abbreviated FDE, was designed and synthesized as a fluorogenic active-site titrant for serine proteases. It is an analogue of p-nitrophenyl p-guanidino-benzoate (NPGB) in which a fluorescein derivative is substituted for p-nitrophenol. FDE and NPGB exhibit similar kinetic characteristics in an active-site titration of trypsin in phosphate-buffered saline, pH 7.2. The rate of acylation with FDE is extremely fast (k2 = 1.05 s-1) and the rate of deacylation extremely slow (k3 = 1.66 X 10(-5) s-1). The Ks is 3.06 X 10(-6) M, and the Km(app) is 4.85 X 10(-11) M. With two of the serine proteases involved in fibrinolysis, the rate of acylation with FDE is also fast, K2 = 0.112 s-1 for urokinase and 0.799 s-1 for plasmin, and the rate of deacylation is slow, k3 = 3.64 X 10(-4) s-1 for urokinase and 6.27 X 10(-6) s-1 for plasmin. The solubility limit of FDE in phosphate-buffered saline is 1.3 X 10(-5) M, and the first-order rate constant for spontaneous hydrolysis is 5.1 X 10(-6) s-1. The major difference between FDE and NPGB is the detectability of the product in an active-site titration. p-Nitrophenol can be detected at concentrations no lower than 10(-6) M whereas fluorescein can be detected at concentrations as low as 10(-12) M. Thus, FDE should be useful in quantitatively assaying serine proteases as very low concentrations.

  7. Novel metal chelating molecules with anticancer activity. Striking effect of the imidazole substitution of the histidine-pyridine-histidine system.

    PubMed

    Ali, Taha F S; Iwamaru, Kana; Ciftci, Halil Ibrahim; Koga, Ryoko; Matsumoto, Masahiro; Oba, Yasunori; Kurosaki, Hiromasa; Fujita, Mikako; Okamoto, Yoshinari; Umezawa, Kazuo; Nakao, Mitsuyoshi; Hide, Takuichiro; Makino, Keishi; Kuratsu, Jun-ichi; Abdel-Aziz, Mohamed; Abuo-Rahma, Gamal El-Din A A; Beshr, Eman A M; Otsuka, Masami

    2015-09-01

    Previously we have reported a metal chelating histidine-pyridine-histidine system possessing a trityl group on the histidine imidazole, namely HPH-2Trt, which induces apoptosis in human pancreatic adenocarcinoma AsPC-1 cells. Herein the influence of the imidazole substitution of HPH-2Trt was examined. Five related compounds, HPH-1Trt, HPH-2Bzl, HPH-1Bzl, HPH-2Me, and HPH-1Me were newly synthesized and screened for their activity against AsPC-1 and brain tumor cells U87 and U251. HPH-1Trt and HPH-2Trt were highly active among the tested HPH compounds. In vitro DNA cleavage assay showed both HPH-1Trt and HPH-2Trt completely disintegrate pUC19 DNA. The introduction of trityl group decisively potentiated the activity.

  8. 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.

  9. A comprehensive search for calcium binding sites critical for TMEM16A calcium-activated chloride channel activity

    PubMed Central

    Tien, Jason; Peters, Christian J; Wong, Xiu Ming; Cheng, Tong; Jan, Yuh Nung; Jan, Lily Yeh; Yang, Huanghe

    2014-01-01

    TMEM16A forms calcium-activated chloride channels (CaCCs) that regulate physiological processes such as the secretions of airway epithelia and exocrine glands, the contraction of smooth muscles, and the excitability of neurons. Notwithstanding intense interest in the mechanism behind TMEM16A-CaCC calcium-dependent gating, comprehensive surveys to identify and characterize potential calcium sensors of this channel are still lacking. By aligning distantly related calcium-activated ion channels in the TMEM16 family and conducting systematic mutagenesis of all conserved acidic residues thought to be exposed to the cytoplasm, we identify four acidic amino acids as putative calcium-binding residues. Alterations of the charge, polarity, and size of amino acid side chains at these sites alter the ability of different divalent cations to activate the channel. Furthermore, TMEM16A mutant channels containing double cysteine substitutions at these residues are sensitive to the redox potential of the internal solution, providing evidence for their physical proximity and solvent accessibility. DOI: http://dx.doi.org/10.7554/eLife.02772.001 PMID:24980701

  10. 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.

  11. [Synthesis and antiinflammatory and anticancer activities of 2-(E)-(un)substituted benzylidene cyclopentanones and their Mannich base hydro chlorides].

    PubMed

    Chen, H T; Jing, Y K; Ji, Z Z; Zhang, B F

    1991-01-01

    It has been known that non-steroidal antiinflammatory drugs (NSAIDs) act by preventing cyclooxygenase products of arachidonic acid. In recent years, research on non-steroid dual inhibitors of cyclooxygenase (CO) and 5-lipoxygenase (5-LO), which should represent a novel class of antiinflammatory drugs with a wider spectrum of activity than classical NSAIDs, has been carried out. In the present paper, a total of 29 compounds including 18 compounds of 2-(E)-(un) substituted benzylidene cyclopentanone (I) derivatives and 11 compounds of 2-(E)-(un)substituted benzylidene-5-dimethylaminomethyl cyclopentanone (II) derivatives were synthesized as dual inhibitors of CO/5-LO. Preliminary pharmacological test showed that I4, I12 and I13 given orally have significant inhibiting effect on carrageenan induced rat paw edema and most compounds of type II exhibited potent effect when given subcutaneously. In particular, II3, which inhibited by 95.8%, 70.34%, and 44.2% at 50, 25, and 12.5 mg/kg respectively, was similar to Ibuprofen. Some compounds of type II exhibited anticancer activity both in vitro (IC50 ranging from 2.93 to 18.06 mumol/L on L1210) and in vivo (maximum increase of life span was 97.5% on EAC in mice).

  12. Indol-3-ylcycloalkyl ketones: effects of N1 substituted indole side chain variations on CB(2) cannabinoid receptor activity.

    PubMed

    Frost, Jennifer M; Dart, Michael J; Tietje, Karin R; Garrison, Tiffany R; Grayson, George K; Daza, Anthony V; El-Kouhen, Odile F; Yao, Betty B; Hsieh, Gin C; Pai, Madhavi; Zhu, Chang Z; Chandran, Prasant; Meyer, Michael D

    2010-01-14

    Several 3-acylindoles with high affinity for the CB(2) cannabinoid receptor and selectivity over the CB(1) receptor have been prepared. A variety of 3-acyl substituents were investigated, and the tetramethylcyclopropyl group was found to lead to high affinity CB(2) agonists (5, 16). Substitution at the N1-indole position was then examined. A series of aminoalkylindoles was prepared and several substituted aminoethyl derivatives were active (23-27, 5) at the CB(2) receptor. A study of N1 nonaromatic side chain variants provided potent agonists at the CB(2) receptor (16, 35-41, 44-47, 49-54, and 57-58). Several polar side chains (alcohols, oxazolidinone) were well-tolerated for CB(2) receptor activity (41, 50), while others (amide, acid) led to weaker or inactive compounds (55 and 56). N1 aromatic side chains also afforded several high affinity CB(2) receptor agonists (61, 63, 65, and 69) but were generally less potent in an in vitro CB(2) functional assay than were nonaromatic side chain analogues.

  13. 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.

  14. Vitamin K epoxide reductase: homology, active site and catalytic mechanism.

    PubMed

    Goodstadt, Leo; Ponting, Chris P

    2004-06-01

    Vitamin K epoxide reductase (VKOR) recycles reduced vitamin K, which is used subsequently as a co-factor in the gamma-carboxylation of glutamic acid residues in blood coagulation enzymes. VKORC1, a subunit of the VKOR complex, has recently been shown to possess this activity. Here, we show that VKORC1 is a member of a large family of predicted enzymes that are present in vertebrates, Drosophila, plants, bacteria and archaea. Four cysteine residues and one residue, which is either serine or threonine, are identified as likely active-site residues. In some plant and bacterial homologues the VKORC1 homologous domain is fused with domains of the thioredoxin family of oxidoreductases. These might reduce disulfide bonds of VKORC1-like enzymes as a prerequisite for their catalytic activities.

  15. CDK1 substitutes for mTOR kinase to activate mitotic cap-dependent protein translation

    PubMed Central

    Shuda, Masahiro; Velásquez, Celestino; Cheng, Erdong; Cordek, Daniel G.; Kwun, Hyun Jin; Chang, Yuan; Moore, Patrick S.

    2015-01-01

    Mitosis is commonly thought to be associated with reduced cap-dependent protein translation. Here we show an alternative control mechanism for maintaining cap-dependent translation during mitosis revealed by a viral oncoprotein, Merkel cell polyomavirus small T (MCV sT). We find MCV sT to be a promiscuous E3 ligase inhibitor targeting the anaphase-promoting complex, which increases cell mitogenesis. MCV sT binds through its Large T stabilization domain region to cell division cycle protein 20 (Cdc20) and, possibly, cdc20 homolog 1 (Cdh1) E3 ligase adapters. This activates cyclin-dependent kinase 1/cyclin B1 (CDK1/CYCB1) to directly hyperphosphorylate eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP1) at authentic sites, generating a mitosis-specific, mechanistic target of rapamycin (mTOR) inhibitor-resistant δ phospho-isoform not present in G1-arrested cells. Recombinant 4E-BP1 inhibits capped mRNA reticulocyte translation, which is partially reversed by CDK1/CYCB1 phosphorylation of 4E-BP1. eIF4G binding to the eIF4E–m7GTP cap complex is resistant to mTOR inhibition during mitosis but sensitive during interphase. Flow cytometry, with and without sT, reveals an orthogonal pH3S10+ mitotic cell population having higher inactive p4E-BP1T37/T46+ saturation levels than pH3S10– interphase cells. Using a Click-iT flow cytometric assay to directly measure mitotic protein synthesis, we find that most new protein synthesis during mitosis is cap-dependent, a result confirmed using the eIF4E/4G inhibitor drug 4E1RCat. For most cell lines tested, cap-dependent translation levels were generally similar between mitotic and interphase cells, and the majority of new mitotic protein synthesis was cap-dependent. These findings suggest that mitotic cap-dependent translation is generally sustained during mitosis by CDK1 phosphorylation of 4E-BP1 even under conditions of reduced mTOR signaling. PMID:25883264

  16. CDK1 substitutes for mTOR kinase to activate mitotic cap-dependent protein translation.

    PubMed

    Shuda, Masahiro; Velásquez, Celestino; Cheng, Erdong; Cordek, Daniel G; Kwun, Hyun Jin; Chang, Yuan; Moore, Patrick S

    2015-05-12

    Mitosis is commonly thought to be associated with reduced cap-dependent protein translation. Here we show an alternative control mechanism for maintaining cap-dependent translation during mitosis revealed by a viral oncoprotein, Merkel cell polyomavirus small T (MCV sT). We find MCV sT to be a promiscuous E3 ligase inhibitor targeting the anaphase-promoting complex, which increases cell mitogenesis. MCV sT binds through its Large T stabilization domain region to cell division cycle protein 20 (Cdc20) and, possibly, cdc20 homolog 1 (Cdh1) E3 ligase adapters. This activates cyclin-dependent kinase 1/cyclin B1 (CDK1/CYCB1) to directly hyperphosphorylate eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP1) at authentic sites, generating a mitosis-specific, mechanistic target of rapamycin (mTOR) inhibitor-resistant δ phospho-isoform not present in G1-arrested cells. Recombinant 4E-BP1 inhibits capped mRNA reticulocyte translation, which is partially reversed by CDK1/CYCB1 phosphorylation of 4E-BP1. eIF4G binding to the eIF4E-m(7)GTP cap complex is resistant to mTOR inhibition during mitosis but sensitive during interphase. Flow cytometry, with and without sT, reveals an orthogonal pH3(S10+) mitotic cell population having higher inactive p4E-BP1(T37/T46+) saturation levels than pH3(S10-) interphase cells. Using a Click-iT flow cytometric assay to directly measure mitotic protein synthesis, we find that most new protein synthesis during mitosis is cap-dependent, a result confirmed using the eIF4E/4G inhibitor drug 4E1RCat. For most cell lines tested, cap-dependent translation levels were generally similar between mitotic and interphase cells, and the majority of new mitotic protein synthesis was cap-dependent. These findings suggest that mitotic cap-dependent translation is generally sustained during mitosis by CDK1 phosphorylation of 4E-BP1 even under conditions of reduced mTOR signaling.

  17. Ser-substituted mutations of Cys residues in Bacillus thuringiensis Vip3Aa7 exert a negative effect on its insecticidal activity.

    PubMed

    Dong, Fang; Zhang, Shanshan; Shi, Ruiping; Yi, Shuyuan; Xu, Fangyan; Liu, Ziduo

    2012-11-01

    Vegetative insecticidal proteins (VIPs), which were produced by Bacillus thuringiensis during its vegetative growth stage, display a broad insecticidal spectrum to Lepidoptera larvae. Sequence alignment of the Vip3A-type indicates that three cysteine residues were conserved in Vip3A-type proteins. To determine whether these conserved cysteine residues contributed to the insecticidal activity, the three residues were respectively substituted with serine in the Vip3Aa7 protein by site-directed mutagenesis. Bioassays using the third instar larvae of Plutella xylostella showed that the toxicity of C401S and C507S mutants were completely abolished. To find out the inactivity reason of mutants, three mutants and the wild-type Vip3Aa7 were treated with trypsin. The results indicated that the C507S mutant was rapidly cleaved and resulted in decrease of the 62 kDa toxic core fragment. These results indicated that the replacement of the Cys(507) with a Ser(507) caused decrease in C507S resistance against trypsin degradation. It is suggesting a possible association between insecticidal activity and trypsin sensitivity of Vip3A proteins. This study serves a guideline for the study of Vip3A protein structure and active mechanism.

  18. Synthesis and antiviral activity of N9-[3-fluoro-2-(phosphonomethoxy)propyl] analogues derived from N6-substituted adenines and 2,6-diaminopurines.

    PubMed

    Baszczyňski, Ondřej; Jansa, Petr; Dračínský, Martin; Klepetářová, Blanka; Holý, Antonín; Votruba, Ivan; de Clercq, Erik; Balzarini, Jan; Janeba, Zlatko

    2011-04-01

    An efficient method for the synthesis of N(9)-[3-fluoro-2-(phosphonomethoxy)propyl] (FPMP) derivatives of purine bases has been developed. Both (R)- and (S)-enantiomers of the N(6)-substituted FPMP derivatives of adenine and 2,6-diaminopurine were prepared and their anti-human immunodeficiency virus (HIV) and anti-Moloney murine sarcoma virus (MSV) activity was evaluated. Whereas none of the 6-substituted FPMPA derivatives showed any antiviral activity, several FPMPDAP derivatives had a moderate antiretroviral activity. Moreover, the data obtained from the study of the substrate activity of the active derivatives towards N(6)-methyl-AMP aminohydrolase support the notion that the studied N(6)-substituted FPMPDAP derivatives act as prodrugs of the antiretroviral FPMPG analogues.

  19. Synthesis, Characterization and Antimicrobial Activity of d8-10 Metal Complexes of some 2-Substituted-1H-Benzimidazoles

    PubMed Central

    Tavman, Aydin; Ötük, Gülten

    1999-01-01

    The metal complexes of nine 2-substituted-1H-benzimidazoles (I-IX) with Ni(II), Pd(II), Cu(II), Ag(I), Zn(II) salts were synthesized. The compounds were characterized by melting point, analytical data, IR spectroscopy and magnetic susceptibility. The antimicrobial activity of the compounds was determined by the disk diffusion method in Mueller-Hinton Agar on Staphylococcus aureus ATCC 6538, Staphylococcus epidermidis ATCC 12228, Escherichia coli ATCC 8739, Klebsiella pneumoniae ATCC 4352, Pseudomonas aeruginosa ATCC 1539, Salmonella typhi, Shigella flexneri, Proteus mirabilis, Candida albicans ATCC 10231. Cu(II)and Ag(I)complexes of II, III and IV showed considerable activity against S. aureus, S. epidermidis, Ps. aeruginosa, S. typhi, Sh. flexneri and C. albicans microorganisms, the ligands themselves having no effect. PMID:18472907

  20. Biological Activity of sym-Triazines with Acetylcholine-like Substitutions as Multitarget Modulators of Alzheimer’s Disease

    PubMed Central

    2013-01-01

    The bioactivities of two novel compounds (TAE-1 and TAE-2) that contain a sym-triazine scaffold with acetylcholine-like substitutions are examined as promising candidate agents against Alzheimer’s disease. Inhibition of amyloid-β fibril formation in the presence of Aβ1–42, evaluated by Thioflavin T fluorescence, demonstrated comparable or improved activity to a previously reported pentapeptide-based fibrillogenesis inhibitor, iAβ5p. Destabilization of Aβ1–42 assemblies by TAE-1 and TAE-2 was confirmed by scanning electron microscopy imaging. sym-Triazine inhibition of acetylcholinesterase (AChE) activity was observed in cytosol extracted from differentiated human SH-SY5Y neuronal cells and also using human erythrocyte AChE. The sym-triazine derivatives were well tolerated by these cells and promoted beneficial effects on human neurons, upregulating expression of synaptophysin, a synaptic marker protein, and MAP2, a neuronal differentiation marker. PMID:23472585

  1. 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.

  2. Synthesis, spectral properties, and antitumor activity of a new axially substituted phthalocyanine complex of zirconium(IV) with citric acid.

    PubMed

    Tomachynski, Larisa A; Chernii, Victor Y; Gorbenko, Helena N; Filonenko, Valeriy V; Volkov, Sergey V

    2004-06-01

    The new axially substituted phthalocyanine (pc) complex of zirconium(IV) with citric acid is reported. It has been shown that the replacement of two Cl-atoms with two citric acid fragments takes place as the result of the reaction between [ZrCl2(pc)] and citric acid. The complex [Zr(citrate)2(pc)] was formed. The spectroscopic properties of the synthesized compound in DMSO, RPMI 1640 medium with and without fetal calf serum (FCS), H2O, and buffer (Tris) solutions have been described. Antitumor activity of this compound has been studied. The cytostatic activity was observed in the concentration range of 6.1-9.0x10(9) molecules [Zr(citrate)2(pc)]/cell and occurred in 4-6 h after treatment with [Zr(citrate)2(pc)] solution.

  3. Synthesis and biological evaluation of diaryl-substituted carboranes as inhibitors of hypoxia inducible factor (HIF)-1 transcriptional activity.

    PubMed

    Minegishi, Hidemitsu; Matsukawa, Takuya; Nakamura, Hiroyuki

    2013-02-01

    Diaromatic-substituted ortho- and meta-carboranes were synthesized as mimics of manassantin A. Among the carboranes synthesized, compounds 1 and 2 showed significant inhibition of hypoxia-induced HIF-1 transcriptional activity, with IC(50) values of 3.2 and 2.2 μM, respectively. Compounds 1 and 2 similarly suppressed hypoxia-induced HIF-1α accumulation in a concentration-dependent manner without affecting the expression level of HIF-1α mRNA. The hypoxia-induced accumulation and translocation of HIF-1α into nuclei were not observed in HeLa cells treated with compounds 1 and 2 by immunofluorescence analysis, revealing that the inhibition of hypoxia-induced HIF-1 transcriptional activity is induced by compounds 1 and 2 through a degradation pathway of the HIF-1α protein under hypoxic conditions.

  4. Identification of covalent active site inhibitors of dengue virus protease

    PubMed Central

    Koh-Stenta, Xiaoying; Joy, Joma; Wang, Si Fang; Kwek, Perlyn Zekui; Wee, John Liang Kuan; Wan, Kah Fei; Gayen, Shovanlal; Chen, Angela Shuyi; Kang, CongBao; Lee, May Ann; Poulsen, Anders; Vasudevan, Subhash G; Hill, Jeffrey; Nacro, Kassoum

    2015-01-01

    Dengue virus (DENV) protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described. PMID:26677315

  5. Bone graft substitutes.

    PubMed

    Bhatt, Reena A; Rozental, Tamara D

    2012-11-01

    Replacement of missing bone stock is a reconstructive challenge to upper extremity surgeons and decision-making with regards to available choices remains difficult. Preference is often given to autograft in the form of cancellous, cortical, or corticocancellous grafts from donor sites. However, the available volume from such donor sites is limited and fraught with potential complications. Advances in surgical management and medical research have produced a wide array of potential substances that can be used for bone graft substitute. Considerations in selecting bone grafts and substitutes include characteristic capabilities, availability, patient morbidity, immunogenicity, potential disease transmission, and cost variability.

  6. Effects of isoelectronic Ru substitution at the Fe site on the energy gaps of optimally F-doped SmFeAsO

    NASA Astrophysics Data System (ADS)

    Daghero, D.; Tortello, M.; Ummarino, G. A.; Stepanov, V. A.; Bernardini, F.; Tropeano, M.; Putti, M.; Gonnelli, R. S.

    2012-08-01

    We studied the effects of isoelectronic Ru substitution at the Fe site on the energy gaps of optimally F-doped SmFeAsO by means of point-contact Andreev-reflection spectroscopy. The results show that the SmFe1-xRuxAsO0.85F0.15 system keeps a multigap character at least up to x = 0.50, and that the gap amplitudes Δ1 and Δ2 scale almost linearly with the local critical temperature {T}_{{c}}^{A}. The gap ratios 2Δi/kBTc remain approximately constant only as long as Tc ≥ 30 K, and increase dramatically when Tc decreases further. This trend seems to be common to many Fe-based superconductors, irrespective of their family. Based on first-principle calculations of the bandstructure and of the density of states projected on the different bands, we show that this trend, as well as the Tc dependence of the gaps and the reduction of Tc upon Ru doping, can be explained within an effective three-band Eliashberg model as being due to a suppression of the superfluid density at finite temperature that, in turn, modifies the temperature dependence of the characteristic spin-fluctuation energy.

  7. 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.

  8. Synthesis and structure-activity relationships of 2-substituted-6-(dimethylamino)-9-(4-methylbenzyl)-9H-purines with antirhinovirus activity.

    PubMed

    Kelley, J L; Linn, J A; Selway, J W

    1989-01-01

    A series of 2-substituted-6-(dimethylamino)-9-(4-methylbenzyl)-9H-purines where the 2-substituent was H, F, Cl, CF3, CH3, CH2CH3, NH2, NHCH3, N(CH3)2, SCH3, or SO2CH3 was synthesized and tested for antirhinovirus activity to evaluate the effect of 2-substituents on antiviral activity. Intuitive and quantitative structure-activity relationship (QSAR) analysis showed that optimum antirhinovirus serotype 1B activity was associated with 9-benzylpurines that contained a C-2 lipophilic, electron-withdrawing substituent. The most active compound, 6-(dimethylamino)-9-(4-methylbenzyl)-2-(trifluoromethyl)-9H-purine (14), had an IC50 = 0.03 microM against serotype 1B, but its activity against 18 other serotypes was not uniform; the IC50s ranged over 260-fold.

  9. MUTATION OF THE ACTIVE SITE CARBOXY-LYSINE (K70) OF OXA-1 β-LACTAMASE RESULTS IN A DEACYLATION-DEFICIENT ENZYME†

    PubMed Central

    Schneider, Kyle D.; Bethel, Christopher R.; Distler, Anne M.; Hujer, Andrea M.; Bonomo, Robert A.; Leonard, David A.

    2009-01-01

    Class D β-lactamases hydrolyze β-lactam antibiotics by using an active site serine nucleophile to form a covalent acyl-enzyme intermediate, and subsequently employ water to deacylate the β-lactam and release product. Class D β-lactamases are carboxylated on the ε-amino group of an active site lysine, with the resulting carbamate functional group serving as a general base. We discovered that substitutions of the active site serine and lysine in OXA-1 β-lactamase, a monomeric class D enzyme, significantly disrupt catalytic turnover. Substitution of glycine for the nucleophilic serine (S67G) results in an enzyme that can still bind substrate but is unable to form a covalent acyl-enzyme intermediate. Substitution of the carboxylated lysine (K70), on the other hand, results in enzyme that can be acylated by substrate, but is impaired for deacylation. We employed the fluorescent penicillin BOCILLIN FL™ to show that three different substitutions for K70 (alanine, aspartate and glutamate) accumulate significant acyl-enzyme intermediate. Interestingly, BOCILLIN FL™ deacylation rates vary depending on the identity of the substituting residue, from t1/2 ≈ 60 min for K70A to undetectable deacylation for K70D. Tryptophan fluorescence spectroscopy was used to confirm that these results are applicable to natural (i.e. non-fluorescent) substrates. Deacylation by K70A, but not K70D or K70E, can be partially restored by the addition of short-chain carboxylic acid mimetics of the lysine carbamate. In conclusion, we establish the functional role of the carboxylated lysine in OXA-1 and highlight its specific role in acylation and deacylation. PMID:19485421

  10. 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.

  11. 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.

  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. Pyridinium substituted tetraphenylethylene entailing alkyne moiety: enhancement of photosensitizing efficiency and antimicrobial activity.

    PubMed

    You, Xue; Ma, Huili; Wang, Yuancheng; Zhang, Guanxin; Peng, Qian; Liu, Libing; Wang, Shu; Zhang, Deqing

    2017-03-14

    Apart from sensing and imaging, luminogens with aggregation-induced emission are also interesting for photosensitizing. In this paper, we report the photosensitizing behavior and bacteria-killing performance of a new pyridinium substituted tetraphenylethylene with an alkyne group (TPE-A-Py+). Interestingly, TPE-A-Py+ exhibits higher photosensitizing efficiency than TPE-Py+ (without alkyne group) with I- as the counter anion. This is well explained by the fact that the energy gap between the excited singlet state (S1) and triplet state (T1) is lower for TPE-A-Py+ in comparison with that of TPE-Py+ on the basis of theoretical calculations. Moreover, the replacement of I- with other anions (PF6-, N(SO2CF3)2- and BPh4-) leads to the decrease of photosensitizing efficiency for TPE-A-Py+. Notably, TPE-A-Py+ can be utilized as an efficient photosensitizer to photo-inactivate Ampr E. coli at low concentration under white light irradiation for just a few minutes.

  14. Target-classification approach applied to active UXO sites

    NASA Astrophysics Data System (ADS)

    Shubitidze, F.; Fernández, J. P.; Shamatava, Irma; Barrowes, B. E.; O'Neill, K.

    2013-06-01

    This study is designed to illustrate the discrimination performance at two UXO active sites (Oklahoma's Fort Sill and the Massachusetts Military Reservation) of a set of advanced electromagnetic induction (EMI) inversion/discrimination models which include the orthonormalized volume magnetic source (ONVMS), joint diagonalization (JD), and differential evolution (DE) approaches and whose power and flexibility greatly exceed those of the simple dipole model. The Fort Sill site is highly contaminated by a mix of the following types of munitions: 37-mm target practice tracers, 60-mm illumination mortars, 75-mm and 4.5'' projectiles, 3.5'', 2.36'', and LAAW rockets, antitank mine fuzes with and without hex nuts, practice MK2 and M67 grenades, 2.5'' ballistic windshields, M2A1-mines with/without bases, M19-14 time fuzes, and 40-mm practice grenades with/without cartridges. The site at the MMR site contains targets of yet different sizes. In this work we apply our models to EMI data collected using the MetalMapper (MM) and 2 × 2 TEMTADS sensors. The data for each anomaly are inverted to extract estimates of the extrinsic and intrinsic parameters associated with each buried target. (The latter include the total volume magnetic source or NVMS, which relates to size, shape, and material properties; the former includes location, depth, and orientation). The estimated intrinsic parameters are then used for classification performed via library matching and the use of statistical classification algorithms; this process yielded prioritized dig-lists that were submitted to the Institute for Defense Analyses (IDA) for independent scoring. The models' classification performance is illustrated and assessed based on these independent evaluations.

  15. Design, synthesis, crystal structure, insecticidal activity, molecular docking, and QSAR studies of novel N3-substituted imidacloprid derivatives.

    PubMed

    Wang, Mei-Juan; Zhao, Xiao-Bo; Wu, Dan; Liu, Ying-Qian; Zhang, Yan; Nan, Xiang; Liu, Huanxiang; Yu, Hai-Tao; Hu, Guan-Fang; Yan, Li-Ting

    2014-06-18

    Three novel series of N3-substituted imidacloprid derivatives were designed and synthesized, and their structures were identified on the basis of satisfactory analytical and spectral ((1)H NMR, (13)C NMR, MS, elemental analysis, and X-ray) data. Preliminary bioassays indicated that all of the derivatives exhibited significant insecticidal activities against Aphis craccivora, with LC50 values ranging from 0.00895 to 0.49947 mmol/L, and the insecticidal activities of some of them were comparable to those of the control imidacloprid. Some key structural features related to their insecticidal activities were identified, and the binding modes between target compounds and nAChR model were also further explored by molecular docking. By comparing the interaction features of imidacloprid and compound 26 with highest insecticidal activity, the origin of the high insecticidal activity of compound 26 was identified. On the basis of the conformations generated by molecular docking, a satisfactory 2D-QSAR model with six selected descriptors was built using genetic algorithm-multiple linear regression (GA-MLR) method. The analysis of the built model showed the molecular size, shape, and the ability to form hydrogen bond were important for insecticidal potency. The information obtained in the study will be very helpful for the design of new derivatives with high insecticidal activities.

  16. Identification of Phosphorylation Sites Altering Pollen Soluble Inorganic Pyrophosphatase Activity.

    PubMed

    Eaves, Deborah J; Haque, Tamanna; Tudor, Richard L; Barron, Yoshimi; Zampronio, Cleidiane G; Cotton, Nicholas P J; de Graaf, Barend H J; White, Scott A; Cooper, Helen J; Franklin, F Christopher H; Harper, Jeffery F; Franklin-Tong, Vernonica E

    2017-03-01

    Protein phosphorylation regulates numerous cellular processes. Identifying the substrates and protein kinases involved is vital to understand how these important posttranslational modifications modulate biological function in eukaryotic cells. Pyrophosphatases catalyze the hydrolysis of inorganic phosphate (PPi) to inorganic phosphate Pi, driving biosynthetic reactions; they are essential for low cytosolic inorganic phosphate. It was suggested recently that posttranslational regulation of Family I soluble inorganic pyrophosphatases (sPPases) may affect their activity. We previously demonstrated that two pollen-expressed sPPases, Pr-p26.1a and Pr-p26.1b, from the flowering plant Papaver rhoeas were inhibited by phosphorylation. Despite the potential significance, there is a paucity of data on sPPase phosphorylation and regulation. Here, we used liquid chromatographic tandem mass spectrometry to map phosphorylation sites to the otherwise divergent amino-terminal extensions on these pollen sPPases. Despite the absence of reports in the literature on mapping phosphorylation sites on sPPases, a database survey of various proteomes identified a number of examples, suggesting that phosphorylation may be a more widely used mechanism to regulate these enzymes. Phosphomimetic mutants of Pr-p26.1a/b significantly and differentially reduced PPase activities by up to 2.5-fold at pH 6.8 and 52% in the presence of Ca(2+) and hydrogen peroxide over unmodified proteins. This indicates that phosphoregulation of key sites can inhibit the catalytic responsiveness of these proteins in concert with key intracellular events. As sPPases are essential for many metabolic pathways in eukaryotic cells, our findings identify the phosphorylation of sPPases as a potential master regulatory mechanism that could be used to attenuate metabolism.

  17. Evidence for segmental mobility in the active site of pepsin

    SciTech Connect

    Pohl, J.; Strop, P.; Senn, H.; Foundling, S.; Kostka, V.

    1986-05-01

    The low hydrolytic activity (k/sub cat/ < 0.001 s/sup -1/) of chicken pepsin (CP) towards tri- and tetrapeptides is enhanced at least 100 times by modification of its single sulfhydryl group of Cys-115, with little effect on K/sub m/-values. Modification thus simulates the effect of secondary substrate binding on pepsin catalysis. The rate of Cys-115 modification is substantially decreased in the presence of some competitive inhibitors, suggesting its active site location. Experiments with CP alkylated at Cys-115 with Acrylodan as a fluorescent probe or with N-iodoacetyl-(4-fluoro)-aniline as a /sup 19/F-nmr probe suggest conformation change around Cys-115 to occur on substrate or substrate analog binding. The difference /sup 1/H-nmr spectra (500 MHz) of unmodified free and inhibitor-complexed CP reveal chemical shifts almost exclusively in the aromatic region. The effects of Cu/sup + +/ on /sup 19/F- and /sup 1/H-nmr spectra have been studied. Examination of a computer graphics model of CP based on E. parasitica pepsin-inhibitor complex X-ray coordinates suggests that Cys-115 is located near the S/sub 3//S/sub 5/ binding site. The results are interpreted in favor of segmental mobility of this region important for pepsin substrate binding and catalysis.

  18. First Principles Computational Study of the Active Site of Arginase

    SciTech Connect

    Ivanov, Ivaylo; Klien, Micheal

    2004-01-14

    Ab initio density functional theory (DFT) methods were used to investigate the structural features of the active site of the binuclear enzyme rat liver arginase. Special emphasis was placed on the crucial role of the second shell ligand interactions. These interactions were systematically studied by performing calculations on models of varying size. It was determined that a water molecule, and not hydroxide, is the bridging exogenous ligand. The carboxylate ligands facilitate the close approach of the Mn (II) ions by attenuating the metal-metal electrostatic repulsion. Of the two metals, MnA was shown to carry a larger positive charge. Analysis of the electronic properties of the active site revealed that orbitals involving the terminal Asp234 residue, as well as the flexible -1,1 bridging Asp232, lie at high energies, suggesting weaker coordination. This is reflected in certain structural variability present in our models and is also consistent with recent experimental findings. Finally, implications of our findings for the biological function of the enzyme are delineated.

  19. Site-directed mutagenesis of human beta-adrenergic receptors: substitution of aspartic acid-130 by asparagine produces a receptor with high-affinity agonist binding that is uncoupled from adenylate cyclase.

    PubMed Central

    Fraser, C M; Chung, F Z; Wang, C D; Venter, J C

    1988-01-01

    By using oligonucleotide-directed mutagenesis, we have produced a point mutation (guanine to adenine) at nucleotide 388 of the gene for human beta-adrenergic receptor (beta AR) that results in a substitution of asparagine for the highly conserved aspartic acid at position 130 in the putative third transmembrane domain of the human beta AR ([Asn130]beta AR). We have examined the functional significance of this mutation in B-82 cells continuously expressing the mutant [Asn130]beta AR. The mutant [Asn130]beta AR displayed normal antagonist binding but unusually high-affinity agonist binding (5- to 10-fold higher than wild-type beta AR), consistent with a single class of high-affinity binding sites. The mutant beta AR displayed guanine nucleotide-sensitive changes in agonist affinity (3- to 5-fold shift) implying an interaction between the beta AR and the stimulatory guanine nucleotide-binding regulatory protein; however, the ability of guanine nucleotides to alter agonist affinity was attenuated. Addition of saturating concentrations of isoproterenol to cell cultures expressing mutant [Asn130]-beta ARs had no effect on intracellular levels of cAMP, indicating that the mutant beta AR is unable to affect stimulation of adenylate cyclase. These results indicate that substitution of the aspartic acid with asparagine at residue 130 of the human beta AR dissociates the well-characterized guanine nucleotide effects on agonist affinity from those on activation of the stimulatory guanine nucleotide-binding regulatory protein and adenylate cyclase and suggests the existence of two distinct counterions for the amine portion of catecholamines that are associated with high- and low-affinity agonist binding states of beta AR. Images PMID:2840663

  20. 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.

  1. C-H Activation on Co,O Sites: Isolated Surface Sites versus Molecular Analogs.

    PubMed

    Estes, Deven P; Siddiqi, Georges; Allouche, Florian; Kovtunov, Kirill V; Safonova, Olga V; Trigub, Alexander L; Koptyug, Igor V; Copéret, Christophe

    2016-11-16

    The activation and conversion of hydrocarbons is one of the most important challenges in chemistry. Transition-metal ions (V, Cr, Fe, Co, etc.) isolated on silica surfaces are known to catalyze such processes. The mechanisms of these processes are currently unknown but are thought to involve C-H activation as the rate-determining step. Here, we synthesize well-defined Co(II) ions on a silica surface using a metal siloxide precursor followed by thermal treatment under vacuum at 500 °C. We show that these isolated Co(II) sites are catalysts for a number of hydrocarbon conversion reactions, such as the dehydrogenation of propane, the hydrogenation of propene, and the trimerization of terminal alkynes. We then investigate the mechanisms of these processes using kinetics, kinetic isotope effects, isotopic labeling experiments, parahydrogen induced polarization (PHIP) NMR, and comparison with a molecular analog. The data are consistent with all of these reactions occurring by a common mechanism, involving heterolytic C-H or H-H activation via a 1,2 addition across a Co-O bond.

  2. 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

  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. Structure-activity relation of human beta-defensin 3: influence of disulfide bonds and cysteine substitution on antimicrobial activity and cytotoxicity.

    PubMed

    Klüver, Enno; Schulz-Maronde, Sandra; Scheid, Svenja; Meyer, Bernd; Forssmann, Wolf-Georg; Adermann, Knut

    2005-07-19

    Human beta-defensins form a group of cysteine-rich antimicrobial peptides which have been found in epithelial tissue and, more recently, in the male genital tract. They play a role in the defense against microbial pathogens in innate immunity and display additional chemotactic functions in the adaptive immune system. An important characteristic of antimicrobial peptides is that they also exhibit toxic potential on eukaryotic cells. Very little is known about the structure dependence of antimicrobial and cytotoxic effects. We investigated human beta-defensin 3 (hBD-3), a potent broad-spectrum antimicrobial effector peptide, regarding the influence of structural parameters on the antimicrobial and cytotoxic activity. We have established a structure-activity relation of the hBD-3 using synthetic derivatives differing in length, charge, disulfide connectivity, and overall hydrophobicity. The antimicrobial activity of the peptides was compared to the cyctotoxic effects on monocytic THP-1 cells and the hemolytic activity on human erythrocytes. We found that it is not important for antimicrobial and cytotoxic activity whether and how cysteine residues are arranged to form disulfide bonds. Substitution of half-cystinyl residues by tryptophan resulted in increased activities, while other substitutions did not change activity. Correlation of activities with the structural changes demonstrates that the activity on eukaryotic cells appears to depend strongly on the overall hydrophobicity. In contrast, the antimicrobial potency of hBD-3 peptides is determined by the distribution of positively charged amino acid residues and hydrophobic side chains. The results facilitate the understanding of beta-defensin interaction with different cell types and guide the design of antimicrobially active peptides.

  5. Mixed conductivity, thermochemical expansion and electrochemical activity of Fe-substituted (La,Sr)(Cr,Mg)O3-δ for solid oxide fuel cell anodes

    NASA Astrophysics Data System (ADS)

    Yaremchenko, A. A.; Kharton, V. V.; Kolotygin, V. A.; Patrakeev, M. V.; Tsipis, E. V.; Waerenborgh, J. C.

    2014-03-01

    The effect of iron substitution in perovskite-type (La0.9Sr0.1)0.98Cr0.9-xFexMg0.1O3-δ (x = 0-0.3) is evaluated with emphasis on the properties relevant for solid oxide fuel cell anode application including the phase stability, oxygen nonstoichiometry, mixed ionic-electronic transport, thermochemical expansion and electrochemical activity. Thermogravimetric analysis, Mössbauer spectroscopy and electrical measurements in combination with X-ray diffraction confirm the stability of perovskite phase for x = 0.3 down to p(O2) as low as 10-19 atm at 1223 K. Mössbauer spectroscopy results indicate also that iron cations substitute in 3+ oxidation state in both oxidized and reduced material. The total conductivity is predominantly p-type electronic, with negligible contribution of ionic transport under oxidizing conditions. Substitution with iron decreases electronic transport, but also leads to higher oxygen deficiency and ionic conductivity under reducing conditions. The oxygen nonstoichiometry variations, determined by coulometric titration, and defect chemistry of (La0.9Sr0.1)0.98Cr0.6Fe0.3Mg0.1O3-δ can be described by non-ideal solution model and site-exclusion effects. The materials exhibit moderate thermal expansion coefficients (10.1-11.5) × 10-6 K-1 in air, nearly independent of iron content and p(O2), and favorably small chemical expansion on reduction. Porous (La0.9Sr0.1)0.98Cr0.6Fe0.3Mg0.1O3-δ anodes applied onto LaGaO3-based solid electrolyte with thin Ce0.8Gd0.2O2-δ interlayers show a better electrochemical performance compared to (La0.75Sr0.25)0.95Cr0.5Mn0.5O3-δ under identical conditions.

  6. 1,2-substituted 4-(1H)-quinolones: synthesis, antimalarial and antitrypanosomal activities in vitro.

    PubMed

    Wube, Abraham; Hüfner, Antje; Seebacher, Werner; Kaiser, Marcel; Brun, Reto; Bauer, Rudolf; Bucar, Franz

    2014-09-10

    A diverse array of 4-(1H)-quinolone derivatives bearing substituents at positions 1 and 2 were synthesized and evaluated for antiprotozoal activities against Plasmodium falciparum and Trypanosoma brucei rhodesiense, and cytotoxicity against L-6 cells in vitro. Furthermore, selectivity indices were also determined for both parasites. All compounds tested showed antimalarial activity at low micromolar concentrations, with varied degrees of selectivity against L-6 cells. Compound 5a was found to be the most active against P. falciparum, with an IC50 value of 90 nM and good selectivity for the malarial parasite compared to the L-6 cells. Compound 10a, on the other hand, showed a strong antitrypanosomal effect with an IC50 value of 1.25 µM. In this study side chain diversity was explored by varying the side chain length and substitution pattern on the aliphatic group at position-2 and a structure-antiprotozoal activity study revealed that the aromatic ring introduced at C-2 contributed significantly to the antiprotozoal activities.

  7. In vitro cytotoxic, antibacterial, antifungal and urease inhibitory activities of some N4- substituted isatin-3-thiosemicarbazones.

    PubMed

    Pervez, Humayun; Iqbal, Mohammad S; Tahir, Muhammad Younas; Nasim, Faiz-ul-Hassan; Choudhary, Muhammad Iqbal; Khan, Khalid Mohammed

    2008-12-01

    A series of 15 previously reported N(4)-substituted isatin-3-thiosemicarbazones 3a-o has been screened for cytotoxic, antibacterial, antifungal and urease inhibitory activities. Compounds 3b, 3e and 3n proved to be active in cytotoxicity assay; 3e exhibited a high degree of cytotoxic activity (LD(50) = 1.10 x 10(-5) M). Compound 3h exhibited significant antibacterial activity against B. subtilis, whereas compounds 3a, 3k and 3l displayed significant antifungal activity against one or more fungal strains i.e. T. longifusus, A. flavus and M. canis. In human urease enzyme inhibition assay, compounds 3g, 3k and 3m proved to be the most potent inhibitors, exhibiting relatively pronounced inhibition of the enzyme. These compounds, being non-toxic, could be potential candidates for orally effective therapeutic agents to treat certain clinical conditions induced by bacterial ureases like H. pylori urease. This study presents the first example of inhibition of urease by isatin-thiosemicarbazones and as such provides a solid basis for further research on such compounds to develop more potent inhibitors.

  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. 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.

  10. 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.

  11. An active site water network in the plasminogen activator pla from Yersinia pestis.

    PubMed

    Eren, Elif; Murphy, Megan; Goguen, Jon; van den Berg, Bert

    2010-07-14

    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 A. The structure shows a water molecule located between active site residues D84 and H208, which likely corresponds to the nucleophilic water. A number of other water molecules are present in the active site, linking residues important for enzymatic activity. The R211 sidechain in loop L4 is close to the nucleophilic water and possibly involved in the stabilization of the oxyanion intermediate. Subtle conformational changes of H208 result from the binding of lipopolysaccharide to the outside of the barrel, explaining the unusual dependence of omptins on lipopolysaccharide for activity. The Pla structure suggests a model for the interaction with plasminogen substrate and provides a more detailed understanding of the catalytic mechanism of omptin proteases.

  12. In vivo knee kinematics during stair and deep flexion activities in patients with bicruciate substituting total knee arthroplasty.

    PubMed

    Kuroyanagi, Yuji; Mu, Shang; Hamai, Satoshi; Robb, William J; Banks, Scott A

    2012-01-01

    Orthopedic surgeons and their patients continue to seek better functional outcomes after total knee arthroplasty. The bicruciate substituting (BCS) total knee arthroplasty design has been introduced to achieve more natural knee mechanics. The purpose of this study was to characterize kinematics in knees with BCS arthroplasty during deep flexion and stair activities using fluoroscopy and model-image registration. In 20 patients with 25 BCS knees, we observed average implant flexion of 128° during kneeling and consistent posterior condylar translations with knee flexion. Tibial rotations were qualitatively similar to those observed in the arthritic natural knee. Knee kinematics with BCS arthroplasty were qualitatively more similar to arthritic natural knees than knees with either posterior cruciate-retaining or posterior-stabilized arthroplasty.

  13. 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.

  14. 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…

  15. 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

  16. 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.

  17. Removal potential of toxic 2378-substituted PCDD/F from incinerator flue gases by waste-derived activated carbons.

    PubMed

    Hajizadeh, Yaghoub; Onwudili, Jude A; Williams, Paul T

    2011-06-01

    The application of activated carbons has become a commonly used emission control protocol for the removal or adsorption of persistent organic pollutants from the flue gas streams of waste incinerators. In this study, the 2378-substituted PCDD/F removal efficiency of three types of activated carbons derived from the pyrolysis of refuse derived fuel, textile waste and scrap tyre was investigated and compared with that of a commercial carbon. Experiments were carried out in a laboratory scale fixed-bed reactor under a simulated flue gas at 275°C with a reaction period of four days. The PCDD/F in the solid matrices and exhaust gas, were analyzed using gas chromatography coupled with a triple quadrupole mass spectrometer. In the absence of activated carbon adsorbent, there was a significant increase in the concentration of toxic PCDD/F produced in the reacted flyash, reaching up to 6.6 times higher than in the raw flyash. In addition, there was a substantial release of PCDD/F into the gas phase, which was found in the flue gas trapping system. By application of the different commercial, refuse derived fuel, textile and tyre activated carbons the total PCDD/F toxic equivalent removal efficiencies in the exhaust gas stream were 58%, 57%, 64% and 52%, respectively. In general, the removal of the PCDDs was much higher with an average of 85% compared to PCDFs at 41%. Analysis of the reacted activated carbons showed that there was some formation of PCDD/F, for instance, a total of 60.6 μg I-TEQ kg(-1) toxic PCDD/F was formed in the refuse derived fuel activated carbon compared to 34 μg I-TEQ kg(-1) in the commercial activated carbon. The activated carbons derived from the pyrolysis of waste, therefore, showed good potential as a control material for PCDD/F emissions in waste incinerator flue gases.

  18. 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.

  19. 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

  20. Evaluation of Substituted N,N′-Diarylsulfonamides as Activators of the Tumor Cell Specific M2 Isoform of Pyruvate Kinase

    PubMed Central

    Boxer, Matthew B.; Jiang, Jian-kang; Vander Heiden, Matthew G.; Shen, Min; Skoumbourdis, Amanda P.; Southall, Noel; Veith, Henrike; Leister, William; Austin, Christopher P.; Park, Hee Won; Inglese, James; Cantley, Lewis C.; Auld, Douglas S.; Thomas, Craig J.

    2009-01-01

    The metabolism of cancer cells is altered to support rapid proliferation. Pharmacological activators of a tumor cell specific pyruvate kinase isozyme (PKM2) may be an approach for altering the classic Warburg effect characteristic of aberrant metabolism in cancer cells yielding a novel anti-proliferation strategy. In this manuscript we detail the discovery of a series of substituted N,N′-diarylsulfonamides as activators of PKM2. The synthesis of numerous analogues and the evaluation of structure activity relationships are presented as well as assessments of mechanism and selectivity. Several agents are found that have good potencies and appropriate solubility for use as chemical probes of PKM2 including 55 (AC50 = 43 nM, maximum response = 84%; solubility = 7.3 μg/mL), 56 (AC50 = 99 nM, maximum response = 84%; solubility = 5.7 μg/mL) and 58 (AC50 = 38 nM, maximum response = 82%; solubility = 51.2 μg/mL). The small molecules described here represent first-in-class activators of PKM2 PMID:20017496

  1. Effect of Erbium substitution on temperature and field dependence of thermally activated flux flow resistance in Bi-2212 superconductor

    NASA Astrophysics Data System (ADS)

    Paladhi, D.; Mandal, P.; Sahoo, R. C.; Giri, S. K.; Nath, T. K.

    2016-12-01

    Thermally activated flux flow (TAFF) regime of Er doped Bi2Sr2Ca1-xErxCu2O8+δ (x=0.0, 0.1, 0.3) polycrystalline systems have been investigated using magneto-transport measurements up to 70 kOe magnetic field. High quality single phase samples have been prepared by standard solid state reaction method. The activation energy or pinning strength (U0) have been calculated using thermally activated flux flow (TAFF) model by linear fitting from the semi-logarithmic curve of ln ρ vs 1/T. It has been observed that activation energy (U0) decreases with Er substitution and U0 follows power law dependence with magnetic field for all three samples. Irreversibility lines (IL) have been drawn from the magneto-transport data for all three samples and it is observed that IL shifts to lower temperature with higher Er concentration. It is confirmed from the above results that pinning strength becomes weaker with Er doping. Finally, the variation of U0 have been shown with temperature by re-plotting -T(ln (ρ/ρ100)) vs T for three samples showing non-linear dependence with temperature.

  2. 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

  3. 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

  4. Design, synthesis and cytotoxic activities of novel aliphatic amino-substituted flavonoids.

    PubMed

    Liu, Guannan; Ge, Zhen; Zhao, Mengdan; Zhou, Yifeng

    2013-11-13

    A series of flavonoids 9a-f, 13b, 13d, 13e and 14a-f bearing diverse aliphatic amino moieties were designed, synthesized and evaluated for their cytotoxic activities against the ECA-109, A-549, HL-60, and PC-3 cancer cell lines. Most of the compounds exhibited moderate to good activities. The structure-activity relationships were studied, revealing that the chalcone skeleton is the most preferable for cytotoxic activities. Chalcone 9d was the most promising compound due to its high potency against the examined cancer cell lines (its IC₅₀ values against ECA-109, A549, HL-60 and PC-3 cells were 1.0, 1.5, 0.96 and 3.9 μM, respectively).

  5. Design, synthesis and pharmacophoric model building of novel substituted nicotinic acid hydrazones with potential antiproliferative activity.

    PubMed

    Abdel-Aziz, Hatem A; Aboul-Fadl, Tarek; Al-Obaid, Abdul-Rahman M; Ghazzali, Mohamed; Al-Dhfyan, Abdullah; Contini, Alessandro

    2012-09-01

    Novel 6-aryl-2-methylnicotinic acid hydrazides 4a-c and their corresponding hydrazones 5a-c and 6a-i were synthesized. X-ray single crystal diffraction of 6h confirmed the chemical structure of hydrazones 6a-i. Antiproliferative activity of the synthetic compounds was investigated against K562 leukemia cell lines. Variable cell growth inhibitory activities were obtained with IC₅₀ range from 24.99 to 66.78 μM where the compound 6c exhibited the maximum activity. Structure activity relationship analysis has been performed and a common pharmacophore model for the synthesized derivatives has been obtained by using the pharmacophore elucidation module of the software MOE. The best model obtained is characterized by two projected locations of potential H-bond donors (F 3 and F4) and two Aromatic annotations (F1 and F2).

  6. Structural Characterization of Mutations at the Oxygen Activation Site in Monomeric Sarcosine Oxidase

    SciTech Connect

    Schuman Jorns, Marilyn; Chen, Zhi-wei; Mathews, F. Scott

    2010-04-30

    Oxygen reduction and sarcosine oxidation in monomeric sarcosine oxidase (MSOX) occur at separate sites above the si- and re-faces, respectively, of the flavin ring. Mutagenesis studies implicate Lys265 as the oxygen activation site. Substitution of Lys265 with a neutral (Met, Gln, or Ala) or basic (Arg) residue results in an {approx}10{sup 4}- or 250-fold decrease, respectively, in the reaction rate. The overall structure of MSOX and residue conformation in the sarcosine binding cavity are unaffected by replacement of Lys265 with Met or Arg. The side chain of Met265 exhibits the same configuration in each molecule of Lys265Met crystals and is nearly congruent with Lys265 in wild-type MSOX. The side chain of Arg265 is, however, dramatically shifted (4-5 {angstrom}) compared with Lys265, points in the opposite direction, and exhibits significant conformational variability between molecules of the same crystal. The major species in solutions of Lys265Arg is likely to contain a 'flipped-out' Arg265 and exhibit negligible oxygen activation, similar to Lys265Met. The 400-fold higher oxygen reactivity observed with Lys265Arg is attributed to a minor (<1%) 'flipped-in' Arg265 conformer whose oxygen reactivity is similar to that of wild-type MSOX. A structural water (WAT1), found above the si-face of the flavin ring in all previously determined MSOX structures, is part of an apparent proton relay system that extends from FAD N(5) to bulk solvent. WAT1 is strikingly absent in Lys265Met and Lys265Arg, a feature that may account for the apparent kinetic stabilization of a reductive half-reaction intermediate that is detectable with the mutants but not wild-type MSOX.

  7. 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.

  8. Some recent approaches of biologically active substituted pyridazine and phthalazine drugs.

    PubMed

    Asif, M

    2012-01-01

    Nitrogen atom containing heterocyclic compounds, pyridazines, pyridazinones and phthalazines are important structural feature of many biologically active compounds and show diverse pharmacological properties. Pyridazines and phthalazines hold considerable interest relative to the preparation of organic intermediates and physiologically active compounds. On the basis of literature, pyridazines, pyridazinone and phthalazines further focus our attention because of their easy fictionalization at various ring positions, which makes them attractive synthetic compounds for designing and development of the novel pyridazines and phthalazines drugs in future.

  9. Auxin Activity of Substituted Benzoic Acids and Their Effect on Polar Auxin Transport 1

    PubMed Central

    Keitt, George W.; Baker, Robert A.

    1966-01-01

    Six dichloro-, 3 trichloro-, 2 triiodo-, and 3 heterosubstituted benzoic acids (amiben, dinoben, dicamba), and N-1-naphthylphthalamic acid have been tested for effects on growth and on polar auxin transport. Growth activity with and without kinetin was measured by effects on fresh and dry weights of 30-day cultures of fresh tobacco pith. Transport inhibition was measured by following uptake and output of IAA-2-14C through 10 mm bean epicotyl sections. The distribution of callus growth on vascularized tobacco stem segments was also observed. Avena first internode extension assays established the relative activities: dicamba > amiben > dinoben suggested by pith growth results. Growth effects of active compounds were similar with and without kinetin, except that amiben was less active with kinetin, while 2,3,6-trichlorobenzoic acid was more active with kinetin than alone. The weak auxin activity of NPA was confirmed. Transport experiments showed that NPA was the most inhibitory compound tested, followed by TIBA. Other compounds tested were at least 300 times less inhibitory to IAA transport. The best growth promoters were the least inhibitory to transport, and the most effective transport inhibitors were at best poor auxins. It is suggested that the weak auxin and auxin synergistic activity of TIBA (and perhaps 2,3-dichlorobenzoic acid) in extension growth tests arises from its inhibition of transport of endogenous or added auxin out of the sections, rather than from its intrinsic auxin activity. Chemically induced apolar callus growth on vascularized tobacco stem explants can arise from inhibition of native auxin transport, apolar growth stimulation by auxinic action of the test compound, or both. PMID:16656441

  10. 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

  11. Mutations inducing an active-site aperture in Rhizobium sp. sucrose isomerase confer hydrolytic activity.

    PubMed

    Lipski, Alexandra; Watzlawick, Hildegard; Ravaud, Stéphanie; Robert, Xavier; Rhimi, Moez; Haser, Richard; Mattes, Ralf; Aghajari, Nushin

    2013-02-01

    Sucrose isomerase is an enzyme that catalyzes the production of sucrose isomers of high biotechnological and pharmaceutical interest. Owing to the complexity of the chemical synthesis of these isomers, isomaltulose and trehalulose, enzymatic conversion remains the preferred method for obtaining these products. Depending on the microbial source, the ratio of the sucrose-isomer products varies significantly. In studies aimed at understanding and explaining the underlying molecular mechanisms of these reactions, mutations obtained using a random-mutagenesis approach displayed a major hydrolytic activity. Two of these variants, R284C and F164L, of sucrose isomerase from Rhizobium sp. were therefore crystallized and their crystal structures were determined. The three-dimensional structures of these mutants allowed the identification of the molecular determinants that favour hydrolytic activity compared with transferase activity. Substantial conformational changes resulting in an active-site opening were observed, as were changes in the pattern of water molecules bordering the active-site region.

  12. A base substitution in the donor site of intron 12 of KIT gene is responsible for the dominant white coat colour of blue fox (Alopex lagopus).

    PubMed

    Yan, S Q; Hou, J N; Bai, C Y; Jiang, Y; Zhang, X J; Ren, H L; Sun, B X; Zhao, Z H; Sun, J H

    2014-04-01

    The dominant white coat colour of farmed blue fox is inherited as a monogenic autosomal dominant trait and is suggested to be embryonic lethal in the homozygous state. In this study, the transcripts of KIT were identified by RT-PCR for a dominant white fox and a normal blue fox. Sequence analysis showed that the KIT transcript in normal blue fox contained the full-length coding sequence of 2919 bp (GenBank Acc. No KF530833), but in the dominant white individual, a truncated isoform lacking the entire exon 12 specifically co-expressed with the normal transcript. Genomic DNA sequencing revealed that a single nucleotide polymorphism (c.1867+1G>T) in intron 12 appeared only in the dominant white individuals and a 1-bp ins/del polymorphism in the same intron showed in individuals representing two different coat colours. Genotyping results of the SNP with PCR-RFLP in 185 individuals showed all 90 normal blue foxes were homozygous for the G allele, and all dominant white individuals were heterozygous. Due to the truncated protein with a deletion of 35 amino acids and an amino acid replacement (p.Pro623Ala) located in the conserved ATP binding domain, we propose that the mutant receptor had absent tyrosine kinase activity. These findings reveal that the base substitution at the first nucleotide of intron 12 of KIT gene, resulting in skipping of exon 12, is a causative mutation responsible for the dominant white phenotype of blue fox.

  13. Activation of Methane by the Pyridine Radical Cation and its Substituted Forms in the Gas Phase

    NASA Astrophysics Data System (ADS)

    Wu, Guohua; Stewart, Hamish; Liu, Zeyu; Wang, Yongcheng; Stace, Anthony J.

    2015-08-01

    We present an experimental study of methane activation by pyridine cation and its substituents in the gas phase. Mass spectrometric experiments in an ion trap demonstrate that pyridine cation and some of its substituent cations are able to react with methane. The deuterated methane experiment has confirmed that the hydrogen atom in the ionic product of reaction does come from methane. The collected information about kinetic isotope effects has been used to distinguish the nature of the bond activation as a hydrogen abstraction. Furthermore, experimental results demonstrated that the substituent groups on the pyridine ring can crucially influence their reactivity in methane bond activation processes. Density functional calculation (DFT) was employed to study the electronic structures of the complex and reaction mechanism of CH4+C5H5N+. The calculations confirmed the hypothesis from the experimental observation, namely, the reaction is rapid with no energy barrier.

  14. 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.

  15. 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).

  16. Synthesis, crystal structure, DFT analysis and fungicidal activity of a novel series O-substituted trifluoroatrolactamide derivatives

    NASA Astrophysics Data System (ADS)

    Yang, Hui-Hui; Cui, Can; Zhu, Cong; Li, Jian-Qiang; Wu, Rui; Tian, Lei; Zhao, Wei-Guang

    2017-01-01

    A series of O-substituted trifluoroatrolactamide derivatives has been synthesized and fully characterized by 1H NMR, 13C NMR, 19F NMR, HRMS and X-ray diffraction analyses. The fungicidal activity of these compounds was evaluated and the results showed that some of them exhibited potent in vitro fungicidal activity against Erysiphe graminis and Pyricularia oryzae. Their structure-property relationships were investigated using density functional theory calculations. The X-ray crystal structure of one of these compounds adopted a monoclinic space group with the following unit cell parameters: a = 24.285 (13) Å, b = 9.006 (5) Å, c = 9.794 (5) Å, β = 92.110 (9)º, V = 2140.6 (19) Å3 and Z = 4. A comparison of these experimental results with the theoretical values revealed that there was good agreement between the two sets of data. The subsequent biological evaluation of these compounds showed that some of them exhibited potent in vitro fungicidal activity against Erysiphe graminis and Pyricularia oryzae.

  17. N-Substituted N'-(2-alkylthio-4-chloro-5-methylbenzenesulfonyl)guanidines--Antibacterial, Cytotoxic Activities and Some Structure-Activity Relationships.

    PubMed

    Zołnowska, Beata; Sławiński, Jarosław; Grzonek, Aleksandra; Kędzia, Anna; Kwapisz, Ewa; Kawiak, Anna

    2015-01-01

    A series of N-substituted N'-(2-alkylthio-4-chloro-5-methylbenzenesulfonyl)guanidine derivatives bearing sulfonamide moiety have been screened in vitro for antibacterial activity against isolates from patients with infections of oral cavity, respiratory tract and intestinal tract. The majority of compounds exhibited good antibacterial potency. 1-[4-Chloro-5-methyl-2-(4-trifluoromethylbenzylthio)benzenesulfonyl]-3-(3-sulfamoylphenyl)guanidine (13) showed very strong activity, with MIC ≤ 6.2 μg/ml against eleven bacteria strains belonged to Gram-positive anaerobes and aerobes. Furthermore, compound 13 exhibited promising activity toward highly resistant microorganisms such as methicillin-resistant Staphylococcus aureus and Enterococcusfaecalis. It was found that Parvimonas micra, Finegoldia magna, Peptostreptococcus anaerobius, Propionibacterium acnes showed the highest susceptibility toward the investigated guanidines.

  18. Bidentate ligation of heme analogues; novel biomimetics of peroxidase active site.

    PubMed

    Ashkenasy, Gonen; Margulies, David; Felder, Clifford E; Shanzer, Abraham; Powers, Linda S

    2002-09-02

    The multifunctional nature of proteins that have iron-heme cofactors with noncovalent histidine linkage to the protein is controlled by the heme environment. Previous studies of these active-site structures show that the primary difference is the length of the iron-proximal histidine bond, which can be controlled by the degree of H-bonding to this histidine. Great efforts to mimic these functions with synthetic analogues have been made for more than two decades. The peroxidase models resulted in several catalytic systems capable of a large range of oxidative transformations. Most of these model systems modified the porphyrin ring covalently by directly binding auxiliary elements that control and facilitate reactivity; for example, electron-donating or -withdrawing substituents. A biomimetic approach to enzyme mimicking would have taken a different route, by attempting to keep the porphyrin ring system unaltered, as close as possible to its native form, and introducing all modifications at or close to the axial coordination sites. Such a model system would be less demanding synthetically, would make it easy to study the effect of a single structural modification, and might even provide a way to probe effects resulting from porphyrin exchange. We introduce here an alternative model system based on these principles. It consists of a two component system: a bis-imidazolyl ligand and an iron-porphyrin (readily substituted by a hemin). All modifications were introduced only to the ligand that engulfs the porphyrin and binds to the iron's fifth and sixth coordination sites. We describe the design, synthesis, and characterization of nine different model compounds with increased complexity. The primary tool for characterizing the environment of each complex Fe(III) center was the Extended X-ray Absorption Fine Structure (EXAFS) measurements, supported by UV/Vis, IR, and NMR spectroscopy and by molecular modeling. Introduction of asymmetry, by attaching different imidazoles

  19. 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

  20. Site-specific PEGylation of lidamycin and its antitumor activity

    PubMed Central

    Li, Liang; Shang, Boyang; Hu, Lei; Shao, Rongguang; Zhen, Yongsu

    2015-01-01

    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 (Mw 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

  1. In Vitro Antileishmanial, Trypanocidal, and Mammalian Cell Activities of Diverse N,N′ -Dihetaryl Substituted Diamines and Related Compounds

    PubMed Central

    Leal, Sandra M.; Amado, Diego F.; Kouznetsov, Vladimir V.; Escobar, Patricia

    2013-01-01

    The leishmaniasis and Chagas diseases constitute a serious public health problem worldwide with few and ineffective treatment options. The search for new antiparasitic candidates at the initial steps of drug discovery and development is still necessary. The synthesis of 22 de novo synthetized N,N′-dihetaryl-alkyldiamine derivatives and in vitro antiparasitic activity were evaluated for the first time against intracellular and extracellular forms of Leishmania (Leishmania) infantum, L. (Viannia) panamensis, L. (Leishmania) amazonensis, and Trypanosoma cruzi. Additionally, the toxicity on mammalian cells was determined. Some of these substituted N,N′-diamines (25–35 % of the tested compounds) showed interesting results against free-living forms of parasites with activities at the inhibitory concentration (IC50) level of 1.96 to 28.83 μM for L. (L.) infantum promastigotes and IC50 of 0.02 to 5.31 μM for T. cruzi epimastigotes. No activity at the IC50 level on intracellular amastigotes of T. cruzi was observed. However, N1,N2-dibenzylethane-1,2-diamine 5a revealed an important activity against the intracellular amastigotes of L. infantum (IC50 25.42 μM ±0.33) and L. panamensis (IC50 58.20 μM ±3.23), while their analogue N1,N4-dibenzylbutane-1,4-diamine 5c resulted in activity only against L. panamensis (IC50 11.19 μM ±0.20) without toxicity on Vero and THP-1 mammalian cells. The active compounds against intracellular parasites with low toxicity in mammalian cells may be considered for future studies in experimental models. PMID:23641328

  2. Allosteric site-mediated active site inhibition of PBP2a using Quercetin 3-O-rutinoside and its combination.

    PubMed

    Rani, Nidhi; Vijayakumar, Saravanan; P T V, Lakshmi; Arunachalam, Annamalai

    2016-08-01

    Recent crystallographic study revealed the involvement of allosteric site in active site inhibition of penicillin binding protein (PBP2a), where one molecule of Ceftaroline (Cef) binds to the allosteric site of PBP2a and paved way for the other molecule (Cef) to bind at the active site. Though Cef has the potency to inhibit the PBP2a, its adverse side effects are of major concern. Previous studies have reported the antibacterial property of Quercetin derivatives, a group of natural compounds. Hence, the present study aims to evaluate the effect of Quercetin 3-o-rutinoside (Rut) in allosteric site-mediated active site inhibition of PBP2a. The molecular docking studies between allosteric site and ligands (Rut, Que, and Cef) revealed a better binding efficiency (G-score) of Rut (-7.790318) and Cef (-6.194946) with respect to Que (-5.079284). Molecular dynamic (MD) simulation studies showed significant changes at the active site in the presence of ligands (Rut and Cef) at allosteric site. Four different combinations of Rut and Cef were docked and their G-scores ranged between -6.320 and -8.623. MD studies revealed the stability of the key residue (Ser403) with Rut being at both sites, compared to other complexes. Morphological analysis through electron microscopy confirmed that combination of Rut and Cefixime was able to disturb the bacterial cell membrane in a similar fashion to that of Rut and Cefixime alone. The results of this study indicate that the affinity of Rut at both sites were equally good, with further validations Rut could be considered as an alternative for inhibiting MRSA growth.

  3. 4,6-Substituted-1,3,5-triazin-2(1H)-ones as monocyclic catalytic inhibitors of human DNA topoisomerase IIα targeting the ATP binding site.

    PubMed

    Pogorelčnik, Barbara; Janežič, Matej; Sosič, Izidor; Gobec, Stanislav; Solmajer, Tom; Perdih, Andrej

    2015-08-01

    Human DNA topoisomerase IIα (htIIα) is a validated target for the development of novel anticancer agents. Starting from our discovered 4-amino-1,3,5-triazine inhibitors of htIIα, we investigated a library of 2,4,6-trisubstituted-1,3,5-triazines for novel inhibitors that bind to the htIIα ATP binding site using a combination of structure-based and ligand-based pharmacophore models and molecular docking. 4,6-substituted-1,3,5-triazin-2(1H)-ones 8, 9 and 14 were identified as novel inhibitors with activity comparable to the established drug etoposide (1). Compound 8 inhibits the htIIα decatenation in a superior fashion to etoposide. Cleavage assays demonstrated that selected compounds 8 and 14 do not act as poisons and antagonize the poison effect of etoposide. Microscale thermophoresis (MST) confirmed binding of compound 8 to the htIIα ATPase domain and compound 14 effectively inhibits the htIIα mediated ATP hydrolysis. The molecular dynamics simulation study provides further insight into the molecular recognition. The 4,6-disubstituted-1,3,5-triazin-2(1H)-ones represent the first validated monocyclic class of catalytic inhibitors that bind to the to the htIIα ATPase domain.

  4. QM/MM calculations of kinetic isotope effects in the chorismate mutase active site.

    PubMed

    Martí, Sergio; Moliner, Vincent; Tuñón, Iñaki; Williams, Ian H

    2003-02-07

    Kinetic isotope effects have been computed for the Claisen rearrangement of chorismate to prephenate in aqueous solution and in the active site of chorismate mutase from B. subtilus. These included primary 13C and 18O and secondary 3H effects for substitutions at the bond-making and bond-breaking positions. The initial structures of the putative stationary points on the potential energy surface, required for the calculations of isotope effects using the CAMVIB/CAMISO programs, have been selected from hybrid QM/MM molecular dynamical simulations using the DYNAMO program. Refinement of the reactant complex and transition-state structures has been carried out by means of AM1/CHARMM24/TIP3P calculations using the GRACE program, with full gradient relaxation of the position of > 5200 atoms for the enzymic simulations, and with a box containing 711 water molecules for the corresponding reaction in aqueous solution. Comparison of these results, and of gas phase calculations, with experimental data has shown that the chemical rearrangement is largely rate-determining for the enzyme mechanism. Inclusion of the chorismate conformational pre-equilibrium step in the modelled kinetic scheme leads to better agreement between recent experimental data and theoretical predictions. These results provide new information on an important enzymatic transformation, and the key factors responsible for the kinetics of its molecular mechanism are clarified. Treatment of the enzyme and/or solvent environment by means of a large and flexible model is absolutely essential for prediction of kinetic isotope effects.

  5. Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site

    PubMed Central

    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. DOI: http://dx.doi.org/10.7554/eLife.06181.001 PMID:25902402

  6. Crystal structure, exogenous ligand binding, and redox properties of an engineered diiron active site in a bacterial hemerythrin.

    PubMed

    Okamoto, Yasunori; Onoda, Akira; Sugimoto, Hiroshi; Takano, Yu; Hirota, Shun; Kurtz, Donald M; Shiro, Yoshitsugu; Hayashi, Takashi

    2013-11-18

    A nonheme diiron active site in a 13 kDa hemerythrin-like domain of the bacterial chemotaxis protein DcrH-Hr contains an oxo bridge, two bridging carboxylate groups from Glu and Asp residues, and five terminally ligated His residues. We created a unique diiron coordination sphere containing five His and three Glu/Asp residues by replacing an Ile residue with Glu in DcrH-Hr. Direct coordination of the carboxylate group of E119 to Fe2 of the diiron site in the I119E variant was confirmed by X-ray crystallography. The substituted Glu is adjacent to an exogenous ligand-accessible tunnel. UV-vis absorption spectra indicate that the additional coordination of E119 inhibits the binding of the exogenous ligands azide and phenol to the diiron site. The extent of azide binding to the diiron site increases at pH ≤ 6, which is ascribed to protonation of the carboxylate ligand of E119. The diferrous state (deoxy form) of the engineered diiron site with the extra Glu residue is found to react more slowly than wild type with O2 to yield the diferric state (met form). The additional coordination of E119 to the diiron site also slows the rate of reduction from the met form. All these processes were found to be pH-dependent, which can be attributed to protonation state and coordination status of the E119 carboxylate. These results demonstrate that modifications of the endogenous coordination sphere can produce significant changes in the ligand binding and redox properties in a prototypical nonheme diiron-carboxylate protein active site.

  7. A split active site couples cap recognition by Dcp2 to activation

    PubMed Central

    Floor, Stephen N.; Jones, Brittnee N.; Hernandez, Gail A.; Gross, John D.

    2010-01-01

    Decapping by Dcp2 is an essential step in 5′-3′ mRNA decay. In yeast, decapping requires an open-to-closed transition in Dcp2, though the link between closure and catalysis remains elusive. Here we show using NMR that cap binds conserved residues on both the catalytic and regulatory domains of Dcp2. Lesions in the cap-binding site on the regulatory domain reduce the catalytic step two orders of magnitude and block formation of the closed state whereas Dcp1 enhances the catalytic step by a factor of ten and promotes closure. We conclude that closure occurs during the rate-limiting catalytic step of decapping, juxtaposing the cap-binding region of each domain to form a composite active site. This work suggests a model for regulation of decapping, where coactivators trigger decapping by stabilizing a labile composite active site. PMID:20711189

  8. Synthesis and anticancer activity of N-substituted 2-arylquinazolinones bearing trans-stilbene scaffold.

    PubMed

    Mahdavi, Mohammad; Pedrood, Keyvan; Safavi, Maliheh; Saeedi, Mina; Pordeli, Mahboobeh; Ardestani, Sussan Kabudanian; Emami, Saeed; Adib, Mehdi; Foroumadi, Alireza; Shafiee, Abbas

    2015-05-05

    A novel series of 2-arylquinazolinones 7a-o bearing trans-stilbene moiety were designed, synthesized, and evaluated against human breast cancer cell lines including human breast adenocarcinoma (MCF-7 and MDA-MB-231) and human ductal breast epithelial tumor (T-47D). Among the tested compounds, the sec-butyl derivative 7h showed the best profile of activity (IC50 < 5 μM) against all cell lines, being 2-fold more potent than standard drug, etoposide. Our investigation revealed that the cytotoxic activity was significantly affected by N3-alkyl substituents. Furthermore, the morphological analysis by acridine orange/ethidium bromide double staining test and flow cytometry analysis indicated that the prototype compound 7h can induce apoptosis in MCF-7 and MDA-MB-231 cells.

  9. Synthesis of substituted N-aryl-N'-Sulfamoyloxazolidin-2-ones with potential antibacterial activity.

    PubMed

    Nessaiba, Mounir; Abdaoui, Mohamed; Djahoudi, Abed El Ghani; Kadri, Mekki; Winum, Jean-Yves

    2007-06-01

    Three series of derived compounds of N, Aryl-N',Sulfamoyloxazolidin-2-ones were synthesized starting from chloro-sulfonylisocyanate (CSI) by carbomoylation, sulfamoylation and intramolecular cyclization reactions followed by methylation and heterocyclic reopening reactions. This later is based on a new hydrolysis method which uses a solid support, in order to obtain the correspondent amino-alcohols, which allowed isolating a new amino-alcohol ester. Measurements of the hydro-solubility by determination partition coefficient (log p) in water/octanol system were carried out by spectrophotometry. The antibacterial activities in vitro of some synthesized compounds were evaluated on a "Staphylococus aureus" strain in a Mûller-Hintone medium, showing good activity for some of them. All the synthesized compounds are characterized by IR, (1)H NMR and mass spectroscopy (ESI-MS).

  10. A matrix-focused structure-activity and binding site flexibility study of quinolinol inhibitors of botulinum neurotoxin serotype A.

    PubMed

    Harrell, William A; Vieira, Rebecca C; Ensel, Susan M; Montgomery, Vicki; Guernieri, Rebecca; Eccard, Vanessa S; Campbell, Yvette; Roxas-Duncan, Virginia; Cardellina, John H; Webb, Robert P; Smith, Leonard A

    2017-02-01

    Our initial discovery of 8-hydroxyquinoline inhibitors of BoNT/A and separation/testing of enantiomers of one of the more active leads indicated considerable flexibility in the binding site. We designed a limited study to investigate this flexibility and probe structure-activity relationships; utilizing the Betti reaction, a 36 compound matrix of quinolinol BoNT/A LC inhibitors was developed using three 8-hydroxyquinolines, three heteroaromatic amines, and four substituted benzaldehydes. This study has revealed some of the most effective quinolinol-based BoNT/A inhibitors to date, with 7 compounds displaying IC50 values ⩽1μM and 11 effective at ⩽2μM in an ex vivo assay.

  11. In vitro activity of E-1040, a 3-substituted cephalosporin, against pathogens from cystic fibrosis sputum.

    PubMed

    Stutman, H R; Akaniro, J C; Vidaurre, C E; Marks, M I

    1990-07-01

    On the basis of preliminary in vitro data, we evaluated E-1040, a new cephalosporin, against 188 cystic fibrosis (CF) sputum isolates obtained from 26 CF centers in the United States. These isolates included mucoid and nonmucoid Pseudomonas aeruginosa, Pseudomonas cepacia, Staphylococcus aureus, Haemophilus influenzae, and Escherichia coli. In addition to MICs measured under standard conditions, selected isolates were tested at various pH values, inoculum sizes, and diluent (CF serum and sputum) conditions. E-1040 activities (MICs for 50 and 90% of the strains) against the isolates were as follows: P. aeruginosa (mucoid and nonmucoid), 1 and 4 micrograms/ml; P. cepacia, 4 and 16 micrograms/ml; S. aureus, 8 and 8 micrograms/ml; H. influenzae, 1 and 4 micrograms/ml; and E. coli, less than or equal to 0.12 and less than or equal to 0.12 microgram/ml. E-1040 activity against mucoid P. aeruginosa was 4-fold greater than that of aztreonam, 16-fold greater than that of ceftazidime, and 32-fold greater than that of piperacillin. E-1040 was similar to other broad-spectrum cephalosporins against S. aureus, H. influenzae, and E. coli. Bactericidal activity was less than or equal to 1 dilution of MIC for 88% of the strains, although kinetic studies with mucoid strains of P. aeruginosa demonstrated effective killing only at eight times the MIC. Variations in pH from 5 to 8, in inoculum size from 10(3) to 10(7) CFU/ml, and in diluent (CF serum or CF sputum) did not affect E-1040 activity.

  12. Critical active-site residues identified by site-directed mutagenesis in Pseudomonas aeruginosa phosphorylcholine phosphatase, a new member of the haloacid dehalogenases hydrolase superfamily.

    PubMed

    Beassoni, Paola R; Otero, Lisandro H; Massimelli, Maria J; Lisa, Angela T; Domenech, Carlos E

    2006-12-01

    Pseudomonas aeruginosa phosphorylcholine phosphatase (PChP), the product of the PA5292 gene, is synthesized when the bacteria are grown with choline, betaine, dimethylglycine, or carnitine. In the presence of Mg(2+), PChP catalyzes the hydrolysis of both phosphorylcholine (PCh) and p-nitrophenylphosphate (p-NPP). PCh saturation curve analysis of the enzyme with or without the signal peptide indicated that the peptide was the fundamental factor responsible for decreasing the affinity of the second site of PChP for PCh, either at pH 5.0 or pH 7.4. PChP contained three conserved motifs characteristic of the haloacid dehalogenases superfamily. In the PChP without the signal peptide, motifs I, II, and III correspond to the residues (31)DMDNT(35), (166)SAA(168), and K(242)/(261)GDTPDSD(267), respectively. To determine the catalytic importance of the D31, D33, T35, S166, K242, D262, D265, and D267 on the enzyme activity, site-directed mutagenesis was performed. D31, D33, D262, and D267 were identified as the more important residues for catalysis. D265 and D267 may be involved in the stabilization of motif III, or might contribute to substrate specificity. The substitution of T35 by S35 resulted in an enzyme with a low PChP activity, but conserves the catalytic sites involved in the hydrolysis of PCh (K(m1) 0.03 mM: , K(m2) 0.5 mM: ) or p-NPP (K(m) 2.1 mM: ). Mutating either S166 or K242 revealed that these residues are also important to catalyze the hydrolysis of both substrates. The substitution of lysine by arginine or by glutamine revealed the importance of the positive charged group, either from the amino or guanidinium groups, because K242Q was inactive, whereas K242R was a functional enzyme.

  13. 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.

  14. Substrate specificity engineering of beta-mannosidase and beta-glucosidase from Pyrococcus by exchange of unique active site residues.

    PubMed

    Kaper, Thijs; van Heusden, Hester H; van Loo, Bert; Vasella, Andrea; van der Oost, John; de Vos, Willem M

    2002-03-26

    A beta-mannosidase gene (PH0501) was identified in the Pyrococcus horikoshii genome and cloned and expressed in E. coli. The purified enzyme (BglB) was most specific for the hydrolysis of p-nitrophenyl-beta-D-mannopyranoside (pNP-Man) (Km: 0.44 mM) with a low turnover rate (kcat: 4.3 s(-1)). The beta-mannosidase has been classified as a member of family 1 of glycoside hydrolases. Sequence alignments and homology modeling showed an apparent conservation of its active site region with, remarkably, two unique active site residues, Gln77 and Asp206. These residues are an arginine and asparagine residue in all other known family 1 enzymes, which interact with the catalytic nucleophile and equatorial C2-hydroxyl group of substrates, respectively. The unique residues of P. horikoshii BglB were introduced in the highly active beta-glucosidase CelB of Pyrococcus furiosus and vice versa, yielding two single and one double mutant for each enzyme. In CelB, both substitutions R77Q and N206D increased the specificity for mannosides and reduced hydrolysis rates 10-fold. In contrast, BglB D206N showed 10-fold increased hydrolysis rates and 35-fold increased affinity for the hydrolysis of glucosides. In combination with inhibitor studies, it was concluded that the substituted residues participate in the ground-state binding of substrates with an equatorial C2-hydroxyl group, but contribute most to transition-state stabilization. The unique activity profile of BglB seems to be caused by an altered interaction between the enzyme and C2-hydroxyl of the substrate and a specifically increased affinity for mannose that results from Asp206.

  15. Lanthanide Complexes of Substituted β-Diketone Hydrazone Derivatives: Synthesis, Characterization, and Biological Activities.

    PubMed

    Hegazy, W H; Al-Motawaa, I H

    2011-01-01

    A series of β-diketone hydrazone derivatives have been synthesized through condensation of β-diketone with aromatic aldehydes followed by reaction with phenylhydrazine. The structure of the ligands and intermediates are well defined through elemental and spectroscopic analyses. These hydrazones are potential ligands toward lanthanide metal ions. New complexes of trivalent Scandium, Yttrium, Lanthanum, and Cerium have been synthesized. The composition of these complexes is discussed on the basis of elemental analyses, IR, magnetic moments, and thermal analyses. The prepared complexes were screened for antibacterial and antifungal properties and have exhibited potential activity.

  16. Microplate assay for screening the antibacterial activity of Schiff bases derived from substituted benzopyran-4-one.

    PubMed

    Amin, Rehab M; Abdel-Kader, Nora S; El-Ansary, Aida L

    2012-09-01

    Schiff bases (SB(1)-SB(3)) were synthesized from the condensation of 6-formyl-7-hydroxy-5-methoxy-2-methylbenzopyran-4-one with 2-aminopyridine (SB(1)), p-phenylenediamine (SB(2)) and o-phenylenediamine (SB(3)), while Schiff bases (SB(4)-SB(6)) were synthesized by condensation of 5,7-dihydroxy-6-formyl-2-methylbenzopyran-4-one with 2-aminopyridine (SB(4)), p-phenylenediamine (SB(5)) and o-phenylenediamine (SB(6)). Schiff bases were characterized using elemental analysis, IR, UV-Vis, (1)H NMR, (13)C NMR and mass spectroscopy. These compounds were screened for antibacterial activities by micro-plate assay technique. Escherichia coli and Staphylococcus capitis were exposed to different concentrations of the Schiff bases. Results showed that the antibacterial effect of these Schiff bases on Gram-negative bacteria were higher than that on Gram-positive bacteria moreover, the Schiff bases containing substituent OCH(3) on position five have higher antibacterial activity than that containing hydroxy group on the same position.

  17. Membrane anchoring of diacylglycerol lactones substituted with rigid hydrophobic acyl domains correlates with biological activities.

    PubMed

    Raifman, Or; Kolusheva, Sofiya; Comin, Maria J; Kedei, Noemi; Lewin, Nancy E; Blumberg, Peter M; Marquez, Victor E; Jelinek, Raz

    2010-01-01

    Synthetic diacylglycerol lactones (DAG lactones) are effective modulators of critical cellular signaling pathways downstream of the lipophilic second messenger diacylglycerol that activate a host of protein kinase C (PKC) isozymes as well as other non-kinase proteins that share with PKC similar C1 membrane-targeting domains. A fundamental determinant of the biological activity of these amphiphilic molecules is the nature of their interactions with cellular membranes. This study characterizes the membrane interactions and bilayer anchoring of a series of DAG lactones in which the hydrophobic moiety is a 'molecular rod', namely a rigid 4-[2-(R-phenyl)ethynyl]benzoate moiety in the acyl position. Use of assays employing chromatic biomimetic vesicles and biophysical techniques revealed that the mode of membrane anchoring of the DAG lactone derivatives was markedly affected by the presence of the hydrophobic diphenyl rod and by the size of the functional unit at the terminus of the rod. Two primary mechanisms of interaction were observed: surface binding of the DAG lactones at the lipid/water interface and deep insertion of the ligands into the alkyl core of the lipid bilayer. These membrane-insertion properties could explain the different patterns of the PKC translocation from the cytosol to membranes that is induced by the molecular-rod DAG lactones. This investigation emphasizes that the side residues of DAG lactones, rather than simply conferring hydrophobicity, profoundly influence membrane interactions, and thus may further contribute to the diversity of biological actions of these synthetic biomimetic ligands.

  18. Site-specific microbial communities in three PCB-impacted sediments are associated with different in situ dechlorinating activities.

    PubMed

    Kjellerup, Birthe V; Sun, Xueli; Ghosh, Upal; May, Harold D; Sowers, Kevin R

    2008-05-01

    Competitive PCR and denaturing HPLC analyses together with an assay detecting potential polychlorinated biphenyl (PCB) dechlorinating activities were combined with physical-chemical site characterizations to identify factors affecting the reductive dechlorination of PCBs in the three historically impacted sediments: Grasse and Buffalo Rivers, NY and Anacostia River, DC. In Grasse River sediment an in situ enriched population of Dehalococcoides phylotypes was abundant in high numbers together with a relatively high dechlorination activity and a high concentration of congeners containing unflanked chlorine substitutions. In contrast microbial communities in Anacostia and Buffalo Rivers sediments consisted of similar total numbers of putative dechlorinating bacteria, but the populations consisted of more diverse putative dechlorinating phylotypes and were associated with lower dechlorination activities and higher concentrations of flanked congeners. Differences observed in the PCB dechlorination activity were not influenced by the chemical PCB availability in spiked sediment or physical sediment characteristics, but were consistent with the concentration of PCBs and total organic carbon in the native sediment. Application of molecular methods for selective detection of indigenous microbial dechlorinating communities combined with assessment of the dechlorinating activity and analysis of the in situ congener profiles provided a comprehensive approach for characterization and identification of sites that are amenable to bioremediation, which is essential for the development of in situ treatment strategies.

  19. Mapping the Zap-70 phosphorylation sites on LAT (linker for activation of T cells) required for recruitment and activation of signalling proteins in T cells.

    PubMed Central

    Paz, P E; Wang, S; Clarke, H; Lu, X; Stokoe, D; Abo, A

    2001-01-01

    T-cell-receptor (TCR)-mediated LAT (linker for activation of T cells) phosphorylation is critical for the membrane recruitment of signalling complexes required for T-cell activation. Although tyrosine phosphorylation of LAT is required for recruitment and activation of signalling proteins, the molecular mechanism associated with this event is unclear. In the present study we reconstituted the LAT signalling pathway by demonstrating that a direct tyrosine phosphorylation of LAT with activated protein-tyrosine kinase Zap70 is necessary and sufficient for the association and activation of signalling proteins. Zap-70 efficiently phosphorylates LAT on tyrosine residues at positions 226, 191, 171, 132 and 127. By substituting these tyrosine residues in LAT with phenylalanine and by utilizing phosphorylated peptides derived from these sites, we mapped the tyrosine residues in LAT required for the direct interaction and activation of Vav, p85/p110alpha and phospholipase Cgamma1 (PLCgamma1). Our results indicate that Tyr(226) and Tyr(191) are required for Vav binding, whereas Tyr(171) and Tyr(132) are necessary for association and activation of phosphoinositide 3-kinase activity and PLCgamma1 respectively. Furthermore, by expression of LAT mutants in LAT-deficient T cells, we demonstrate that Tyr(191) and Tyr(171) are required for T-cell activation and Tyr(132) is required for the activation of PLCgamma1 and Ras signalling pathways. PMID:11368773

  20. Identification of BRCA1 missense substitutions that confer partial functional activity: potential moderate risk variants?

    PubMed Central

    Lovelock, Paul K; Spurdle, Amanda B; Mok, Myth TS; Farrugia, Daniel J; Lakhani, Sunil R; Healey, Sue; Arnold, Stephen; Buchanan, Daniel; Investigators, kConFab; Couch, Fergus J; Henderson, Beric R; Goldgar, David E; Tavtigian, Sean V; Chenevix-Trench, Georgia; Brown, Melissa A

    2007-01-01

    Introduction Many of the DNA sequence variants identified in the breast cancer susceptibility gene BRCA1 remain unclassified in terms of their potential pathogenicity. Both multifactorial likelihood analysis and functional approaches have been proposed as a means to elucidate likely clinical significance of such variants, but analysis of the comparative value of these methods for classifying all sequence variants has been limited. Methods We have compared the results from multifactorial likelihood analysis with those from several functional analyses for the four BRCA1 sequence variants A1708E, G1738R, R1699Q, and A1708V. Results Our results show that multifactorial likelihood analysis, which incorporates sequence conservation, co-inheritance, segregation, and tumour immunohistochemical analysis, may improve classification of variants. For A1708E, previously shown to be functionally compromised, analysis of oestrogen receptor, cytokeratin 5/6, and cytokeratin 14 tumour expression data significantly strengthened the prediction of pathogenicity, giving a posterior probability of pathogenicity of 99%. For G1738R, shown to be functionally defective in this study, immunohistochemistry analysis confirmed previous findings of inconsistent 'BRCA1-like' phenotypes for the two tumours studied, and the posterior probability for this variant was 96%. The posterior probabilities of R1699Q and A1708V were 54% and 69%, respectively, only moderately suggestive of increased risk. Interestingly, results from functional analyses suggest that both of these variants have only partial functional activity. R1699Q was defective in foci formation in response to DNA damage and displayed intermediate transcriptional transactivation activity but showed no evidence for centrosome amplification. In contrast, A1708V displayed an intermediate transcriptional transactivation activity and a normal foci formation response in response to DNA damage but induced centrosome amplification. Conclusion

  1. Acidic substitution of the activation loop tyrosines in TrkA supports nerve growth factor-independent cell survival and neuronal differentiation.

    PubMed

    Gryz, E A; Meakin, S O

    2000-01-20

    TrkA is the receptor tyrosine kinase (RTK) for nerve growth factor (NGF) and stimulates NGF-dependent cell survival and differentiation in primary neurons. TrkA expression in neuronal tumors also supports NGF-dependent differentiation of neuroblastomas and apoptosis of medulloblastomas. Phosphorylation of the activation loop tyrosines in RTK's are essential to activation as well as allosteric changes that facilitate substrate interaction and phosphorylation. Acidic amino acid substitution of the activation loop tyrosines in TrkA, Tyr683Tyr684, was performed to mimic the negative charges normally induced by ligand activation and receptor phosphorylation. A total of eight independent mutants containing single or double substitutions were generated for comparison. Herein, we demonstrate that acidic substitution of the activation loop tyrosines is sufficient to induce allosteric changes required for constitutive TrkA kinase activity as well as phosphorylation of TrkA signaling proteins such as Shc, PLCgamma-1, FRS-2 and erk1/2. The strongest constitutively active TrkA mutants, GluAsp and AspGlu, support NGF-independent neuritogenesis and cell survival to levels approximately 65 and 80-100%, respectively, of NGF-activated wild type TrkA. Thus, constitutively active TrkA may provide a useful strategy in future therapeutic approaches to limit the development and progression of neuronal tumors.

  2. Effects of 7-O substitutions on estrogenic and anti-estrogenic activities of daidzein analogues in MCF-7 breast cancer cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Daidzein (1) is a natural estrogenic isoflavone. We report here that 1 can be transformed into an antiestrogenic ligand by simple alkyl substitutions of the 7-hydroxyl hydrogen. To test the effect of such structural modifications on the hormonal activities of the resulting compounds, a series of dai...

  3. Discovery of molluscicidal and cercaricidal activities of 3-substituted quinazolinone derivatives by a scaffold hopping approach using a pseudo-ring based on the intramolecular hydrogen bond formation.

    PubMed

    Guo, Wei; Zheng, Lv-Yin; Li, Yong-Dong; Wu, Ren-Miao; Chen, Qiang; Yang, Ding-Qiao; Fan, Xiao-Lin

    2016-06-10

    Discovery of novel topological agents against Oncomelania hupensis snails and cercariae remains a significant challenge in current Schistosomiasis control. A pseudo-ring formed from salicylanilide by an intramolecular hydrogen bond led to the discovery of 3-substituted quinazolinone derivatives which showed a potent molluscicidal and cercaricidal activities.

  4. Computer-aided active-site-directed modeling of the Herpes Simplex Virus 1 and human thymidine kinase

    NASA Astrophysics Data System (ADS)

    Folkers, Gerd; Trumpp-Kallmeyer, Susanne; Gutbrod, Oliver; Krickl, Sabine; Fetzer, Jürgen; Keil, Günther M.

    1991-10-01

    Thymidine kinase (TK), which is induced by Herpes Simplex Virus 1 (HSV1), plays a key role in the antiviral activity of guanine derivatives such as aciclovir (ACV). In contrast, ACV shows only low affinity to the corresponding host cell enzyme. In order to define the differences in substrate binding of the two enzymes on molecular level, models for the three-dimensional (3-D) structures of the active sites of HSV1-TK and human TK were developed. The reconstruction of the active sites started from primary and secondary structure analysis of various kinases. The results were validated to homologous enzymes with known 3-D structures. The models predict that both enzymes consist of a central core β-sheet structure, connected by loops and α-helices very similar to the overall structure of other nucleotide binding enzymes. The phosphate binding is made up of a highly conserved glycine-rich loop at the N-terminus of the proteins and a conserved region at the C-terminus. The thymidine recognition site was found about 100 amino acids downstream from the phosphate binding loop. The differing substrate specificity of human and HSV1-TK can be explained by amino-acid substitutions in the homologous regions. To achieve a better understanding of the structure of the active site and how the thymidine kinase proteins interact with their substrates, the corresponding complexes of thymidine and dihydroxypropoxyguanine (DHPG) with HSV1 and human TK were built. For the docking of the guanine derivative, the X-ray structure of Elongation Factor Tu (EF-Tu), co-crystallized with guanosine diphosphate, was taken as reference. Fitting of thymidine into the active sites was done with respect to similar interactions found in thymidylate kinase. To complement the analysis of the 3-D structures of the two kinases and the substrate enzyme interactions, site-directed mutagenesis of the thymidine recognition site of HSV1-TK has been undertaken, changing Asp162 in the thymidine recognition site

  5. Characterization of Active Site Residues of Nitroalkane Oxidase†

    PubMed Central

    Valley, Michael P.; Fenny, Nana S.; Ali, Shah R.; Fitzpatrick, Paul F.

    2010-01-01

    The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitrolkanes to the corresponding aldehydes and ketones plus nitrite. The structure of the enzyme shows that Serl71 forms a hydrogen bond to the flavin N5, suggesting that it plays a role in catalysis. Cys397 and Tyr398 were previously identified by chemical modification as potential active site residues. To more directly probe the roles of these residues, the S171A, S171V, S171T, C397S, and Y398F enzymes have been characterized with nitroethane as substrate. The C397S and Y398 enzymes were less stable than the wild-type enzyme, and the C397S enzyme routinely contained a substoichiometric amount of FAD. Analysis of the steady-state kinetic parameters for the mutant enzymes, including deuterium isotope effects, establishes that all of the mutations result in decreases in the rate constants for removal of the substrate proton by ~5-fold and decreases in the rate constant for product release of ~2-fold. Only the S171V and S171T mutations alter the rate constant for flavin oxidation. These results establish that these residues are not involved in catalysis, but rather are required for maintaining the protein structure. PMID:20056514

  6. 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.

  7. 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.

  8. Biorhythms of activities of liver and blood dehydrogenases and changes in body weight of the rats feeding normal diet or excess of sugar substitutes.

    PubMed

    Petrovich, Yu A; Volozhin, A I; Zubtsov, V A; Kichenko, S M

    2007-12-01

    Biorhythms with higher levels of activity of sorbitol dehydrogenase and lactate dehydrogenase in blood plasma, specific activity of sorbitol dehydrogenase, lactate dehydrogenase, and malate dehydrogenase in the liver, and body weight of rats were more pronounced in the spring-summer period than in the autumn-winter period. These specific features were revealed in animals feeding a normal diet or food with 54 and 27% sugar substitute sorbitol. However, specific activity of glucose-6-phosphate dehydrogenase in the liver was higher in the autumn-winter period. Activity of sorbitol dehydrogenase in blood plasma increased by tens of times due to induction of sorbitol synthesis (substrate) in the liver. Sugar substitute xylitol is structurally similar to sorbitol, but is not the substrate for sorbitol dehydrogenase. However, the effect of xylitol on activities of lactate dehydrogenase, malate dehydrogenase, and glucose-6-phosphate dehydrogenase in the spring-summer period was similar to that of sorbitol.

  9. 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

  10. Synthesis, biological evaluation, hydration site thermodynamics, and chemical reactivity analysis of α-keto substituted peptidomimetics for the inhibition of Plasmodium falciparum.

    PubMed

    Weldon, David J; Shah, Falgun; Chittiboyina, Amar G; Sheri, Anjaneyulu; Chada, Raji Reddy; Gut, Jiri; Rosenthal, Philip J; Shivakumar, Develeena; Sherman, Woody; Desai, Prashant; Jung, Jae-Chul; Avery, Mitchell A

    2014-03-01

    A new series of peptidomimetic pseudo-prolyl-homophenylalanylketones were designed, synthesized and evaluated for inhibition of the Plasmodium falciparum cysteine proteases falcipain-2 (FP-2) and falcipain-3 (FP-3). In addition, the parasite killing activity of these compounds in human blood-cultured P. falciparum was examined. Of twenty-two (22) compounds synthesized, one peptidomimetic comprising a homophenylalanine-based α-hydroxyketone linked Cbz-protected hydroxyproline (39) showed the most potency (IC50 80 nM against FP-2 and 60 nM against FP-3). In silico analysis of these peptidomimetic analogs offered important protein-ligand structural insights including the role, by WaterMap, of water molecules in the active sites of these protease isoforms. The pseudo-dipeptide 39 and related compounds may serve as a promising direction forward in the design of competitive inhibitors of falcipains for the effective treatment of malaria.

  11. An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions

    PubMed Central

    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 (–NH2) 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 MC, to make what are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA's difficulty in specifying a unique conformation and highlighting Nature's potential to use local transitions of RNA in complex function. PMID:26567314

  12. 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.

  13. Synthesis, anticonvulsant, antioxidant and binding interaction of novel N-substituted methylquinazoline-2,4(1H, 3H)-dione derivatives to bovine serum albumin: A structure-activity relationship study

    NASA Astrophysics Data System (ADS)

    Prashanth, M. K.; Madaiah, M.; Revanasiddappa, H. D.; Veeresh, B.

    2013-06-01

    A novel class of N-substituted glycosmicine derivatives was synthesized, and their anticonvulsant, antioxidant activity and interaction with bovine serum albumin (BSA) were evaluated. The synthesized compounds 4a-j were examined for anticonvulsant activity by maximal electroshock induced seizures (MESs) test and their neurotoxic effects were determined by rotorod test in mice. The structure-activity relationships (SARs) of these compounds were also investigated. Compounds 4d, 4g, 4i and 4j were found to have good protective effect from seizure. The in vitro antioxidant activity was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide radical scavenging assay. The interaction between novel N-substituted methylquinazoline-2,4(1H, 3H)-dione (NMQ) and BSA was analyzed by fluorescence and ultraviolet spectroscopy at 304 K under simulative physiological conditions. BSA fluorescence quenched by NMQ is discussed according to the Stern-Volmer equation. The binding constant and binding sites of NMQ with BSA were calculated. According to Forster non-radiation energy transfer theory, the binding distance (r) between NMQ and BSA was calculated.

  14. 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

  15. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 9 2012-07-01 2012-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an active... visible emissions to the outside air from any active waste disposal site where asbestos-containing...

  16. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 9 2014-07-01 2014-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an active... visible emissions to the outside air from any active waste disposal site where asbestos-containing...

  17. 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.

  18. Synthesis and Antibacterial Activity of 3-(Substituted)-2-(4-oxo-2-phenylquinazolin-3(4H)-ylamino)quinazolin-4(3H)-one.

    PubMed

    Appani, Ramgopal; Bhukya, Baburao; Gangarapu, Kiran

    2016-01-01

    A series of novel 3-(substituted)-2-(substituted quinazolinylamino)quinazolin-4(3H)-ones were synthesized by the reaction of 3-(substituted)-2-hydrazino-quinazoline-4(3H)-ones with 2-phenyl-3,1-benzoxazin-4-one. The starting materials 3-(substituted)-2-hydrazino-quinazolin-4(3H)-ones were synthesized from various primary amines by a multistep synthesis. All the title compounds were tested for their antibacterial activity using ciprofloxacin as reference standard. Compounds 3-(4-fluorophenyl)-2-(4-oxo-2-phenylquinazolin-3(4H)-ylamino)quinazolin-4(3H)-one (9a) and 3-(4-chlorophenyl)-2-(4-oxo-2-phenylquinazolin-3(4H)-ylamino)quinazolin-4(3H)-one (9h) emerged as the most active compounds of the series. These compounds have shown most potent antibacterial activity against the tested organisms of Proteus vulgaris and Bacillus subtilis having zone of inhibition values of 1.1 cm and 1.4 cm for compound 9a 1.2 cm and 1.0 cm for compound 9h, respectively.

  19. 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

  20. The role of a parasite-specific allosteric site in the distinctive activation behavior of Eimeria tenella cGMP-dependent protein kinase.

    PubMed

    Salowe, Scott P; Wiltsie, Judyann; Liberator, Paul A; Donald, Robert G K

    2002-04-02

    A cGMP-dependent protein kinase (PKG) was recently identified as an anticoccidial target for the apicomplexan parasite Eimeria tenella [Gurnett, A., Liberator, P. A., Dulski, P., Salowe, S., Donald, R. G. K., Anderson, J., Wiltsie, J., Diaz, C., Harris, G., Chang, B., Darkin-Rattray, S. J., Nare, B., Crumley, T., Blum, P., Misura, A., Tamas, T., Sardana, M., Yuan, J., Biftu, T., and Schmatz, D. (2002) J. Biol. Chem. (in press)]. Unlike the PKGs of higher organisms that have two cGMP binding sites in their regulatory domain, the PKG from Eimeria tenella (Et-PKG) contains three putative cGMP binding sites and has distinctive activation properties, including a very large stimulation by cGMP ( approximately 1000-fold) with significant cooperativity (Hill coefficient of 1.7). During our investigation of Et-PKG activation, we found that 8-substituted cGMP analogues are weak partial activators. For example, 8-NBD-cGMP provides a maximal stimulation of activity of only 20-fold with little evident cooperativity, although cGMP can synergize with the analogue to provide full activation. The results suggest that partial activation is a consequence of restricted binding of 8-NBD-cGMP to a subset of cGMP sites in the enzyme. Site-directed mutagenesis of conserved arginine and glutamate residues in the parasite-specific third cGMP site confirms that this site is an important functional participant in the allosteric regulation of the kinase and that it exhibits very high selectivity against 8-NBD-cGMP. Since the results are consistent with full activation of Et-PKG requiring cyclic nucleotide binding in all three allosteric sites, one role for the additional cGMP site may be to establish a stricter regulatory mechanism for the kinase activity than is present in the PKGs of higher organisms containing only two allosteric sites.

  1. 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...

  2. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  3. 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...

  4. 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...

  5. 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...

  6. 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.

  7. 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.

  8. Quantitative structure-activity relationship for the cleavage of C3/C4-substituted catechols by a prototypal extradiol catechol dioxygenase with broad substrate specificity.

    PubMed

    Ishida, Tetsuo; Tanaka, Hiroyuki; Horiike, Kihachiro

    2004-06-01

    Catechol 2,3-dioxygenase [EC 1.13.11.2] from Pseudomonas putida mt-2 (Mpc) catalyzes the extradiol cleavage of catechol to produce 2-hydroxymuconate semialdehyde. The K(m) values for the catecholic substrate (K(mA)) and O(2) (K(mO2)), and catalytic constants (k(cat)) were kinetically determined for eight C3/C4-substituted catechols at 25 degrees C and pH 6.5 or 7.5. The first pK(a) values (pK(1)) were determined for eleven catechols (pK(1) = 7.26-9.47), correlated with Hammett substituent constants, and electron-withdrawing substituents significantly stabilized the monoanionic species of free catechols. Mpc preferred catechols with non-ionic substituents at the C3 or C4 position. 3-Phenylcatechol, a biphenyl, was cleaved, while 4-tert-butylcatechol was not. The logarithm of k(cat)/K(mA) (substrate specificity constant) exhibited a good linear correlation with pK(1), with the exception of those for 4-halocatechols. The logarithm of k(cat)/K(mO2) showed a good linear correlation with pK(1), with the exception of that of 3-phenylcatechol. These results demonstrate that catechol binding to the Mpc active site, the following O(2) binding, and the activation of the bound O(2) are all sensitive to electronic effects of the substituents. However, k(cat) did not correlate significantly with pK(1). The present study distinguishes clearly between the electronic and the steric effects of catecholic substrates in the reactivity of Mpc, and provides important insight into the mechanistic basis for a vast range of substrate specificities of extradiol dioxygenases.

  9. 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

  10. Photo-inducible cytotoxic and clastogenic activities of 3,6-di-substituted acridines obtained by acylation of proflavine.

    PubMed

    Benchabane, Yohann; Di Giorgio, Carole; Boyer, Gérard; Sabatier, Anne-Sophie; Allegro, Diane; Peyrot, Vincent; De Méo, Michel

    2009-06-01

    The cytotoxicity and photo-enhanced cytotoxicity of a series of 18 3,6-di-substituted acridines were evaluated on both tumour CHO cells and human normal keratinocytes, and compared to their corresponding clastogenicity as assessed by the micronucleus assay. Compounds 2f tert-butyl N-[(6-tert-butoxycarbonylamino)acridin-3-yl]carbamate and 2d N-[6-(pivalamino)acridin-3-yl]pivalamide displayed a specific cytotoxicity on CHO cells. These results suggested that the two derivatives could be considered as interesting candidates for anticancer chemotherapy and hypothesized that the presence of 1,1-dimethylethyl substituents was responsible for a strong nonclastogenic cytotoxicity. Compounds 2b and 2c, on the contrary, displayed a strong clastogenicity. They indicated that the presence of nonbranched aliphatic chains on positions 3 and 6 of the acridine rings tended to induce a significant clastogenic effect. Finally, they established that most of the acridine compounds could be photo-activated by UVA-visible rays and focussed on the significant role of light irradiation on their biological properties.

  11. A natural, single-residue substitution yields a less active peptaibiotic: the structure of bergofungin A at atomic resolution.

    PubMed

    Gessmann, Renate; Axford, Danny; Brückner, Hans; Berg, Albrecht; Petratos, Kyriacos

    2017-02-01

    Bergofungin is a peptide antibiotic that is produced by the ascomycetous fungus Emericellopsis donezkii HKI 0059 and belongs to peptaibol subfamily 2. The crystal structure of bergofungin A has been determined and refined to 0.84 Å resolution. This is the second crystal structure of a natural 15-residue peptaibol, after that of samarosporin I. The amino-terminal phenylalanine residue in samarosporin I is exchanged to a valine residue in bergofungin A. According to agar diffusion tests, this results in a nearly inactive antibiotic peptide compared with the moderately active samarosporin I. Crystals were obtained from methanol solutions of purified bergofungin mixed with water. Although there are differences in the intramolecular hydrogen-bonding scheme of samarosporin I, the overall folding is very similar for both peptaibols, namely 310-helical at the termini and α-helical in the middle of the molecules. Bergofungin A and samarosporin I molecules are arranged in a similar way in both lattices. However, the packing of bergofungin A exhibits a second solvent channel along the twofold axis. This latter channel occurs in the vicinity of the N-terminus, where the natural substitution resides.

  12. 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.

  13. Effects of arginine and leucine substitutions on anti-endotoxic activities and mechanisms of action of cationic and amphipathic antimicrobial octadecapeptide from rice α-amylase.

    PubMed

    Taniguchi, Masayuki; Ochiai, Akihito; Toyoda, Ryu; Sato, Teppei; Saitoh, Eiichi; Kato, Tetsuo; Tanaka, Takaaki

    2017-03-01

    Previously, we showed that the antimicrobial cationic and amphipathic octadecapeptide AmyI-1-18 from rice α-amylase (AmyI-1) inhibited the endotoxic activity of lipopolysaccharide (LPS) from Escherichia coli. In addition, we demonstrated that several AmyI-1-18 analogs containing arginine or leucine substitutions, which were designed on the basis of the helical wheel projection of AmyI-1-18, exhibited higher antimicrobial activity against human pathogenic microorganisms than AmyI-1-18. In the present study, anti-inflammatory (anti-endotoxic) activities of five AmyI-1-18 analogs containing arginine or leucine substitutions were investigated. Two single arginine-substituted and two single leucine-substituted AmyI-1-18 analogs inhibited the production of LPS-induced nitric oxide in mouse macrophages (RAW264) more effectively than AmyI-1-18. These data indicate that enhanced cationic and hydrophobic properties of AmyI-1-18 are associated with improved anti-endotoxic activity. In subsequent chromogenic Limulus amebocyte lysate assays, 50% inhibitory concentrations (IC50 ) of the three AmyI-1-18 analogs (G12R, D15R, and E9L) were 0.11-0.13 μm, indicating higher anti-endotoxic activity than that of AmyI-1-18 (IC50, 0.22 μm), and specific LPS binding activity. In agreement, surface plasmon resonance analyses confirmed direct LPS binding of three AmyI-1-18 analogs. In addition, AmyI-1-18 analogs exhibited little or no cytotoxic activity against RAW264 cells, indicating that enhancements of anti-inflammatory and LPS-neutralizing activities following replacement of arginine or leucine did not result in significant increases in cytotoxicity. This study shows that the arginine-substituted and leucine-substituted AmyI-1-18 analogs with improved anti-endotoxic and antimicrobial activities have clinical potential as dual-function host defense agents. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

  14. [The effect of intercultivar substitution of wheat Triticum aestivum L. chromosomes on lipoxygenase activity and its correlation with the technological properties of flour].

    PubMed

    Trufanov, V A; Permiakova, M D; Pshenichnikova, T A; Ermakova, M F; Davydov, V A; Permiakov, A V; Berezovskaia, E V

    2007-01-01

    The effects of intercultivar substitution of individual chromosome pairs (except for 1B, 6D, and 7A) in the wheat cultivars Saratovskaya 29 and Janetzkis Probat, differing in quality, on specific lipoxygenase activity, the grain yield per spike, and the main technological properties of flour and dough were studied. It was demonstrated that the substitution of individual chromosomes of the recipient cultivar Saratovskaya 29 with the homologous chromosomes of the donor cultivar Janetzkis Probat caused significant changes in lipoxygenase activity and several other quality characteristics. The correlations between the lipoxygenase activity and the parameters of physical dough properties were determined. Three molecular forms of lipoxygenase (Lpx-1, Lpx-2, and Lpx-3), differing in the value of surface charge and enzymatic activity, were detected by native PAGE.

  15. Temperature-induced conformational change at the catalytic site of Sulfolobus solfataricus alcohol dehydrogenase highlighted by Asn249Tyr substitution. A hydrogen/deuterium exchange, kinetic, and fluorescence quenching study.

    PubMed

    Secundo, Francesco; Russo, Consiglia; Giordano, Antonietta; Carrea, Giacomo; Rossi, Mosè; Raia, Carlo A

    2005-08-23

    A combination of hydrogen/deuterium exchange, fluorescence quenching, and kinetic studies was used to acquire experimental evidence for the crystallographically hypothesized increase in local flexibility which occurs in thermophilic NAD(+)-dependent Sulfolobus solfataricus alcohol dehydrogenase (SsADH) upon substitution Asn249Tyr. The substitution, located at the adenine-binding site, proved to decrease the affinity for both coenzyme and substrate, rendering the mutant enzyme 6-fold more active when compared to the wild-type enzyme [Esposito et al. (2003) FEBS Lett. 539, 14-18]. The amide H/D exchange data show that the wild-type and mutant enzymes have similar global flexibility at 22 and 60 degrees C. However, the temperature dependence of the Stern-Volmer constant determined by acrylamide quenching shows that the increase in temperature affects the local flexibility differently, since the K(SV) increment is significantly higher for the wild-type than for the mutant enzyme over the range 18-45 degrees C. Interestingly, the corresponding van't Hoff plot (log K(SV) vs 1/T) proves nonlinear for the apo and holo wild-type and apo mutant enzymes, with a break at approximately 45 degrees C in all three cases due to a conformational change affecting the tryptophan microenvironment experienced by the quencher molecules. The Arrhenius and van't Hoff plots derived from the k(cat) and K(M) thermodependence measured with cyclohexanol and NAD(+) at different temperatures display an abrupt change of slope at 45-50 degrees C. This proves more pronounced in the case of the mutant enzyme compared to the wild-type enzyme due to a conformational change in the structure rather than to an overlapping of two or more rate-limiting reaction steps with different temperature dependencies of their rate constants. Three-dimensional analysis indicates that the observed conformational change induced by temperature is associated with the flexible loops directly involved in the substrate and

  16. Lidar research activities and observations at NARL site, Gadanki, India

    NASA Astrophysics Data System (ADS)

    Yellapragada, Bhavani Kumar

    2016-05-01

    The National Atmospheric Research Laboratory (NARL), a unit of Department of Space (DOS), located at Gadanki village (13.5°N, 79.2°E, 370 m AMSL) in India, is involved in the development of lidar remote sensing technologies for atmospheric research. Several advanced lidar technologies employing micropulse, polarization, Raman and scanning have been developed at this site and demonstrated for atmospheric studies during the period between 2008 and 2015. The technology of micropulse lidar, operates at 532 nm wavelength, was successfully transferred to an industry and the commercial version has been identified for Indian Lidar network (I-LINK) programme. Under this lidar network activity, several lidar units were installed at different locations in India to study tropospheric aerosols and clouds. The polarization sensitive lidar technology was realized using a set of mini photomultiplier tube (PMT) units and has the capability to operate during day and night without a pause. The lidar technology uses a compact flashlamp pumped Qswitched laser and employs biaxial configuration between the transmitter and receiver units. The lidar technology has been utilized for understanding the polarization characteristics of boundary layer aerosols during the mixed layer development. The demonstrated Raman lidar technology, uses the third harmonic wavelength of Nd:YAG laser, provides the altitude profiles of aerosol backscattering, extinction and water vapor covering the boundary layer range and allows operation during nocturnal periods. The Raman lidar derived height profiles of aerosol backscattering and extinction coefficient, lidar ratio, and watervapor mixing ratio inform the tropical boundary layer aerosol characteristics. The scanning lidar technology uses a near infrared laser wavelength for probing the lower atmosphere and has been utilized for high resolution cloud profiling during convective periods. The lidar technology is also used for rain rate measurement during

  17. 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.

  18. Active site inhibitors of HCV NS5B polymerase. The development and pharmacophore of 2-thienyl-5,6-dihydroxypyrimidine-4-carboxylic acid.

    PubMed

    Stansfield, Ian; Avolio, Salvatore; Colarusso, Stefania; Gennari, Nadia; Narjes, Frank; Pacini, Barbara; Ponzi, Simona; Harper, Steven

    2004-10-18

    5,6-Dihydroxypyrimidine-4-carboxylic acids are a promising series of hepatitis C virus (HCV) NS5B polymerase inhibitors that bind at the active site of the enzyme. Here we report a simple 2-thienyl substituted analogue that shows 10-fold improved activity over the original lead, and which allowed us to further delineate the key elements of the pharmacophore of this class of inhibitor. This work led to the identification of a trifluoromethyl acylsulfonamide group as a viable replacement for the C4 carboxylic acid in this series.

  19. Mutational analysis of active site contact residues in anti-fluorescein monoclonal antibody 4-4-20.

    PubMed

    Denzin, L K; Gulliver, G A; Voss, E W

    1993-10-01

    The contribution to high affinity Fl binding by each crystallographically defined Mab 4-4-20 (Ka = 1.7 x 10(10) M-1; Qmax = 90%) ligand contact residue (L27dHis, L32Tyr, L34Arg, L91Ser, L96Trp and H33Trp) has been determined by site-specific mutagenesis studies. All six antigen contact residues were changed to Ala in the single-chain derivative of Mab 4-4-20 and following expression in E. coli, denaturation, refolding and purification, each SCA mutant was characterized in terms of Fl binding affinity, Qmax, lambda max and idiotype. Results demonstrated that Ala substitutions at each ligand contact residue reduced the binding affinities and quenching maxima for all residues except L27d which retained wild type characteristics. The SCA TyrL32Ala, SerL91Ala and TrpH33Ala mutants exhibited binding affinities that were approximately 1000-fold lower than the wild type value and greatly reduced Qmax values. Additionally, other amino acid substitutions were performed at three of the six antigen contact residues (L91Ser, L96Trp and H33Trp) to further evaluate the role of each in Fl binding. Therefore, the following mutations were constructed and characterized: SerL91Asn, TrpL96Tyr, TrpL96Phe, TrpL96Leu, TrpH33Tyr and TrpH33Phe. Results of site-specific mutagenesis studies are discussed in terms of Mab active site structure and suggest that L32Tyr, L91Ser and H33Trp are important for high affinity Fl binding and efficient Fl quenching.

  20. Relaxor or classical ferroelectric behavior in A-site substituted perovskite type Ba 1- x(Sm 0.5Na 0.5) xTiO 3

    NASA Astrophysics Data System (ADS)

    Abdelmoula, N.; Chaabane, H.; Khemakhem, H.; Von der Mühll, R.; Simon, A.

    2006-08-01

    New ferroelectric ceramics of ABO 3 perovskite type were synthetized in the Ba 1- x(Sm 0.5Na 0.5) xTiO 3 system by solid state reaction technique. The effect of the replacement of barium by samarium and sodium in the A cationic site on structural and physical properties was investigated. These compounds crystallize with tetragonal or cubic symmetry. The material is classical ferroelectric for 0⩽x⩽0.1 and x⩾0.5, and present a relaxor behavior for 0.2⩽x<0.4. The dielectric behavior depends upon the cationic disorder in the A-site and the cell size. Small rate substitution allows a ferroelectric-paraelectric transition. For higher rate of substitution the possible random position of the Sm-Na cations brings to a relaxor state and when the substitution rate x becomes higher than 0.5, the material comes back to a ferroelectric state due to the reduced cell size. Some of these new compositions are of interest for applications due to their physical properties and environmentally friendly character.

  1. Active-site zinc ligands and activated H2O of zinc enzymes.

    PubMed Central

    Vallee, B L; Auld, D S

    1990-01-01

    The x-ray crystallographic structures of 12 zinc enzymes have been chosen as standards of reference to identify the ligands to the catalytic and structural zinc atoms of other members of their respective enzyme families. Universally, H2O is a ligand and critical component of the catalytically active zinc sites. In addition, three protein side chains bind to the catalytic zinc atom, whereas four protein ligands bind to the structural zinc atom. The geometry and coordination number of zinc can vary greatly to accommodate particular ligands. Zinc forms complexes with nitrogen and oxygen just as readily as with sulfur, and this is reflected in catalytic zinc sites having a binding frequency of His much greater than Glu greater than Asp = Cys, three of which bind to the metal atom. The systematic spacing between the ligands is striking. For all catalytic zinc sites except the coenzyme-dependent alcohol dehydrogenase, the first two ligands are separated by a "short-spacer" consisting of 1 to 3 amino acids. These ligands are separated from the third ligand by a "long spacer" of approximately 20 to approximately 120 amino acids. The spacer enables formation of a primary bidentate zinc complex, whereas the long spacer contributes flexibility to the coordination sphere, which can poise the zinc for catalysis as well as bring other catalytic and substrate binding groups into apposition with the active site. The H2O is activated by ionization, polarization, or poised for displacement. Collectively, the data imply that the preferred mechanistic pathway for activating the water--e.g., zinc hydroxide or Lewis acid catalysis--will be determined by the identity of the other three ligands and their spacing. Images PMID:2104979

  2. QSAR modeling of aromatase inhibitory activity of 1-substituted 1,2,3-triazole analogs of letrozole.

    PubMed

    Nantasenamat, Chanin; Worachartcheewan, Apilak; Prachayasittikul, Supaluk; Isarankura-Na-Ayudhya, Chartchalerm; Prachayasittikul, Virapong

    2013-11-01

    Aromatase is an estrogen biosynthesis enzyme belonging to the cytochrome P450 family that catalyzes the rate-limiting step of converting androgens to estrogens. As it is pertinent toward tumor cell growth promotion, aromatase is a lucrative therapeutic target for breast cancer. In the pursuit of robust aromatase inhibitors, a set of fifty-four 1-substituted mono- and bis-benzonitrile or phenyl analogs of 1,2,3-triazole letrozole were employed in quantitative structure-activity relationship (QSAR) study using multiple linear regression (MLR), artificial neural network (ANN) and support vector machine (SVM). Such QSAR models were developed using a set of descriptors providing coverage of the general characteristics of a molecule encompassing molecular size, flexibility, polarity, solubility, charge and electronic properties. Important physicochemical properties giving rise to good aromatase inhibition were obtained by means of exploring its chemical space as a function of the calculated molecular descriptors. The optimal subset of 3 descriptors (i.e. number of rings, ALogP and HOMO-LUMO) was further used for QSAR model construction. The predicted pIC₅₀ values were in strong correlation with their experimental values displaying correlation coefficient values in the range of 0.72-0.83 for the cross-validated set (QCV) while the external test set (Q(Ext)) afforded values in the range of 0.65-0.66. Insights gained from the present study are anticipated to provide pertinent information contributing to the origins of aromatase inhibitory activity and therefore aid in our on-going quest for aromatase inhibitors with robust properties.

  3. Active site of the mRNA-capping enzyme guanylyltransferase from Saccharomyces cerevisiae: similarity to the nucleotidyl attachment motif of DNA and RNA ligases.

    PubMed Central

    Fresco, L D; Buratowski, S

    1994-01-01

    Nascent mRNA chains are capped at the 5' end by the addition of a guanylyl residue to form a G(5')ppp(5')N ... structure. During the capping reaction, the guanylyltransferase (GTP:mRNA guanylyltransferase, EC 2.7.7.50) is reversibly and covalently guanylylated. In this enzyme-GMP (E-GMP) intermediate, GMP is linked to the epsilon-amino group of a lysine residue via a phosphoamide bond. Lys-70 was identified as the GMP attachment site of the Saccharomyces cerevisiae guanylyltransferase (encoded by the CEG1 gene) by guanylylpeptide sequencing. CEG1 genes with substitutions at Lys-70 were unable to support viability in yeast and produced proteins that were not guanylylated in vitro. The CEG1 active site exhibits sequence similarity to the active sites of viral guanylyltransferases and polynucleotide ligases, suggesting similarity in the mechanisms of nucleotidyl transfer catalyzed by these enzymes. Images PMID:8022828

  4. Oxalate decarboxylase and oxalate oxidase activities can be interchanged with a specificity switch of up to 282,000 by mutating an active site lid.

    PubMed

    Burrell, Matthew R; Just, Victoria J; Bowater, Laura; Fairhurst, Shirley A; Requena, Laura; Lawson, David M; Bornemann, Stephen

    2007-10-30

    Oxalate decarboxylases and oxalate oxidases are members of the cupin superfamily of proteins that have many common features: a manganese ion with a common ligand set, the substrate oxalate, and dioxygen (as either a unique cofactor or a substrate). We have hypothesized that these enzymes share common catalytic steps that diverge when a carboxylate radical intermediate becomes protonated. The Bacillus subtilis decarboxylase has two manganese binding sites, and we proposed that Glu162 on a flexible lid is the site 1 general acid. We now demonstrate that a decarboxylase can be converted into an oxidase by mutating amino acids of the lid that include Glu162 with specificity switches of 282,000 (SEN161-3DAS), 275,000 (SENS161-4DSSN), and 225,000 (SENS161-4DASN). The structure of the SENS161-4DSSN mutant showed that site 2 was not affected. The requirement for substitutions other than of Glu162 was, at least in part, due to the need to decrease the Km for dioxygen for the oxidase reaction. Reversion of decarboxylase activity could be achieved by reintroducing Glu162 to the SENS161-4DASN mutant to give a relative specificity switch of 25,600. This provides compelling evidence for the crucial role of Glu162 in the decarboxylase reaction consistent with it being the general acid, for the role of the lid in controlling the Km for dioxygen, and for site 1 being the sole catalytically active site. We also report the trapping of carboxylate radicals produced during turnover of the mutant with the highest oxidase activity. Such radicals were also observed with the wild-type decarboxylase.

  5. How RNase HI (Escherichia coli) promoted site-selective hydrolysis works on RNA in duplex with carba-LNA and LNA substituted antisense strands in an antisense strategy context?

    PubMed

    Plashkevych, Oleksandr; Li, Qing; Chattopadhyaya, Jyoti

    2017-03-29

    A detailed kinetic study of 36 single modified AON-RNA heteroduplexes shows that substitution of a single native nucleotide in the antisense strand (AON) by locked nucleic acid (LNA) or by diastereomerically pure carba-LNA results in site-dependent modulation of RNase H promoted cleavage of complementary mRNA strands by 2 to 5 fold at 5'-GpN-3' cleavage sites, giving up to 70% of the RNA cleavage products. The experiments have been performed using RNase HI of Escherichia coli. The 2nd best cleavage site, being the 5'-ApN-3' sites, cleaves up to 23%, depending upon the substitution site in 36 isosequential complementary AONs. A comparison of the modified AON promoted RNA cleavage rates with that of the native AON shows that sequence-specificity is considerably enhanced as a result of modification. Clearly, relatively weaker 5'-purine (Pu)-pyrimidine (Py)-3' stacking in the complementary RNA strand is preferred (giving ∼90% of total cleavage products), which plays an important role in RNase H promoted RNA cleavage. A plausible mechanism of RNase H mediated cleavage of the RNA has been proposed to be two-fold, dictated by the balancing effect of the aromatic character of the purine aglycone: first, the locally formed 9-guanylate ion (pKa 9.3, ∼18-20% N1 ionized at pH 8) alters the adjoining sugar-phosphate backbone around the scissile phosphate, transforming its sugar N/S conformational equilibrium, to preferential S-type, causing preferential cleavage at 5'-GpN-3' sites around the center of 20 mer complementary mRNA. Second, the weaker nearest-neighbor strength of 5'-Pu-p-Py-3' stacking promotes preferential 5'-GpN-3' and 5'-ApN-3' cleavage, providing ∼90% of the total products, compared to ∼50% in that of the native one, because of the cLNA/LNA substituent effect on the neighboring 5'-Pu-p-Py-3' sites, providing both local steric flexibility and additional hydration. This facilitates both the water and water/Mg(2+) ion availability at the cleavage site

  6. The yeast regulator of transcription protein Rtr1 lacks an active site and phosphatase activity.

    PubMed

    Xiang, Kehui; Manley, James L; Tong, Liang

    2012-07-10

    The activity of RNA polymerase II (Pol II) is controlled in part by the phosphorylation state of the C-terminal domain (CTD) of its largest subunit. Recent reports have suggested that yeast regulator of transcription protein, Rtr1, and its human homologue RPAP2, possess Pol II CTD Ser5 phosphatase activity. Here we report the crystal structure of Kluyveromyces lactis Rtr1, which reveals a new type of zinc finger protein and does not have any close structural homologues. Importantly, the structure does not show evidence of an active site, and extensive experiments to demonstrate its CTD phosphatase activity have been unsuccessful, suggesting that Rtr1 has a non-catalytic role in CTD dephosphorylation.

  7. Mutational analysis of the active site flap (20s loop) of mandelate racemase.

    PubMed

    Bourque, Jennifer R; Bearne, Stephen L

    2008-01-15

    with kcat/Km values approximately 2-fold greater than those observed for wild-type mandelate racemase. Although minor changes in substrate specificity were achieved through alterations of the active site flap of mandelate racemase, our results suggest that hydrophobic residues that reside on a flexible flap and define the topology of an active site through their van der Waals contacts with the substrate are quite tolerant of a variety of steric substitutions.

  8. 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.

  9. 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.

  10. 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.

  11. Primary amino acid derivatives: substitution of the 4'-N'-benzylamide site in (R)-N'-benzyl 2-amino-3-methylbutanamide, (R)-N'-benzyl 2-amino-3,3-dimethylbutanamide, and (R)-N'-benzyl 2-amino-3-methoxypropionamide provides potent anticonvulsants with pain-attenuating properties.

    PubMed

    King, Amber M; Salomé, Christophe; Salomé-Grosjean, Elise; De Ryck, Marc; Kaminski, Rafal; Valade, Anne; Stables, James P; Kohn, Harold

    2011-10-13

    Recently, we reported that select N'-benzyl 2-substituted 2-amino acetamides (primary amino acid derivatives (PAADs)) exhibited pronounced activities in established whole animal anticonvulsant (i.e., maximal electroshock seizure (MES)) and neuropathic pain (i.e., formalin) models. The anticonvulsant activities of C(2)-hydrocarbon N'-benzyl 2-amino acetamides (MES ED(50) = 13-21 mg/kg) exceeded those of phenobarbital (ED(50) = 22 mg/kg). Two additional studies defining the structure-activity relationship of PAADs are presented in this issue of the journal. In this study, we demonstrated that the anticonvulsant activities of (R)-N'-benzyl 2-amino-3-methylbutanamide and (R)-N'-benzyl 2-amino-3,3-dimethylbutanamide were sensitive to substituents at the 4'-N'-benzylamide site; electron-withdrawing groups retained activity, electron-donating groups led to a loss of activity, and incorporating either a 3-fluorobenzyloxy or 3-fluorophenoxymethyl group using a rationally designed multiple ligand approach improved activity. Additionally, we showed that substituents at the 4'-N'-benzylamide site of (R)-N'-benzyl 2-amino-3-methoxypropionamide also improved anticonvulsant activity, with the 3-fluorophenoxymethyl group providing the largest (∼4-fold) increase in activity (ED(50) = 8.9 mg/kg), a value that surpassed phenytoin (ED(50) = 9.5 mg/kg). Collectively, the pharmacological findings provided new information that C(2)-hydrocarbon PAADs represent a novel class of anticonvulsants.

  12. The Ala95-to-Gly substitution in Aerococcus viridans l-lactate oxidase revisited - structural consequences at the catalytic site and effect on reactivity with O2 and other electron acceptors.

    PubMed

    Stoisser, Thomas; Rainer, Daniela; Leitgeb, Stefan; Wilson, David K; Nidetzky, Bernd

    2015-02-01

    Aerococcus viridansl-lactate oxidase (avLOX) is a biotechnologically important flavoenzyme that catalyzes the conversion of L-lactate and O₂ into pyruvate and H₂O₂. The enzymatic reaction underlies different biosensor applications of avLOX for blood L-lactate determination. The ability of avLOX to replace O₂ with other electron acceptors such as 2,6-dichlorophenol-indophenol (DCIP) allows the possiblity of analytical and practical applications. The A95G variant of avLOX was previously shown to exhibit lowered reactivity with O₂ compared to wild-type enzyme and therefore was employed in a detailed investigation with respect to the specificity for different electron acceptor substrates. From stopped-flow experiments performed at 20 °C (pH 6.5), we determined that the A95G variant (fully reduced by L-lactate) was approximately three-fold more reactive towards DCIP (1.0 ± 0.1 × 10(6) M(-1) ·s(-1) ) than O₂, whereas avLOX wild-type under the same conditions was 14-fold more reactive towards O₂(1.8 ± 0.1 × 10(6) m(-1) ·s(-1)) than DCIP. Substituted 1,4-benzoquinones were up to five-fold better electron acceptors for reaction with L-lactate-reduced A95G variant than wild-type. A 1.65-Å crystal structure of oxidized A95G variant bound with pyruvate was determined and revealed that the steric volume created by removal of the methyl side chain of Ala95 and a slight additional shift in the main chain at position Gly95 together enable the accomodation of a new active-site water molecule within hydrogen-bond distance to the N5 of the FMN cofactor. The increased steric volume available in the active site allows the A95G variant to exhibit a similar trend with the related glycolate oxidase in electron acceptor substrate specificities, despite the latter containing an alanine at the analogous position.

  13. Synthesis, analgesic, anti-inflammatory and antibacterial activities of some novel 2-phenyl-3-substituted quinazolin-4(3H) ones.

    PubMed

    Alagarsamy, Veerachamy; Salomon, Viswas Raja; Vanikavitha, Gnanavel; Paluchamy, Veeran; Chandran, Muniyandi Ravi; Sujin, Augustin Arnald; Thangathiruppathy, Arunachalam; Amuthalakshmi, Sivaperuman; Revathi, Rajappan

    2002-11-01

    A series of novel 2-phenyl-3-substituted quinazolin-4(3H)-ones have been synthesized by treating methyl-N-(2-phenyl quinazolin-3-yl-4(3H)-one) dithiocarbamate with different amines, the starting material dithiocarbamate was synthesized from anthranilic acid. The title compounds were investigated for analgesic, anti-inflammatory and antibacterial activities. All the test compounds exhibited significant activity, the compounds A1, A2 and A3 shown more potent analgesic activity, and the compound A3 shown more potent anti-inflammatory activity than the reference standard diclofenac sodium.

  14. Cobalt triarylcorroles containing one, two or three nitro groups. Effect of NO₂ substitution on electrochemical properties and catalytic activity for reduction of molecular oxygen in acid media.

    PubMed

    Li, Bihong; Ou, Zhongping; Meng, Deying; Tang, Jijun; Fang, Yuanyuan; Liu, Rui; Kadish, Karl M

    2014-07-01

    Cobalt(III) triarylcorroles containing 0-3 nitro groups on the para-position of the three meso-phenyl rings of the macrocycle were synthesized and characterized by electrochemistry, mass spectrometry, (UV-vis) and (1)H NMR spectroscopy. The examined compounds are represented as (NO2Ph)(n)Ph(3-n)CorCo(PPh3), where n varies from 0 to 3 and Cor represents the core of the corrole. Each compound can undergo two metal-centered one-electron reductions leading to formation of Co(II) and Co(I) derivatives in CH2Cl2 or pyridine containing 0.1 M tetra-n-butylammonium perchlorate (TBAP). A stepwise two electron reduction of each NO2Ph group of the compound is also observed. The first is reversible and occurs in a single overlapping step at the same potential which involves an overall one-, two- or three-electron transfer process for compounds 2-4, respectively. This indicates the lack of an interaction between these redox active sites on the corroles. The second reduction of the NO2Ph groups is irreversible and located at a potential which overlaps the Co(II)/Co(I) process of the compounds. Thin-layer UV-visible spectroelectrochemical measurements in CH2Cl2, 0.1 M TBAP demonstrate the occurrence of an equilibrium between a Co(III) π-anion radical and a Co(II) derivative with an uncharged macrocycle after the first controlled potential reduction of the nitro-substituted corroles. All four cobalt corroles were also examined as catalysts for the electroreduction of O2 when coated on an edge-plane pyrrolytic graphite electrode in 1.0 M HClO4. This study indicates that the larger the number of nitro-substituents on the cobalt corrole, the better the compound acts as a catalyst.

  15. Iron Acyl Thiolato Carbonyls: Structural Models for the Active Site of the [Fe]-Hydrogenase (Hmd)

    PubMed Central

    Royer, Aaron M.; Salomone-Stagni, Marco

    2012-01-01

    Phosphine-modified thioester derivatives are shown to serve as efficient precursors to phosphine-stabilized ferrous acyl thiolato carbonyls via the reaction of phosphine thioesters and sources of Fe(0). The reaction generates both Fe(SPh)(Ph2PC6H4CO)(CO)3 (1) and the diferrous diacyl Fe2(SPh)2(CO)3(Ph2PC6H4CO)2, which carbonylates to give 1. For the extremely bulky arylthioester Ph2PC6H4C(O)SC6H4-2,6-(2,4,6-trimethylphenyl)2, oxidative addition is arrested and the Fe(0) adduct of the phosphine is obtained. Complex 1 reacts with cyanide to give Et4N[Fe(SPh)(Ph2PC6H4CO)(CN)(CO)2] (Et4N[2]). 13C and 31P NMR spectra indicate that substitution is stereospecific and cis to P. The IR spectrum of [2]− in CH2Cl2 solution very closely matches that for HmdCN. XANES and EXAFS measurements also indicate close structural and electronic similarity of Et4N[2] to the active site of wild-type Hmd. Complex 1 also stereospecifically forms a derivative with TsCH2NC, but the adduct is more labile than Et4N[2]. Tricarbonyl 1 was found to reversibly protonate to give a thermally labile derivative, IR measurements of which indicate that the acyl and thiolate ligands are probably not protonated in Hmd. PMID:21062066

  16. Four novel mutations in mucopolysaccharidosis type VII including a unique base substitution in exon 10 of the beta-glucuronidase gene that creates a novel 5'-splice site.

    PubMed

    Yamada, S; Tomatsu, S; Sly, W S; Islam, R; Wenger, D A; Fukuda, S; Sukegawa, K; Orii, T

    1995-04-01

    Mucopolysaccharidosis type VII (MPS VII), or Sly syndrome, is a lysosomal storage disorder caused by a deficiency in the enzyme beta-glucuronidase. Various clinical phenotypes of this autosomal recessively inherited disease have been described. Recent isolation and characterization of human beta-glucuronidase cDNA and the genomic sequences facilitate analysis of molecular defects underlying the different phenotypes, and eight mutations in the beta-glucuronidase gene have been described. This report summarizes studies characterizing four new mutations in two Caucasian patients with a severe form of MPS VII. Three are point mutations, resulting in two missense and one nonsense change, and one is a 38 bp deletion. The first patient was a compound heterozygote having P148S and Y495C alleles. The second patient was a compound heterozygote of W507X and a 38 bp deletion at position 1642-1679 in exon 10(1642 delta 38nt). The 38 bp deletion was caused by a single base change mutation in exon 10 that generates a new, premature 5' splice site. Expression of mutant cDNAs encoding each of the four mutations showed that all four resulted in a severe reduction of beta-glucuronidase activity, indicating that these mutations are responsible for the reduced enzyme activity in patient cells. These four previously undescribed mutations provide further evidence for the broad molecular heterogeneity in Sly syndrome.

  17. 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.

  18. Ground motion estimation for the elevated bridges of the Kyushu Shinkansen derailment caused by the foreshock of the 2016 Kumamoto earthquake based on the site-effect substitution method

    NASA Astrophysics Data System (ADS)

    Hata, Yoshiya; Yabe, Masaaki; Kasai, Akira; Matsuzaki, Hiroshi; Takahashi, Yoshikazu; Akiyama, Mitsuyoshi

    2016-12-01

    An earthquake of JMA magnitude 6.5 (first event) hit Kumamoto Prefecture, Japan, at 21:26 JST, April 14, 2016. Subsequently, an earthquake of JMA magnitude 7.3 (second event) hit Kumamoto and Oita Prefectures at 01:46 JST, April 16, 2016. An out-of-service Kyushu Shinkansen train carrying no passengers traveling on elevated bridges was derailed by the first event. This was the third derailment caused by an earthquake in the history of the Japanese Shinkansen, after one caused by the 2004 Mid-Niigata Prefecture Earthquake and another triggered by the 2011 Tohoku Earthquake. To analyze the mechanism of this third derailment, it is crucial to evaluate the strong ground motion at the derailment site with high accuracy. For this study, temporary earthquake observations were first carried out at a location near the bridge site; these observations were conducted because although the JMA Kumamoto Station site and the derailment site are closely located, the ground response characteristics at these sites differ. Next, empirical site amplification and phase effects were evaluated based on the obtained observation records. Finally, seismic waveforms during the first event at the bridge site of interest were estimated based on the site-effect substitution method. The resulting estimated acceleration and velocity waveforms for the derailment site include much larger amplitudes than the waveforms recorded at the JMA Kumamoto and MLIT Kumamoto station sites. The reliability of these estimates is confirmed by the finding that the same methods reproduce strong ground motions at the MLIT Kumamoto Station site accurately. These estimated ground motions will be useful for reasonable safety assessment of anti-derailment devices on elevated railway bridges.[Figure not available: see fulltext.

  19. DNA, protein binding, cytotoxicity, cellular uptake and antibacterial activities of new palladium(II) complexes of thiosemicarbazone ligands: effects of substitution on biological activity.

    PubMed

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

    2012-01-01

    The coordination propensities of 4(N,N')-diethylaminosalicylaldehyde-4(N)-substituted thiosemicarbazones (H(2)L(1-4)) were investigated by reacting with an equimolar amount of [PdCl(2)(PPh(3))(2)]. The new complexes were characterized by various spectroscopic techniques. The structure determination of the complexes [Pd(DeaSal-tsc)(PPh(3))] (1), [Pd(DeaSal-mtsc)(PPh(3))] (2) and [Pd(DeaSal-etsc)(PPh(3))] (3) by X-ray crystallography showed that ligands are coordinated in a dibasic tridentate ONS donor fashion forming stable five and six membered chelate rings. The binding ability of complexes (1-4) to calf-thymus DNA (CT DNA) has been explored by absorption and emission titration methods. Based on the observations, an electrostatic and an intercalative binding mode have been proposed. The protein binding studies have been monitored by quenching of tryptophan and tyrosine residues in the presence of complexes using lysozyme as a model protein. As determined by MTT assays, complex 3 exhibited a higher cytotoxic effect towards human lung cancer cell line (A549) and liver cancer cells (HepG2). LDH, NO assay and cellular uptake of the complexes have been studied. Further, antibacterial activity studies of the complexes have been screened against the pathogenic bacteria such as Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, MIC50 values of the complexes showed that the complexes exhibited significant activity against the pathogens and among the complexes, 3 exhibited higher activity.

  20. Substituted phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonamides as antimitotics. Antiproliferative, antiangiogenic and antitumoral activity, and quantitative structure-activity relationships.

    PubMed

    Fortin, Sébastien; Wei, Lianhu; Moreau, Emmanuel; Lacroix, Jacques; Côté, Marie-France; Petitclerc, Eric; Kotra, Lakshmi P; Gaudreault, René C

    2011-11-01

    The importance of the bridge linking the two phenyl moieties of substituted phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates (PIB-SOs) was assessed using a sulfonamide group, which is a bioisostere of sulfonate and ethenyl groups. Forty one phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonamide (PIB-SA) derivatives were prepared and biologically evaluated. PIB-SAs exhibit antiproliferative activities at the nanomolar level against sixteen cancer cell lines, block the cell cycle progression in G(2)/M phase, leading to cytoskeleton disruption and anoikis. These results were subjected to CoMFA and CoMSIA analyses to establish quantitative structure-activity relationships. These results evidence that the sulfonate and sulfonamide moieties are reciprocal bioisosteres and that phenylimidazolidin-2-one could mimic the trimethoxyphenyl moiety found in the structure of numerous potent antimicrotubule agents. Finally, compounds 16 and 17 exhibited potent antitumor and antiangiogenic activities on HT-1080 fibrosarcoma cells grafted onto chick chorioallantoic membrane similar to CA-4 without significant toxicity for the chick embryos, making this class of compounds a promising class of anticancer agents.

  1. 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.

  2. Design synthesis and structure–activity relationship of 5-substituted (tetrahydronaphthalen-2yl)methyl with N-phenyl-N-(piperidin-2-yl) propionamide derivatives as opioid ligands

    PubMed Central

    Deekonda, Srinivas; Rankin, David; Davis, Peg; Lai, Josephine; Vanderah, Todd. W.; Porecca, Frank; Hruby, Victor J.

    2016-01-01

    Here, we report the design, synthesis and structure activity relationship of novel small molecule opioid ligands based on 5-amino substituted (tetrahydronaphthalen-2-yl)methyl moiety with N-phenyl-N-(piperidin-2-yl)propionamide derivatives. We synthesized various molecules including amino, amide and hydroxy substitution on the 5th position of the (tetrahydronaphthalen-2-yl)methyl moiety. In our further designs we replaced the (tetrahydronaphthalen-2-yl)methyl moiety with benzyl and phenethyl moiety. These N-phenyl-N-(piperidin-2-yl)propionamide analogues showed moderate to good binding affinities (850–4 nM) and were selective towards the μ opioid receptor over the δ opioid receptors. From the structure activity relationship studies, we found that a hydroxyl substitution at the 5th position of (tetrahydronapthalen-2yl)methyl group, ligands 19 and 20, showed excellent binding affinities 4 and 5 nM, respectively, and 1000 fold selectivity towards the μ opioid relative to the delta opioid receptor. The ligand 19 showed potent agonist activities 75 ± 21 nM, and 190 ± 42 nM in the GPI and MVD assays. Surprisingly the fluoro analogue 20 showed good agonist activities in MVD assays 170 ± 42 nM, in contrast to its binding affinity results. PMID:26712115

  3. Synthesis of 3' '-substituted TSAO derivatives with anti-HIV-1 and anti-HIV-2 activity through an efficient palladium-catalyzed cross-coupling approach.

    PubMed

    Lobatón, Esther; Rodríguez-Barrios, Fátima; Gago, Federico; Pérez-Pérez, María-Jesús; De Clercq, Erik; Balzarini, Jan; Camarasa, María-José; Velázquez, Sonsoles

    2002-08-29

    Various synthetic studies for the introduction of several functional groups at position 3' ' of the spiro moiety of TSAO derivatives have been explored. Among them, Stille cross-coupling of 3' '-iodo-TSAO derivatives with different stannanes provided an efficient and straightforward route for the direct and selective functionalization of the 3' '-position of the sultone spiro moiety via carbon-carbon bond formation. The compounds synthesized were evaluated for their inhibitory effect on HIV-1 and HIV-2 replication in cell culture. The introduction of a bromine and particularly an iodine at the 3' '-position conferred the highest anti-HIV-1 activity. In contrast, the presence at this position of (un)substituted vinyl, alkynyl, phenyl, or thienyl groups markedly diminished the anti-HIV-1 activity. Surprisingly, several of the 3' '-alkenyl-substituted TSAO derivatives also gained anti-HIV-2 activity at subtoxic concentrations, an observation that is very unusual for NNRTIs and never observed before for TSAO derivatives. Finally, the anti-HIV-1 activity of some of the 3' '-substituted TSAO derivatives is discussed in light of our recently proposed molecular model of interaction of TSAO derivatives with the interphase between the two subunits of HIV-1 reverse transcriptase.

  4. Mechanism and site of inhibition of AMPA receptors: