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Sample records for acid covalently bound

  1. Analytical laser induced liquid beam desorption mass spectrometry of protonated amino acids and their non-covalently bound aggregates

    NASA Astrophysics Data System (ADS)

    Charvat, A.; Lugovoj, E.; Faubel, M.; Abel, B.

    2002-09-01

    We have used analytical laser induced liquid beam desorption in combination with high resolution mass spectrometry (m/Δ mgeq 1 000) for the study of protonated amino acids (ornithine, citrulline, lysine, arginine) and their non-covalently bound complexes in the gas phase desorbed from water solutions. We report studies in which the desorption mechanism has been investigated. The results imply that biomolecule desorption at our conditions is a single step process involving laser heating of the solvent above its supercritical temperature, a rapid expansion, ion recombination and finally isolation and desorption of only a small fraction of preformed ions and charged aggregates. In addition, we report an investigation of the aqueous solution concentration and pH-dependence of the laser induced desorption of protonated species (monomers and dimers). The experimental findings suggest that the desorption process depends critically upon the proton affinity of the molecules, the concentration of other ions, and of the pH value of the solution. Therefore the ion concentrations measured in the gas phase very likely reflect solution properties (equilibrium concentrations). Arginine self-assembles large non-covalent singly protonated multimers (n=1...8) when sampled by IR laser induced water beam desorption mass spectrometry. The structures of these aggregates may resemble those of the solid state and may be preformed in solution prior to desorption. A desorption of mixtures of amino acids in water solution enabled us to study (mixed) protonated dimers, one of the various applications of the present technique. Reasons for preferred dimerization leading to simple cases of molecular recognition as well as less preferred binding is discussed in terms of the number of specific H-bonds that can be established in the clusters.

  2. Hydrogen-bond acidic functionalized carbon nanotubes (CNTs) with covalently-bound hexafluoroisopropanol groups

    SciTech Connect

    Fifield, Leonard S.; Grate, Jay W.

    2010-06-01

    Fluorinated hydrogen-bond acidic groups are directly attached to the backbone of single walled carbon nanotubes (SWCNTs) without the introduction of intermediate electron donating surface groups. Hexafluoroalcohol functional groups are exceptionally strong hydrogen bond acids, and are added to the nanotube surface using the aryl diazonium approach to create hydrogen-bond acidic carbon nanotube (CNT) surfaces. These groups can promote strong hydrogen-bonding interactions with matrix materials in composites or with molecular species to be concentrated and sensed. In the latter case, this newly developed material is expected to find useful application in chemical sensors and in CNT-based preconcentrator devices for the detection of pesticides, chemical warfare agents and explosives.

  3. Fluoride-Mediated Capture of a Noncovalent Bound State of a Reversible Covalent Enzyme Inhibitor: X-ray Crystallographic Analysis of an Exceptionally Potent α-Ketoheterocycle Inhibitor of Fatty Acid Amide Hydrolase

    PubMed Central

    Mileni, Mauro; Garfunkle, Joie; Ezzili, Cyrine; Cravatt, Benjamin F.; Stevens, Raymond C.; Boger, Dale L.

    2011-01-01

    Two cocrystal X-ray structures of the exceptionally potent α-ketoheterocycle inhibitor 1 (Ki = 290 pM) bound to a humanized variant of rat fatty acid amide hydrolase (FAAH) are disclosed, representing noncovalently and covalently bound states of the same inhibitor with the enzyme. Key to securing the structure of the noncovalently bound state of the inhibitor was the inclusion of fluoride ion in the crystallization conditions that is proposed to bind the oxyanion hole precluding inhibitor covalent adduct formation with stabilization of the tetrahedral hemiketal. This permitted the opportunity to detect important noncovalent interactions stabilizing the binding of the inhibitor within the FAAH active site independent of the covalent reaction. Remarkably, noncovalently bound 1 in the presence of fluoride appears to capture the active site in the same “in action” state with the three catalytic residues Ser241–Ser217–Lys142 occupying essentially identical positions observed in the covalently bound structure of 1, suggesting that this technique of introducing fluoride may have important applications in structural studies beyond inhibiting substrate or inhibitor oxyanion hole binding. Key insights to emerge from the studies include the observations that noncovalently bound 1 binds in its ketone (not gem diol) form, that the terminal phenyl group in the acyl side chain of the inhibitor serves as the key anchoring interaction overriding the intricate polar interactions in the cytosolic port, and that the role of the central activating heterocycle is dominated by its intrinsic electron-withdrawing properties. These two structures are also briefly compared with five X-ray structures of α-ketoheterocycle-based inhibitors bound to FAAH recently disclosed. PMID:21355555

  4. Fluoride-Mediated Capture of a Noncovalent Bound State of a Reversible Covalent Enzyme Inhibitor: X-ray Crystallographic Analysis of an Exceptionally Potent [alpha]-Ketoheterocycle Inhibitor of Fatty Acid Amide Hydrolase

    SciTech Connect

    Mileni, Mauro; Garfunkle, Joie; Ezzili, Cyrine; Cravatt, Benjamin F.; Stevens, Raymond C.; Boger, Dale L.

    2011-11-02

    Two cocrystal X-ray structures of the exceptionally potent {alpha}-ketoheterocycle inhibitor 1 (K{sub i} = 290 pM) bound to a humanized variant of rat fatty acid amide hydrolase (FAAH) are disclosed, representing noncovalently and covalently bound states of the same inhibitor with the enzyme. Key to securing the structure of the noncovalently bound state of the inhibitor was the inclusion of fluoride ion in the crystallization conditions that is proposed to bind the oxyanion hole precluding inhibitor covalent adduct formation with stabilization of the tetrahedral hemiketal. This permitted the opportunity to detect important noncovalent interactions stabilizing the binding of the inhibitor within the FAAH active site independent of the covalent reaction. Remarkably, noncovalently bound 1 in the presence of fluoride appears to capture the active site in the same 'in action' state with the three catalytic residues Ser241-Ser217-Lys142 occupying essentially identical positions observed in the covalently bound structure of 1, suggesting that this technique of introducing fluoride may have important applications in structural studies beyond inhibiting substrate or inhibitor oxyanion hole binding. Key insights to emerge from the studies include the observations that noncovalently bound 1 binds in its ketone (not gem diol) form, that the terminal phenyl group in the acyl side chain of the inhibitor serves as the key anchoring interaction overriding the intricate polar interactions in the cytosolic port, and that the role of the central activating heterocycle is dominated by its intrinsic electron-withdrawing properties. These two structures are also briefly compared with five X-ray structures of {alpha}-ketoheterocycle-based inhibitors bound to FAAH recently disclosed.

  5. Spin Labeling ESR Investigation of Covalently Bound Residues in Soil

    NASA Astrophysics Data System (ADS)

    Aleksandrova, Olga; Steinhoff, Heinz-Juergen; Klasmeier, Joerg; Schulz, Marcus; Matthies, Michael

    2013-04-01

    Organic xenobiotic chemicals, such as pesticides, biocides and veterinary pharmaceuticals, interact with soil, which results in the simultaneous formations of metabolites, mineralization products, and bound or non-extractable residues (NER). Substances or metabolites with reactive functional groups, such as aniline or phenol, have a tendency to give a larger proportion of NER. Despite numerous studies on NER, the majority of their chemical structures is still unknown. Reversible sequestration and irreversible formation of NER were also observed for veterinary antibiotic pharmaceuticals, after their application to soil with and without manure. For this purpose, we hypothesized a key role of specific functional groups of soil contaminants, via which contaminants are covalently bound to soil constituents, and advance a method of spin labeling ESR investigation of reaction products using a membrane method. Spin labels (SL) represent chemically stable paramagnetic molecules used as molecular labels and molecular probes for testing the covalent binding, structural properties, and molecular mobility of different physical, chemical, and biological systems. In the case of covalent binding of SL, their ESR spectra become broadened. We used stable nitroxide radicals (NR) as SL. These radicals modeled organic chemical contaminants and differed only in one functional group. The paramagnetic SL 4-Amino Tempo (4-amino-2,2,6,6-tetramethyl-1-piperidinylox) differed from Tempo (2,2,6,6-Tetramethylpiperidinooxy) in a substituent at the para-position of the piperidine ring, whereas Aniline Tempo (1-Piperidinyloxy, 2,2,6,-tetramethyl, 6-Aniline) differed from Tempo in an Aniline substituting one CH3 functional group. Before experimental analysis, we tested temporal changes in the concentration of both NR incubated with soil and found that the life-times of them in soil exceeded 3 days. We contaminated and labeled soil samples with NR, adding to soil the aqueous solution, which already

  6. Selective Nucleic Acid Capture with Shielded Covalent Probes

    PubMed Central

    2013-01-01

    Nucleic acid probes are used for diverse applications in vitro, in situ, and in vivo. In any setting, their power is limited by imperfect selectivity (binding of undesired targets) and incomplete affinity (binding is reversible, and not all desired targets bound). These difficulties are fundamental, stemming from reliance on base pairing to provide both selectivity and affinity. Shielded covalent (SC) probes eliminate the longstanding trade-off between selectivity and durable target capture, achieving selectivity via programmable base pairing and molecular conformation change, and durable target capture via activatable covalent cross-linking. In pure and mixed samples, SC probes covalently capture complementary DNA or RNA oligo targets and reject two-nucleotide mismatched targets with near-quantitative yields at room temperature, achieving discrimination ratios of 2–3 orders of magnitude. Semiquantitative studies with full-length mRNA targets demonstrate selective covalent capture comparable to that for RNA oligo targets. Single-nucleotide DNA or RNA mismatches, including nearly isoenergetic RNA wobble pairs, can be efficiently rejected with discrimination ratios of 1–2 orders of magnitude. Covalent capture yields appear consistent with the thermodynamics of probe/target hybridization, facilitating rational probe design. If desired, cross-links can be reversed to release the target after capture. In contrast to existing probe chemistries, SC probes achieve the high sequence selectivity of a structured probe, yet durably retain their targets even under denaturing conditions. This previously incompatible combination of properties suggests diverse applications based on selective and stable binding of nucleic acid targets under conditions where base-pairing is disrupted (e.g., by stringent washes in vitro or in situ, or by enzymes in vivo). PMID:23745667

  7. Synthesis and characterization of covalently bound benzocaine graphite oxide derivative

    NASA Astrophysics Data System (ADS)

    Kabbani, Ahmad; Kabbani, Mohamad; Safadi, Khadija

    2015-09-01

    Graphite oxide (GO) derived materials include chemically functionalize or reduced graphene oxide (exfoliated from GO) sheets, assembled paper-like forms , and graphene-based composites GO consists of intact graphitic regions interspersed with sp3-hybridized carbons containing hydroxyl and epoxide functional groups on the top and bottom surfaces of each sheet and sp2-hybridized carbons containing carboxyl and carbonyl groups mostly at the sheet edges. Hence, GO is hydrophilic and readily disperses in water to form stable colloidal suspensions Due to the attached oxygen functional groups, GO was used to prepare different derivatives which result in some physical and chemical properties that are dramatically different from their bulk counterparts .The present work discusses the covalent cross linking of graphite oxide to benzocaine or ethyl ester of para-aminobenzoic acid,structure I,used in many over-the-counter ointment drug.Synthesis is done via diazotization of the amino group.The product is characterized via IR,Raman, X-ray photoelectron spectroscopy as well as electron microscopy.

  8. Review: radiolabeled polymers containing covalently bound (3) H and (14) C.

    PubMed

    Wolf, Jeremy R

    2016-02-01

    Radiolabeled compounds are invaluable tools used to study synthetic and biological processes. Radiolabeled polymers find uses in mechanistic pathway elucidation, bioincorporation studies, biodegradation studies, and drug delivery applications. This literature review examines the syntheses (or biosyntheses), physical properties, and applications of radiolabeled polymers which contain covalently bound tritium and carbon-14 atoms. PMID:26762187

  9. Nucleic acid duplexes incorporating a dissociable covalent base pair

    NASA Technical Reports Server (NTRS)

    Gao, K.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    1999-01-01

    We have used molecular modeling techniques to design a dissociable covalently bonded base pair that can replace a Watson-Crick base pair in a nucleic acid with minimal distortion of the structure of the double helix. We introduced this base pair into a potential precursor of a nucleic acid double helix by chemical synthesis and have demonstrated efficient nonenzymatic template-directed ligation of the free hydroxyl groups of the base pair with appropriate short oligonucleotides. The nonenzymatic ligation reactions, which are characteristic of base paired nucleic acid structures, are abolished when the covalent base pair is reduced and becomes noncoplanar. This suggests that the covalent base pair linking the two strands in the duplex is compatible with a minimally distorted nucleic acid double-helical structure.

  10. Nucleic acid duplexes incorporating a dissociable covalent base pair.

    PubMed

    Gao, K; Orgel, L E

    1999-12-21

    We have used molecular modeling techniques to design a dissociable covalently bonded base pair that can replace a Watson-Crick base pair in a nucleic acid with minimal distortion of the structure of the double helix. We introduced this base pair into a potential precursor of a nucleic acid double helix by chemical synthesis and have demonstrated efficient nonenzymatic template-directed ligation of the free hydroxyl groups of the base pair with appropriate short oligonucleotides. The nonenzymatic ligation reactions, which are characteristic of base paired nucleic acid structures, are abolished when the covalent base pair is reduced and becomes noncoplanar. This suggests that the covalent base pair linking the two strands in the duplex is compatible with a minimally distorted nucleic acid double-helical structure. PMID:10611299

  11. Structure-based virtual screening approach for discovery of covalently bound ligands.

    PubMed

    Toledo Warshaviak, Dora; Golan, Gali; Borrelli, Kenneth W; Zhu, Kai; Kalid, Ori

    2014-07-28

    We present a fast and effective covalent docking approach suitable for large-scale virtual screening (VS). We applied this method to four targets (HCV NS3 protease, Cathepsin K, EGFR, and XPO1) with known crystal structures and known covalent inhibitors. We implemented a customized "VS mode" of the Schrödinger Covalent Docking algorithm (CovDock), which we refer to as CovDock-VS. Known actives and target-specific sets of decoys were docked to selected X-ray structures, and poses were filtered based on noncovalent protein-ligand interactions known to be important for activity. We were able to retrieve 71%, 72%, and 77% of the known actives for Cathepsin K, HCV NS3 protease, and EGFR within 5% of the decoy library, respectively. With the more challenging XPO1 target, where no specific interactions with the protein could be used for postprocessing of the docking results, we were able to retrieve 95% of the actives within 30% of the decoy library and achieved an early enrichment factor (EF1%) of 33. The poses of the known actives bound to existing crystal structures of 4 targets were predicted with an average RMSD of 1.9 Å. To the best of our knowledge, CovDock-VS is the first fully automated tool for efficient virtual screening of covalent inhibitors. Importantly, CovDock-VS can handle multiple chemical reactions within the same library, only requiring a generic SMARTS-based predefinition of the reaction. CovDock-VS provides a fast and accurate way of differentiating actives from decoys without significantly deteriorating the accuracy of the predicted poses for covalent protein-ligand complexes. Therefore, we propose CovDock-VS as an efficient structure-based virtual screening method for discovery of novel and diverse covalent ligands. PMID:24932913

  12. Bottom-up synthesis of chiral covalent organic frameworks and their bound capillaries for chiral separation

    PubMed Central

    Qian, Hai-Long; Yang, Cheng-Xiong; Yan, Xiu-Ping

    2016-01-01

    Covalent organic frameworks (COFs) are a novel class of porous materials, and offer great potential for various applications. However, the applications of COFs in chiral separation and chiral catalysis are largely underexplored due to the very limited chiral COFs available and their challenging synthesis. Here we show a bottom-up strategy to construct chiral COFs and an in situ growth approach to fabricate chiral COF-bound capillary columns for chiral gas chromatography. We incorporate the chiral centres into one of the organic ligands for the synthesis of the chiral COFs. We subsequently in situ prepare the COF-bound capillary columns. The prepared chiral COFs and their bound capillary columns give high resolution for the separation of enantiomers with excellent repeatability and reproducibility. The proposed strategy provides a promising platform for the synthesis of chiral COFs and their chiral separation application. PMID:27401541

  13. Bottom-up synthesis of chiral covalent organic frameworks and their bound capillaries for chiral separation

    NASA Astrophysics Data System (ADS)

    Qian, Hai-Long; Yang, Cheng-Xiong; Yan, Xiu-Ping

    2016-07-01

    Covalent organic frameworks (COFs) are a novel class of porous materials, and offer great potential for various applications. However, the applications of COFs in chiral separation and chiral catalysis are largely underexplored due to the very limited chiral COFs available and their challenging synthesis. Here we show a bottom-up strategy to construct chiral COFs and an in situ growth approach to fabricate chiral COF-bound capillary columns for chiral gas chromatography. We incorporate the chiral centres into one of the organic ligands for the synthesis of the chiral COFs. We subsequently in situ prepare the COF-bound capillary columns. The prepared chiral COFs and their bound capillary columns give high resolution for the separation of enantiomers with excellent repeatability and reproducibility. The proposed strategy provides a promising platform for the synthesis of chiral COFs and their chiral separation application.

  14. Bottom-up synthesis of chiral covalent organic frameworks and their bound capillaries for chiral separation.

    PubMed

    Qian, Hai-Long; Yang, Cheng-Xiong; Yan, Xiu-Ping

    2016-01-01

    Covalent organic frameworks (COFs) are a novel class of porous materials, and offer great potential for various applications. However, the applications of COFs in chiral separation and chiral catalysis are largely underexplored due to the very limited chiral COFs available and their challenging synthesis. Here we show a bottom-up strategy to construct chiral COFs and an in situ growth approach to fabricate chiral COF-bound capillary columns for chiral gas chromatography. We incorporate the chiral centres into one of the organic ligands for the synthesis of the chiral COFs. We subsequently in situ prepare the COF-bound capillary columns. The prepared chiral COFs and their bound capillary columns give high resolution for the separation of enantiomers with excellent repeatability and reproducibility. The proposed strategy provides a promising platform for the synthesis of chiral COFs and their chiral separation application. PMID:27401541

  15. Dynamic nuclear polarization of membrane proteins: covalently bound spin-labels at protein-protein interfaces.

    PubMed

    Wylie, Benjamin J; Dzikovski, Boris G; Pawsey, Shane; Caporini, Marc; Rosay, Melanie; Freed, Jack H; McDermott, Ann E

    2015-04-01

    We demonstrate that dynamic nuclear polarization of membrane proteins in lipid bilayers may be achieved using a novel polarizing agent: pairs of spin labels covalently bound to a protein of interest interacting at an intermolecular interaction surface. For gramicidin A, nitroxide tags attached to the N-terminal intermolecular interface region become proximal only when bimolecular channels forms in the membrane. We obtained signal enhancements of sixfold for the dimeric protein. The enhancement effect was comparable to that of a doubly tagged sample of gramicidin C, with intramolecular spin pairs. This approach could be a powerful and selective means for signal enhancement in membrane proteins, and for recognizing intermolecular interfaces. PMID:25828256

  16. Electron transport in DNA initiated by diaminonaphthalene donors alternatively bound by non-covalent and covalent association.

    PubMed

    Campbell, Neil P; Rokita, Steven E

    2014-02-21

    Covalent conjugation is typically used to fix a potential charge donor to a chosen site for studying either hole or excess electron transport in duplex DNA. A model system based on oligonucleotides containing an abasic site and (Br)dU was previously developed to provide a rapid method of screening new donors without the need of synthetic chemistry. While this strategy is effective for discovering important lead compounds, it is not appropriate for establishing extensive correlations between molecular structure and donor efficiency as demonstrated with a series of closely related electron donors based on diaminonaphthalene. The non-covalent system accurately identified the ability of the donors to reduce a distal (Br)dU in DNA, but their varying efficiencies were not recapitulated when attached covalently to an equivalent sequence of DNA. Reduction within the covalent system was not sensitive to the strong donor potentials as consistent with charge recombination dominating the net migration of charge. PMID:24398596

  17. Surface with antimicrobial activity obtained through silane coating with covalently bound polymyxin B.

    PubMed

    Mohorcič, M; Jerman, I; Zorko, M; Butinar, L; Orel, B; Jerala, R; Friedrich, J

    2010-10-01

    Surfaces exhibiting antimicrobial activity were prepared for potential medical application. A polycationic lipopeptide polymyxin B was selected as the bioactive agent for covalent immobilization onto the surface. First, by using sol-gel technology the inert glass substrate was functionalized by a silane coating with epoxide rings to which the peptide was coupled by means of a catalyst. Preparation of the coating and presence of the peptide on the surface were followed by FTIR, XPS and AFM analyses. The obtained material showed antimicrobial effect indicating that in spite of immobilization the peptide has retained its bioactivity. The coated surface was able to reduce bacterial cell counts of the Gram-negative bacterium Escherichia coli by more than five orders of magnitude in 24 h of incubation. It can be concluded that bioactive coatings with covalently bound polycationic peptides have potential for application on medical devices where leakage into the surrounding is not allowed in order to prevent bacterial growth and biofilm formation. PMID:20665235

  18. Dynamic Nuclear Polarization of membrane proteins: covalently bound spin-labels at protein-protein interfaces

    PubMed Central

    Wylie, Benjamin J; Dzikovski, Boris G.; Pawsey, Shane; Caporini, Marc; Rosay, Melanie; Freed, Jack H.; McDermott, Ann E.

    2016-01-01

    We demonstrate that dynamic nuclear polarization (DNP) of membrane proteins in lipid bilayers may be achieved using a novel polarizing agent: pairs of spin labels covalently bound to a protein of interest interacting at an intermolecular interaction surface. For gramicidin A, nitroxide tags attached to the N-terminal intermolecular interface region become proximal only when bimolecular channels forms in the membrane. We obtained signal enhancements of 6-fold for the dimeric protein. The enhancement affect was comparable to that of a doubly tagged sample of gramicidin C, with intramolecular spin pairs. This approach could be a powerful and selective means for signal enhancement in membrane proteins, and for recognizing intermolecular interfaces. PMID:25828256

  19. Covalent and non-covalent curcumin loading in acid-responsive polymeric micellar nanocarriers

    NASA Astrophysics Data System (ADS)

    Gao, Min; Chen, Chao; Fan, Aiping; Zhang, Ju; Kong, Deling; Wang, Zheng; Zhao, Yanjun

    2015-07-01

    Poor aqueous solubility, potential degradation, rapid metabolism and elimination lead to low bioavailability of pleiotropic impotent curcumin. Herein, we report two types of acid-responsive polymeric micelles where curcumin was encapsulated via both covalent and non-covalent modes for enhanced loading capacity and on-demand release. Biodegradable methoxy poly(ethylene glycol)-poly(lactic acid) copolymer (mPEG-PLA) was conjugated with curcumin via a hydrazone linker, generating two conjugates differing in architecture (single-tail versus double-tail) and free curcumin was encapsulated therein. The two micelles exhibited similar hydrodynamic size at 95 ± 3 nm (single-tail) and 96 ± 3 nm (double-tail), but their loading capacities differed significantly at 15.0 ± 0.5% (w/w) (single-tail) and 4.8 ± 0.5% (w/w) (double-tail). Under acidic sink conditions (pH 5.0 and 6.0), curcumin displayed a faster release from the single-tail nanocarrier, which was correlated to a low IC50 of 14.7 ± 1.6 (μg mL-1) compared to the value of double-tail micelle (24.9 ± 1.3 μg mL-1) in HeLa cells. The confocal imaging and flow cytometry analysis demonstrated a superior capability of single-tail micelle for intracellular curcumin delivery, which was a consequence of the higher loading capacity and lower degree of mPEG surface coverage. In conclusion, the dual loading mode is an effective means to increase the drug content in the micellar nanocarriers whose delivery efficiency is highly dependent on its polymer-drug conjugate architecture. This strategy offers an alternative nanoplatform for intracellularly delivering impotent hydrophobic agents (i.e. curcumin) in an efficient stimuli-triggered way, which is valuable for the enhancement of curcumin’s efficacy in managing a diverse range of disorders.

  20. Covalent and non-covalent binding in the ion/ion charge inversion of peptide cations with benzene-disulfonic acid anions.

    PubMed

    Stutzman, John R; Luongo, Carl A; McLuckey, Scott A

    2012-06-01

    Protonated angiotensin II and protonated leucine enkephalin-based peptides, which included YGGFL, YGGFLF, YGGFLH, YGGFLK and YGGFLR, were subjected to ion/ion reactions with the doubly deprotonated reagents 4-formyl-1,3-benzenedisulfonic acid (FBDSA) and 1,3-benzenedisulfonic acid (BDSA). The major product of the ion/ion reaction is a negatively charged complex of the peptide and reagent. Following dehydration of [M + FBDSA-H](-) via collisional-induced dissociation (CID), angiotensin II (DRVYIHPF) showed evidence for two product populations, one in which a covalent modification has taken place and one in which an electrostatic modification has occurred (i.e. no covalent bond formation). A series of studies with model systems confirmed that strong non-covalent binding of the FBDSA reagent can occur with subsequent ion trap CID resulting in dehydration unrelated to the adduct. Ion trap CID of the dehydration product can result in cleavage of amide bonds in competition with loss of the FBDSA adduct. This scenario is most likely for electrostatically bound complexes in which the peptide contains both an arginine residue and one or more carboxyl groups. Otherwise, loss of the reagent species from the complex, either as an anion or as a neutral species, is the dominant process for electrostatically bound complexes. The results reported here shed new light on the nature of non-covalent interactions in gas phase complexes of peptide ions that can be used in the rationale design of reagent ions for specific ion/ion reaction applications. PMID:22707160

  1. Covalent interaction of ascorbic acid with natural products

    PubMed Central

    Kesinger, Nicholas G.; Stevens, Jan F.

    2009-01-01

    While ascorbic acid (Vitamin C) is mostly known as a cofactor for proline hydroxylase and as a biological antioxidant, it also forms covalent bonds with natural products which we here refer to as ‘ascorbylation’. A number of natural products containing an ascorbate moiety has been isolated and characterized from a variety of biological sources, ranging from marine algae to flowering plants. Most of these compounds are formed as a result of nucleophilic substitution or addition by ascorbate, e.g. the ascorbigens from Brassica species are ascorbylated indole derivatives. Some ascorbylated tannins appear to be formed from electrophilic addition to dehydroascorbic acid. There are also examples of annulations of ascorbate with dietary polyphenols, e.g., epigallocatechin gallate (EGCG) and resveratrol derivatives. Herein is a survey of thirty-three ascorbylated natural products and their reported biological activities. PMID:19875138

  2. Covalent binding of sulfamethazine to natural and synthetic humic acids: assessing laccase catalysis and covalent bond stability.

    PubMed

    Gulkowska, Anna; Sander, Michael; Hollender, Juliane; Krauss, Martin

    2013-07-01

    Sulfonamide antibiotics form stable covalent bonds with quinone moieties in organic matter via nucleophilic addition reactions. In this work, we combined analytical electrochemistry with trace analytics to assess the catalytic role of the oxidoreductase laccase in the binding of sulfamethazine (SMZ) to Leonardite humic acid (LHA) and to four synthetic humic acids (SHAs) polymerized from low molecular weight precursors and to determine the stability of the formed bonds. In the absence of laccase, a significant portion of the added SMZ formed covalent bonds with LHA, but only a very small fraction (<0.4%) of the total quinone moieties in LHA reacted. Increasing absolute, but decreasing relative concentrations of SMZ-LHA covalent bonds with increasing initial SMZ concentration suggested that the quinone moieties in LHA covered a wide distribution in reactivity for the nucleophilic addition of SMZ. Laccase catalyzed the formation of covalent bonds by oxidizing unreactive hydroquinone moieties in LHA to reactive, electrophilic quinone moieties, of which a large fraction (5%) reacted with SMZ. Compared to LHA, the SHA showed enhanced covalent bond formation in the absence of laccase, suggesting a higher reactivity of their quinone moieties toward nucleophilic addition. This work supports that binding to soil organic matter (SOM) is an important process governing the fate, bioactivity, and extractability of sulfonamides in soils. PMID:23384282

  3. Covalently Bound Tetracoordinated Organoborons are Superhalogens: A Combined Negative Ion Photoelectron Spectroscopy and Theoretical Study

    SciTech Connect

    Zhang, Jian; Yang, Ping; Sun, Zhenrong; Wang, Xue B.

    2014-09-18

    Molecular species with electron affinities (EAs) larger than that of the chlorine atom (3.6131 eV) are superhalogens. The corresponding negative ions, namely, superhalogen anions, are intrinsically very stable with high electron binding energies (EBEs), and widely exist as building blocks of bulk materials and ionic liquids. The most common superhalogen anions proposed and confirmed to date are either ionic salts or compact inorganic species. Herein we report a new class of superhalogen species, a series of tetracoordinated organoboron anions [BL4]– (L = phenyl (1), 4-fluorophenyl (2), 1-imidazolyl (3), L4 = H(pyrazolyl)3 (4)) with bulky organic ligands covalently bound to the central B atom. Negative ion photoelectron spectroscopy (NIPES) reveals all of these anions possessing EBEs higher than that of Cl- with the adiabatic / vertical detachment energy (ADE / VDE) of 4.44/4.8 (1), 4.78/5.2 (2), 5.08/5.4 (3), and 4.59/4.9 eV (4), respectively. First-principles calculations confirmed high EBEs of [BL4]– and predicted that these anions are thermodynamically stable against fragmentation. The unraveled superhalogen nature of these species provides a molecular basis to explain the wide-range applications of tetraphenylborate (TPB) (1) and trispyrazolylborate (Tp) (4) in many areas spanning from industrial waste treatment to soft material synthesis and organometallic chemistry

  4. Lability of copper bound to humic acid.

    PubMed

    Mao, Lingchen; Young, Scott D; Bailey, Elizabeth H

    2015-07-01

    Geochemical speciation models generally include the assumption that all metal bound to humic acid and fulvic acid (HA, FA) is labile. However, in the current study, we determined the presence of a soluble 'non-labile' Cu fraction bound to HA extracted from grassland and peat soils. This was quantified by determining isotopically-exchangeable Cu (E-value) and EDTA-extraction of HA-bound Cu, separated by size-exclusion chromatography (SEC) and assayed by coupled ICP-MS. Evidence of time-dependent Cu fixation by HA was found during the course of an incubation study (160 d); up to 50% of dissolved HA-bound Cu was not isotopically exchangeable. This result was supported by extraction with EDTA where approximately 40% of Cu remained bound to HA despite dissolution in 0.05 M Na2-EDTA. The presence of a substantial non-labile metal fraction held by HA challenges the assumption of wholly reversible equilibrium which is central to current geochemical models of metal binding to humic substances. PMID:25863164

  5. Major membrane surface proteins of Mycoplasma hyopneumoniae selectively modified by covalently bound lipid.

    PubMed Central

    Wise, K S; Kim, M F

    1987-01-01

    Surface protein antigens of Mycoplasma hyopneumoniae were identified by direct antibody-surface binding or by radioimmunoprecipitation of surface 125I-labeled proteins with a series of monoclonal antibodies (MAbs). Surface proteins p70, p65, p50, and p44 were shown to be integral membrane components by selective partitioning into the hydrophobic phase during Triton X-114 (TX-114)-phase fractionation, whereas p41 was concomitantly identified as a surface protein exclusively partitioning into the aqueous phase. Radioimmunoprecipitation of TX-114-phase proteins from cells labeled with [35S]methionine, 14C-amino acids, or [3H] palmitic acid showed that proteins p65, p50, and p44 were abundant and (with one other hydrophobic protein, p60) were selectively labeled with lipid. Covalent lipid attachment was established by high-performance liquid chromatography identification of [3H]methyl palmitate after acid methanolysis of delipidated proteins. An additional, unidentified methanolysis product suggested conversion of palmitate to another form of lipid also attached to these proteins. Alkaline hydroxylamine treatment of labeled proteins indicated linkage of lipids by amide or stable O-linked ester bonds. Proteins p65, p50, and p44 were highly immunogenic in the natural host as measured by immunoblots of TX-114-phase proteins with antisera from swine inoculated with whole organisms. These proteins were antigenically and structurally unrelated, since hyperimmune mouse antibodies to individual gel-purified proteins were monospecific and gave distinct proteolytic epitope maps. Intraspecies size variants of one surface antigen of M. hyopneumoniae were revealed by a MAb to p70 (defined in strain J, ATCC 25934), which recognized a larger p73 component on strain VPP11 (ATCC 25617). In addition, MAb to internal, aqueous-phase protein p82 of strain J failed to bind an analogous antigen in strain VPP11. These studies establish that a highly restricted set of distinct, lipid

  6. Probing Protein Structure by Amino Acid-Specific Covalent Labeling and Mass Spectrometry

    PubMed Central

    Mendoza, Vanessa Leah; Vachet, Richard W.

    2009-01-01

    For many years, amino acid-specific covalent labeling has been a valuable tool to study protein structure and protein interactions, especially for systems that are difficult to study by other means. These covalent labeling methods typically map protein structure and interactions by measuring the differential reactivity of amino acid side chains. The reactivity of amino acids in proteins generally depends on the accessibility of the side chain to the reagent, the inherent reactivity of the label and the reactivity of the amino acid side chain. Peptide mass mapping with ESI- or MALDI-MS and peptide sequencing with tandem MS are typically employed to identify modification sites to provide site-specific structural information. In this review, we describe the reagents that are most commonly used in these residue-specific modification reactions, details about the proper use of these covalent labeling reagents, and information about the specific biochemical problems that have been addressed with covalent labeling strategies. PMID:19016300

  7. Detection and Time Course of Formation of Major Thiamin Diphosphate-Bound Covalent Intermediates Derived from a Chromophoric Substrate Analogue on Benzoylformate Decarboxylase

    SciTech Connect

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

    2009-04-02

    The mechanism of the enzyme benzoylformate decarboxylase (BFDC), which carries out a typical thiamin diphosphate (ThDP)-dependent nonoxidative decarboxylation reaction, was studied with the chromophoric alternate substrate (E)-2-oxo-4(pyridin-3-yl)-3-butenoic acid (3-PKB). Addition of 3-PKB resulted in the appearance of two transient intermediates formed consecutively, the first one to be formed a predecarboxylation ThDP-bound intermediate with {lambda}{sub max} at 477 nm, and the second one corresponding to the first postdecarboxylation intermediate the enamine with {lambda}{sub max} at 437 nm. The time course of formation/depletion of the PKB-ThDP covalent complex and of the enamine showed that decarboxylation was slower than formation of the PKB-ThDP covalent adduct. When the product of decarboxylation 3-(pyridin-3-yl)acrylaldehyde (PAA) was added to BFDC, again an absorbance with {lambda}{sub max} at 473 nm was formed, corresponding to the tetrahedral adduct of PAA with ThDP. Addition of well-formed crystals of BFDC to a solution of PAA resulted in a high resolution (1.34 {angstrom}) structure of the BFDC-bound adduct of ThDP with PAA confirming the tetrahedral nature at the C2{alpha} atom, rather than of the enamine, and supporting the assignment of the {lambda}{sub max} at 473 nm to the PAA-ThDP adduct. The structure of the PAA-ThDP covalent complex is the first example of a product-ThDP adduct on BFDC. Similar studies with 3-PKB indicated that decarboxylation had taken place. Evidence was also obtained for the slow formation of the enamine intermediate when BFDC was incubated with benzaldehyde, the product of the decarboxylation reaction thus confirming its presence on the reaction pathway.

  8. Detection and Time Course of Formation of Major Thiamin Diphosphate-Bound Covalent Intermediates Derived from a Chromophoric Substrate Analogue on Benzoylformate Decarboxylase†

    PubMed Central

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

    2009-01-01

    The mechanism of the enzyme benzoylformate decarboxylase (BFDC), which carries out a typical thiamin diphosphate (ThDP)-dependent nonoxidative decarboxylation reaction, was studied with the chromophoric alternate substrate (E)-2-oxo-4(pyridin-3-yl)-3-butenoic acid (3-PKB). Addition of 3-PKB resulted in the appearance of two transient intermediates formed consecutively, the first one to be formed a predecarboxylation ThDP-bound intermediate with λmax at 477 nm, and the second one corresponding to the first postdecarboxylation intermediate the enamine with λmax at 437 nm. The time course of formation/depletion of the PKB–ThDP covalent complex and of the enamine showed that decarboxylation was slower than formation of the PKB–ThDP covalent adduct. When the product of decarboxylation 3-(pyridin-3-yl)acrylaldehyde (PAA) was added to BFDC, again an absorbance with λmax at 473 nm was formed, corresponding to the tetrahedral adduct of PAA with ThDP. Addition of well-formed crystals of BFDC to a solution of PAA resulted in a high resolution (1.34 Å) structure of the BFDC-bound adduct of ThDP with PAA confirming the tetrahedral nature at the C2α atom, rather than of the enamine, and supporting the assignment of the λmax at 473 nm to the PAA–ThDP adduct. The structure of the PAA–ThDP covalent complex is the first example of a product–ThDP adduct on BFDC. Similar studies with 3-PKB indicated that decarboxylation had taken place. Evidence was also obtained for the slow formation of the enamine intermediate when BFDC was incubated with benzaldehyde, the product of the decarboxylation reaction thus confirming its presence on the reaction pathway. PMID:19140682

  9. Studies on the incorporation of a covalently bound disubstituted phosphate residue into Azotobacter vinelandii flavodoxin in vivo.

    PubMed

    Boylan, M H; Edmondson, D E

    1990-06-15

    Previous studies have shown the flavodoxin from Azotobacter vinelandii (strain OP, Berkeley) to contain a covalently bound disubstituted phosphate residue [Edmondson & James (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3786-3789]. Phosphorylation of the protein in vivo was investigated by the addition of [32P]phosphate to cells grown under N2-fixing conditions, under conditions of nif-gene repression and under conditions of nif-gene de-repression. Rocket immunoelectrophoresis of cell extracts showed an approx. 5-fold decrease in the concentration of flavodoxin expressed in cells grown in the presence of NH4+ as compared with those grown under N2-fixing conditions. A similar increase in flavodoxin concentration was observed on nif-gene de-repression. Incorporation of [32P]phosphate occurs only into newly synthesized flavodoxin, as observed on SDS/PAGE of immunoprecipitates of cell extracts. Western blots demonstrated no observable precursor forms of flavodoxin. These data provide conclusive evidence for the phosphorylation of Azotobacter strain OP flavodoxin in vivo and suggest that the covalently bound phosphate residue does not exchange with cellular phosphate pools. Thus the role of this phosphodiester cross-link is proposed to be structural rather than regulatory. PMID:2363708

  10. Determination of the amino acid residue involved in [3H]beta-funaltrexamine covalent binding in the cloned rat mu-opioid receptor.

    PubMed

    Chen, C; Yin, J; Riel, J K; DesJarlais, R L; Raveglia, L F; Zhu, J; Liu-Chen, L Y

    1996-08-30

    We previously demonstrated that [3H]beta-funaltrexamine ([3H]beta-FNA) labeled the rat mu opioid receptor expressed in Chinese hamster ovary cells with high specificity, and [3H]beta-FNA-labeled receptors migrated as one broad band with a mass of 80 kDa. In this study, we determined the region and then the amino acid residue of the mu receptor involved in the covalent binding of [3H]beta-FNA. [3H]beta-FNA-labeled receptors were solubilized and purified to approximately 10% purity by immunoaffinity chromatography with antibodies against a C-terminal domain peptide. The site of covalent bond formation was determined to be within Ala206-Met243 by CNBr cleavage of partially purified labeled mu receptors and determinations of sizes of labeled receptor fragments. The amino acid residue of beta-FNA covalent incorporation was then determined by site-directed mutagenesis studies within this region. Mutation of Lys233 to Ala, Arg, His, and Leu completely eliminated covalent binding of [3H]beta-FNA, although these mutants bound beta-FNA with high affinity. Mutations of other amino acid residues did not affect covalent binding of [3H]beta-FNA. These results indicate that [3H]beta-FNA binds covalently to Lys233. Since [3H]beta-FNA is a rigid molecule, the information will be very useful for molecular modeling of interaction between morphinans and the mu receptor. PMID:8702924

  11. Genetically Encoding an Electrophilic Amino Acid for Protein Stapling and Covalent Binding to Native Receptors

    PubMed Central

    2015-01-01

    Covalent bonds can be generated within and between proteins by an unnatural amino acid (Uaa) reacting with a natural residue through proximity-enabled bioreactivity. Until now, Uaas have been developed to react mainly with cysteine in proteins. Here we genetically encoded an electrophilic Uaa capable of reacting with histidine and lysine, thereby expanding the diversity of target proteins and the scope of the proximity-enabled protein cross-linking technology. In addition to efficient cross-linking of proteins inter- and intramolecularly, this Uaa permits direct stapling of a protein α-helix in a recombinant manner and covalent binding of native membrane receptors in live cells. The target diversity, recombinant stapling, and covalent targeting of endogenous proteins enabled by this versatile Uaa should prove valuable in developing novel research tools, biological diagnostics, and therapeutics by exploiting covalent protein linkages for specificity, irreversibility, and stability. PMID:25010185

  12. Aryloxyalkanoic Acids as Non-Covalent Modifiers of the Allosteric Properties of Hemoglobin.

    PubMed

    Omar, Abdelsattar M; Mahran, Mona A; Ghatge, Mohini S; Bamane, Faida H A; Ahmed, Mostafa H; El-Araby, Moustafa E; Abdulmalik, Osheiza; Safo, Martin K

    2016-01-01

    Hemoglobin (Hb) modifiers that stereospecifically inhibit sickle hemoglobin polymer formation and/or allosterically increase Hb affinity for oxygen have been shown to prevent the primary pathophysiology of sickle cell disease (SCD), specifically, Hb polymerization and red blood cell sickling. Several such compounds are currently being clinically studied for the treatment of SCD. Based on the previously reported non-covalent Hb binding characteristics of substituted aryloxyalkanoic acids that exhibited antisickling properties, we designed, synthesized and evaluated 18 new compounds (KAUS II series) for enhanced antisickling activities. Surprisingly, select test compounds showed no antisickling effects or promoted erythrocyte sickling. Additionally, the compounds showed no significant effect on Hb oxygen affinity (or in some cases, even decreased the affinity for oxygen). The X-ray structure of deoxygenated Hb in complex with a prototype compound, KAUS-23, revealed that the effector bound in the central water cavity of the protein, providing atomic level explanations for the observed functional and biological activities. Although the structural modification did not lead to the anticipated biological effects, the findings provide important direction for designing candidate antisickling agents, as well as a framework for novel Hb allosteric effectors that conversely, decrease the protein affinity for oxygen for potential therapeutic use for hypoxic- and/or ischemic-related diseases. PMID:27529207

  13. Protected Graft Copolymer (PGC) in Imaging and Therapy: A Platform for the Delivery of Covalently and Non-Covalently Bound Drugs

    PubMed Central

    Bogdanov Jr, Alexei A.; Mazzanti, Mary; Castillo, Gerardo; Bolotin, Elijah

    2012-01-01

    Initially developed in 1992 as an MR imaging agent, the family of protected graft copolymers (PGC) is based on a conjugate of polylysine backbone to which methoxypoly(ethylene glycol) (MPEG) chains are covalently linked in a random fasion via N-ε-amino groups. While PGC is relatively simple in terms of its chemcial composition and structure, it has proved to be a versatile platform for in vivo drug delivery. The advantages of poly amino acid backbone grafting include multiple available linking sites for drug and adaptor molecules. The grafting of PEG chains to PGC does not compromise biodegradability and does not result in measurable toxicity or immunogenicity. In fact, the biocompatablility of PGC has resulted in its being one of the few 100% synthetic non-proteinaceous macromolecules that has suceeded in passing the initial safety phase of clinical trials. PGC is capable of long circulation times after injection into the blood stream and as such found use early on as a carrier system for delivery of paramagnetic imaging compounds for angiography. Other PGC types were later developed for use in nuclear medicine and optical imaging applications in vivo. Recent developments in PGC-based drug carrier formulations include the use of zinc as a bridge between the PGC carrier and zinc-binding proteins and re-engineering of the PGC carrier as a covalent amphiphile that is capabe of binding to hydrophobic residues of small proteins and peptides. At present, PGC-based formulations have been developed and tested in various disease models for: 1) MR imaging local blood circulation in stroke, cancer and diabetes; 2) MR and nuclear imaging of blood volume and vascular permeability in inflammation; 3) optical imaging of proteolytic activity in cancer and inflammation; 4) delivery of platinum(II) compounds for treating cancer; 5) delivery of small proteins and peptides for treating diabetes, obesity and myocardial infarction. This review summarizes the experience accumulated by

  14. Fiber optic monitoring of carbamate pesticides using porous glass with covalently bound chlorophenol red.

    PubMed

    Xavier, M P; Vallejo, B; Marazuela, M D; Moreno-Bondi, M C; Baldini, F; Falai, A

    2000-02-01

    An optical fiber biosensor for the determination of the pesticides propoxur (Baygon) and carbaryl, two of the most commonly used carbamate insecticides in vegetable crops, is described. A pH indicator, chlorophenol red, is used as optical transducer of the inhibition of the enzyme acetylcholinesterase by the analytes. The biorecognition element is covalently immobilized onto controlled pore glass beads (CPG) and packed in a thermostatized bioreactor connected to a flow-through cell that contains CPG-immobilized chlorophenol red placed at the common end of a bifurcated fiber optic bundle. In the presence of a constant acetylcholine concentration, the colour of the pH sensitive layer changes and the measured reflectance signal can be related to the carbamate concentration in the sample solution. The performance of the biosensor has been optimized using a flow injection system. The linear dynamic range for the determination of carbaryl and propoxur spans from 0.8 to 3.0 mg l(-1) and from 0.03 to 0.50 mg l(-1), respectively. The detection limit (3 s) of the biosensor for propoxur (0.4 ng) is lower than that measured for carbaryl (25 ng). Reproducibility, stability and interference studies of the optical device are reported. The biosensor has been applied to the determination of propoxur in spiked vegetables (onion and lettuce) using ultrasound extraction, achieving recovery values between 93 and 95% for onion samples at the different concentration levels assayed. PMID:10722147

  15. Fluorescent silica nanoparticles containing covalently bound dyes for reporter, marker, and sensor applications

    NASA Astrophysics Data System (ADS)

    Patonay, Gabor; Henary, Maged; Chapman, Gala; Emer, Kyle; Crow, Sidney

    2016-03-01

    Silica nanoparticles have proven to be useful in many bioanalytical and medical applications and have been used in numerous applications during the last decade. Combining the properties of silica nanoparticles and fluorescent dyes that may be used as chemical probes or labels can be relatively easy by simply soaking porous silica nanoparticles in a solution of the dye of interest. Under proper conditions the entrapped dye can stay inside the silica nanoparticle for several hours resulting in a useful probe. In spite of the relative durability of these probes, leaching can still occur. A much better approach is to synthesize silica nanoparticles that have the fluorescent dye covalently attached to the backbone structure of the silica nanoparticle. This can be achieved by using appropriately modified tetraethyl orthosilicate (TEOS) analogues during the silica nanoparticle synthesis. The molar ratio of TEOS and modified TEOS will determine the fluorescent dye load in the silica nanoparticle. Dependent on the chemical stability of the reporting dye either reverse micellar (RM) or Stöber method can be used for silica nanoparticle synthesis. If dye stability allows RM procedure is preferred as it results in a much easier control of the silica nanoparticle reaction itself. Also controlling the size and uniformity of the silica nanoparticles are much easier using RM method. Dependent on the functional groups present in the reporting dye used in preparation of the modified TEOS, the silica nanoparticles can be utilized in many applications such as pH sensor, metal ion sensors, labels, etc. In addition surface activated silica nanoparticles with reactive moieties are also excellent reporters or they can be used as bright fluorescent labels. Many different fluorescent dyes can be used to synthesize silica nanoparticles including visible and NIR dyes. Several bioanalytical applications are discussed including studying amoeba phagocytosis.

  16. Covalent Small Molecule Inhibitors of Ca2+-Bound S100B

    PubMed Central

    2015-01-01

    Elevated levels of the tumor marker S100B are observed in malignant melanoma, and this EF-hand-containing protein was shown to directly bind wild-type (wt) p53 in a Ca2+-dependent manner, dissociate the p53 tetramer, and inhibit its tumor suppression functions. Likewise, inhibiting S100B with small interfering RNA (siRNAS100B) is sufficient to restore wild-type p53 levels and its downstream gene products and induce the arrest of cell growth and UV-dependent apoptosis in malignant melanoma. Therefore, it is a goal to develop S100B inhibitors (SBiXs) that inhibit the S100B–p53 complex and restore active p53 in this deadly cancer. Using a structure–activity relationship by nuclear magnetic resonance approach (SAR by NMR), three persistent binding pockets are found on S100B, termed sites 1–3. While inhibitors that simultaneously bind sites 2 and 3 are in place, no molecules that simultaneously bind all three persistent sites are available. For this purpose, Cys84 was used in this study as a potential means to bridge sites 1 and 2 because it is located in a small crevice between these two deeper pockets on the protein. Using a fluorescence polarization competition assay, several Cys84-modified S100B complexes were identified and examined further. For five such SBiX–S100B complexes, crystallographic structures confirmed their covalent binding to Cys84 near site 2 and thus present straightforward chemical biology strategies for bridging sites 1 and 3. Importantly, one such compound, SC1982, showed an S100B-dependent death response in assays with WM115 malignant melanoma cells, so it will be particularly useful for the design of SBiX molecules with improved affinity and specificity. PMID:25268459

  17. Covalent small molecule inhibitors of Ca(2+)-bound S100B.

    PubMed

    Cavalier, Michael C; Pierce, Adam D; Wilder, Paul T; Alasady, Milad J; Hartman, Kira G; Neau, David B; Foley, Timothy L; Jadhav, Ajit; Maloney, David J; Simeonov, Anton; Toth, Eric A; Weber, David J

    2014-10-28

    Elevated levels of the tumor marker S100B are observed in malignant melanoma, and this EF-hand-containing protein was shown to directly bind wild-type (wt) p53 in a Ca(2+)-dependent manner, dissociate the p53 tetramer, and inhibit its tumor suppression functions. Likewise, inhibiting S100B with small interfering RNA (siRNA(S100B)) is sufficient to restore wild-type p53 levels and its downstream gene products and induce the arrest of cell growth and UV-dependent apoptosis in malignant melanoma. Therefore, it is a goal to develop S100B inhibitors (SBiXs) that inhibit the S100B-p53 complex and restore active p53 in this deadly cancer. Using a structure-activity relationship by nuclear magnetic resonance approach (SAR by NMR), three persistent binding pockets are found on S100B, termed sites 1-3. While inhibitors that simultaneously bind sites 2 and 3 are in place, no molecules that simultaneously bind all three persistent sites are available. For this purpose, Cys84 was used in this study as a potential means to bridge sites 1 and 2 because it is located in a small crevice between these two deeper pockets on the protein. Using a fluorescence polarization competition assay, several Cys84-modified S100B complexes were identified and examined further. For five such SBiX-S100B complexes, crystallographic structures confirmed their covalent binding to Cys84 near site 2 and thus present straightforward chemical biology strategies for bridging sites 1 and 3. Importantly, one such compound, SC1982, showed an S100B-dependent death response in assays with WM115 malignant melanoma cells, so it will be particularly useful for the design of SBiX molecules with improved affinity and specificity. PMID:25268459

  18. An electrochemical immunosensor based on covalent immobilization of okadaic acid onto screen printed carbon electrode via diazotization-coupling reaction.

    PubMed

    Hayat, Akhtar; Barthelmebs, Lise; Sassolas, Audrey; Marty, Jean-Louis

    2011-07-15

    In this work, an electrochemical method based on the diazonium-coupling reaction mechanism for the immobilization of okadaic acid (OA) on screen printed carbon electrode was developed. At first, 4-carboxyphenyl film was grafted by electrochemical reduction of 4-carboxyphenyl diazonium salt, followed by terminal carboxylic group activation by N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyle)-N'-ethyle-carbodiimide hydrochloride (EDC). Hexamethyldiamine was then covalently bound by one of its terminal amine group to the activated carboxylic group. The carboxyl group of okadaic acid was activated by EDC/NHS and then conjugated to the second terminal amine group on other side of the hexamethyldiamine through amide bond formation. After immobilization of OA, an indirect competitive immunoassay format was employed to detect OA. The immunosensor obtained using this novel approach allowed detection limit of 1.44 ng/L of OA, and was also validated with certified reference mussel samples. PMID:21645734

  19. Covalently linked deoxyribonucleic acid with multi-walled carbon nanotubes: synthesis and characterization.

    PubMed

    Chen, Weiwei; Yi, Changqing; Chi-Hung, Tzang; Lee, Shuit-Tong; Yang, Mengsu

    2010-01-01

    In this chapter, a multi-step protocol for covalently linking functionalized multi-walled carbon nanotubes (MWCNT) to deoxyribonucleic acid (DNA) oligonucleotides is provided. X-ray photoelectron spectroscopy (XPS) is used to characterize the initially formed amine-terminated MWCNTs, to which DNA is covalently anchored. Atomic force microscopy (AFM) investigation of the DNA-MWCNT conjugates reveals that the chemical functionalization occurs at both the ends and sidewalls of the nanotubes. The described methodology represents an important step toward the realization of DNA-guided self-assembly for carbon nanotubes. PMID:20422378

  20. Major membrane surface proteins of Mycoplasma hyopneumoniae selectively modified by covalently bound lipid

    SciTech Connect

    Wise K.S.; Kim, M.F.

    1987-12-01

    Surface protein antigens of Mycoplasma hyopneumoniae were identified by direct antibody-surface binding or by radioimmunoprecipitation of surface /sup 125/I-labeled proteins with a series of monoclonal antibodies (MAbs). Radioimmunoprecipitation of TX-114-phase proteins from cells labeled with (/sup 35/S) methionine, /sup 14/C-amino acids, or (/sup 3/H) palmitic acid showed that proteins p65, p50, and p44 were abundant and (with one other hydrophobic protein, p60) were selectively labeled with lipid. Alkaline hydroxylamine treatment of labeled proteins indicated linkage of lipids by amide or stable O-linked ester bonds. Proteins p65, p50, and p44 were highly immunogenic in the natural host as measured by immunoblots of TX-114-phase proteins with antisera from swine inoculated with whole organisms. These proteins were antigenically and structurally unrelated, since hyperimmune mouse antibodies to individual gel-purified proteins were monospecific and gave distinct proteolytic epitope maps. Intraspecies size variants of one surface antigen of M. hyopneumoniae were revealed by a MAb to p70 (defined in strain J, ATCC 25934), which recognized a large p73 component on strain VPP11 (ATCC 25617). In addition, MAb to internal, aqueous-phase protein p82 of strain J failed to bind an analogous antigen in strain VPP11.

  1. Ionic and Covalent Stabilization of Intermediates and Transition States in Catalysis by Solid Acids

    SciTech Connect

    Deshlahra, Prashant; Carr, Robert T.; Iglesia, Enrique

    2014-10-29

    Reactivity descriptors describe catalyst properties that determine the stability of kinetically relevant transition states and adsorbed intermediates. Theoretical descriptors, such as deprotonation energies (DPE), rigorously account for Brønsted acid strength for catalytic solids with known structure. Here, mechanistic interpretations of methanol dehydration turnover rates are used to assess how charge reorganization (covalency) and electrostatic interactions determine DPE and how such interactions are recovered when intermediates and transition states interact with the conjugate anion in W and Mo polyoxometalate (POM) clusters and gaseous mineral acids. Turnover rates are lower and kinetically relevant species are less stable on Mo than W POM clusters with similar acid strength, and such species are more stable on mineral acids than that predicted from W-POM DPE–reactivity trends, indicating that DPE and acid strength are essential but incomplete reactivity descriptors. Born–Haber thermochemical cycles indicate that these differences reflect more effective charge reorganization upon deprotonation of Mo than W POM clusters and the much weaker reorganization in mineral acids. Such covalency is disrupted upon deprotonation but cannot be recovered fully upon formation of ion pairs at transition states. Predictive descriptors of reactivity for general classes of acids thus require separate assessments of the covalent and ionic DPE components. Here, we describe methods to estimate electrostatic interactions, which, taken together with energies derived from density functional theory, give the covalent and ionic energy components of protons, intermediates, and transition states. In doing so, we provide a framework to predict the reactive properties of protons for chemical reactions mediated by ion-pair transition states.

  2. Dynamics of human acetylcholinesterase bound to non-covalent and covalent inhibitors shedding light on changes to the water network structure.

    PubMed

    Peters, Judith; Martinez, Nicolas; Trovaslet, Marie; Scannapieco, Kévin; Koza, Michael Marek; Masson, Patrick; Nachon, Florian

    2016-05-14

    We investigated the effects of non-covalent reversible and covalent irreversible inhibitors on human acetylcholinesterase and human butyrylcholinesterase. Remarkably a non-covalent inhibitor, Huperzine A, has almost no effect on the molecular dynamics of the protein, whereas the covalently binding nerve agent soman renders the molecular structure stiffer in its aged form. The modified movements were studied by incoherent neutron scattering on different time scales and they indicate a stabilization and stiffening of aged human acetylcholinesterase. It is not straightforward to understand the forces leading to this strong effect. In addition to the specific interactions of the adduct within the protein, some indications point towards an extensive water structure change for the aged conjugate as water Bragg peaks appeared at cryogenic temperature despite an identical initial hydration state for all samples. Such a change associated to an apparent increase in free water volume upon aging suggests higher ordering of the hydration shell that leads to the stiffening of protein. Thus, several additive contributions seem responsible for the improved flexibility or stiffening effect of the inhibitors rather than a single interaction. PMID:27109895

  3. Participation of the iron-sulphur cluster and of the covalently bound coenzyme of trimethylamine dehydrogenase in catalysis.

    PubMed Central

    Steenkamp, D J; Singer, T P

    1978-01-01

    Bacterial trimethylamine dehydrogenase contains a novel type of covalently bound flavin mononucleotide and a tetrameric iron-sulphur centre. The dehydrogenase takes up 1.5mol of dithionite/mol of enzyme and is thereby converted into the flavin quinol-reduced (4Fe-4S) form, with the expected bleaching of the visible absorption band of the flavin and the emergence of signals of typical reduced ferredoxin in the electronparamagnetic-resonance spectrum. On reduction with a slight excess of substrate, however, unusual absorption and electron-paramagnetic-resonance spectra appear quite rapidly. The latter is attributed to extensive interaction between the reduced (4Fe-4S) centre and the flavin semiquinone. The species of enzyme arising during the catalytic cycle were studied by a combination of rapid-freeze e.p.r. and stopped-flow spectophotometry. The initial reduction of the flavin to the quinol form is far too rapid to be rate-limiting in catalysis, as is the reoxidation of the substrate-reduced enzyme by phenazine methosulphate. Formation of the spin-spin-interacting species from the dihydroflavin is considerably slower, however, and it may be the rate-limiting step in the catalytic cycle, since its rate of formation agrees reasonably well with the catalytic-centre activity determined in steady-state kinetic assays. In addition to the interacting form, a second form of the enzyme was noted during reduction by trimethylamine, differing in absorption spectrum, the structure of which remains to be determined. PMID:204297

  4. Participation of the iron-sulphur cluster and of the covalently bound coenzyme of trimethylamine dehydrogenase in catalysis.

    PubMed

    Steenkamp, D J; Singer, T P

    1978-02-01

    Bacterial trimethylamine dehydrogenase contains a novel type of covalently bound flavin mononucleotide and a tetrameric iron-sulphur centre. The dehydrogenase takes up 1.5mol of dithionite/mol of enzyme and is thereby converted into the flavin quinol-reduced (4Fe-4S) form, with the expected bleaching of the visible absorption band of the flavin and the emergence of signals of typical reduced ferredoxin in the electronparamagnetic-resonance spectrum. On reduction with a slight excess of substrate, however, unusual absorption and electron-paramagnetic-resonance spectra appear quite rapidly. The latter is attributed to extensive interaction between the reduced (4Fe-4S) centre and the flavin semiquinone. The species of enzyme arising during the catalytic cycle were studied by a combination of rapid-freeze e.p.r. and stopped-flow spectophotometry. The initial reduction of the flavin to the quinol form is far too rapid to be rate-limiting in catalysis, as is the reoxidation of the substrate-reduced enzyme by phenazine methosulphate. Formation of the spin-spin-interacting species from the dihydroflavin is considerably slower, however, and it may be the rate-limiting step in the catalytic cycle, since its rate of formation agrees reasonably well with the catalytic-centre activity determined in steady-state kinetic assays. In addition to the interacting form, a second form of the enzyme was noted during reduction by trimethylamine, differing in absorption spectrum, the structure of which remains to be determined. PMID:204297

  5. Structure of the red fluorescent protein from a lancelet (Branchiostoma lanceolatum): a novel GYG chromophore covalently bound to a nearby tyrosine

    SciTech Connect

    Pletnev, Vladimir Z. Pletneva, Nadya V.; Lukyanov, Konstantin A.; Souslova, Ekaterina A.; Fradkov, Arkady F.; Chudakov, Dmitry M.; Chepurnykh, Tatyana; Yampolsky, Ilia V.; Wlodawer, Alexander; Dauter, Zbigniew; Pletnev, Sergei

    2013-09-01

    The crystal structure of the novel red emitting fluorescent protein from lancelet Branchiostoma lanceolatum (Chordata) revealed an unusual five residues cyclic unit comprising Gly58-Tyr59-Gly60 chromophore, the following Phe61 and Tyr62 covalently bound to chromophore Tyr59. A key property of proteins of the green fluorescent protein (GFP) family is their ability to form a chromophore group by post-translational modifications of internal amino acids, e.g. Ser65-Tyr66-Gly67 in GFP from the jellyfish Aequorea victoria (Cnidaria). Numerous structural studies have demonstrated that the green GFP-like chromophore represents the ‘core’ structure, which can be extended in red-shifted proteins owing to modifications of the protein backbone at the first chromophore-forming position. Here, the three-dimensional structures of green laGFP (λ{sub ex}/λ{sub em} = 502/511 nm) and red laRFP (λ{sub ex}/λ{sub em} ≃ 521/592 nm), which are fluorescent proteins (FPs) from the lancelet Branchiostoma lanceolatum (Chordata), were determined together with the structure of a red variant laRFP-ΔS83 (deletion of Ser83) with improved folding. Lancelet FPs are evolutionarily distant and share only ∼20% sequence identity with cnidarian FPs, which have been extensively characterized and widely used as genetically encoded probes. The structure of red-emitting laRFP revealed three exceptional features that have not been observed in wild-type fluorescent proteins from Cnidaria reported to date: (i) an unusual chromophore-forming sequence Gly58-Tyr59-Gly60, (ii) the presence of Gln211 at the position of the conserved catalytic Glu (Glu222 in Aequorea GFP), which proved to be crucial for chromophore formation, and (iii) the absence of modifications typical of known red chromophores and the presence of an extremely unusual covalent bond between the Tyr59 C{sup β} atom and the hydroxyl of the proximal Tyr62. The impact of this covalent bond on the red emission and the large Stokes shift (

  6. Coffee bean arabinogalactans: acidic polymers covalently linked to protein.

    PubMed

    Redgwell, Robert J; Curti, Delphine; Fischer, Monica; Nicolas, Pierre; Fay, Laurent B

    2002-02-11

    The arabinogalactan content of green coffee beans (Coffea arabica var. Yellow Caturra) was released by a combination of chemical extraction and enzymatic hydrolysis of the mannan-cellulose component of the wall. Several arabinogalactan fractions were isolated, purified by gel-permeation and ion-exchange chromatography and characterised by compositional and linkage analysis. The AG fractions contained between 6 and 8% glucuronic acid, and gave a positive test for the beta-glucosyl-Yariv reagent, a stain specific for arabinogalactan-proteins. The protein component accounted for between 0.5 and 2.0% of the AGPs and contained between 7 and 12% hydroxyproline. The AG moieties displayed considerable heterogeneity with regard to their degree of arabinosylation and the extent and composition of their side-chains. They possessed a MW average of 650 kDa which ranged between 150 and 2000 kDa. An investigation of the structural features of the major AG fraction, released following enzymatic hydrolysis of the mannan-cellulose polymers, allowed a partial structure of coffee arabinogalactan to be proposed. PMID:11844494

  7. Isoreticular two-dimensional covalent organic frameworks synthesized by on-surface condensation of diboronic acids.

    PubMed

    Dienstmaier, Jürgen F; Medina, Dana D; Dogru, Mirjam; Knochel, Paul; Bein, Thomas; Heckl, Wolfgang M; Lackinger, Markus

    2012-08-28

    On-surface self-condensation of 1,4-benzenediboronic acid was previously shown to yield extended surface-supported, long-range-ordered two-dimensional covalent organic frameworks (2D COFs). The most important prerequisite for obtaining high structural quality is that the polycondensation (dehydration) reaction is carried out under slightly reversible reaction conditions, i.e., in the presence of water. Only then can the subtle balance between kinetic and thermodynamic control of the polycondensation be favorably influenced, and defects that are unavoidable during growth can be corrected. In the present study we extend the previously developed straightforward preparation protocol to a variety of para-diboronic acid building blocks with the aim to tune lattice parameters and pore sizes of 2D COFs. Scanning tunneling microscopy is employed for structural characterization of the covalent networks and of noncovalently self-assembled structures that form on the surface prior to the thermally activated polycondensation reaction. PMID:22775491

  8. GC-{sup 13}C IRMS characterisation of extractable and covalently bound alkanes in petroleum source rocks to reveal compositional fractionation effects

    SciTech Connect

    Love, G.D.; Fallick, A.E.; Taylor, C.

    1995-12-31

    The application of a sequential extraction/degradation scheme to differentiate between molecular alkanes (both easily extractable and physically-trapped) and covalently-bound alkyl moieties to a number of vitrinite concentrates and petroleum source rocks has been reported previously. Gas chromatography-isotope ratio mass spectrometry GC-s{sup 13}C IRMS has now been applied to the different awe fractions to probe compositional fractionation effects that might occur from the different initial biological inputs. For a Turkish oil shale (Goynuk - Type I kerogen), inputs from diverse sources, including phytoplanktron, higher plants and bacteria were implied from analysis of solvent-extractable alkanes. However, the much larger quantities of covalently-bound alkanes had an isotopic signature typical of eukarytoic (freshwater) algae. The isotopic uniformity of alkanes/alkenes released from sequential hydropyrolysis of a torbanite (Duunet shale) confirmed that this sample was largely derived from the selective preservation of resistant aliphatic biopolymers found in Botryococcus cell walls.

  9. Spin Labeling ESR Investigation of a Role of Humic Acids at Covalent Binding of Xenobiotics to Soil

    NASA Astrophysics Data System (ADS)

    Aleksandrova, Olga

    2014-05-01

    The environmental risk of organic xenobiotic chemicals released into soils is controlled by their sorption and binding processes. However, the molecular mechanisms of reversible and irreversible interactions of xenobiotics with soil constituents and an influence of humic substances on this interaction are only partly understood. New methods and approaches aimed at understanding of molecular mechanisms in the soil environment and a role of humic substances in the sorption and binding processes are today required to manage and keep the quality of soil used and fertilized in agricultural industry. The paper presents a new approach of using stable ESR spin labels to investigate a role of humic substances in the interactions of organic xenobiotic chemicals with constituents of natural soil via the typical functional groups of xenobiotics, such as Amines. At the experiment, the nitroxide spin labels, such as TEMPO (2,2,6,6-Tetramethylpiperidin-1-oxyl), Amino-TEMPO (4-amino-2,2,6,6-Tetramethylpiperidin-1-oxyl) and Aniline spin labels (2,5,5-Trimethyl-2-(3-aminophenyl)pyrrolidin-1-oxyl), were added to samples of different natural soils, such luvisol, cambisol and chernozem. Amino-TEMPO and Aniline spin labels include the aliphatic amino and aromatic amino functional groups, respectively. A significant broadening of the ESR spectrum of Aniline spin labels incubated in different soils indicated a stable effect of covalent binding of the spin labels to soil constituents via the aromatic amino, whereas the ESR spectra of the other two spin labels were not broadened that pointed at the absence of covalent binding of spin labels via the aliphatic amino. As shown, a part of bound spin labels via the aromatic amino increased with increasing of the concentration of humic acids in soil. The same broadened signals were also be detected with the humic acids extracted from the investigated soils. A strong covalent binding of spin labels to humic substances via the aromatic amines was

  10. The contribution of a covalently bound cofactor to the folding and thermodynamic stability of an integral membrane protein.

    PubMed

    Curnow, Paul; Booth, Paula J

    2010-11-01

    The factors controlling the stability, folding, and dynamics of integral membrane proteins are not fully understood. The high stability of the membrane protein bacteriorhodopsin (bR), an archetypal member of the rhodopsin photoreceptor family, has been ascribed to its covalently bound retinal cofactor. We investigate here the role of this cofactor in the thermodynamic stability and folding kinetics of bR. Multiple spectroscopic probes were used to determine the kinetics and energetics of protein folding in mixed lipid/detergent micelles in the presence and absence of retinal. The presence of retinal increases extrapolated values for the overall unfolding free energy from 6.3 ± 0.4 kcal mol(-1) to 23.4 ± 1.5 kcal mol(-1) at zero denaturant, suggesting that the cofactor contributes 17.1 kcal mol(-1) towards the overall stability of bR. In addition, the cooperativity of equilibrium unfolding curves is markedly reduced in the absence of retinal with overall m-values decreasing from 31.0 ± 2.0 kcal mol(-1) to 10.9 ± 1.0 kcal mol(-1), indicating that the folded state of the apoprotein is less compact than the equivalent for the holoprotein. This change in the denaturant response means that the difference in the unfolding free energy at a denaturant concentration midway between the two unfolding curves is only ca 3-6 kcal mol(-1). Kinetic data show that the decrease in stability upon removal of retinal is associated with an increase in the apparent intrinsic rate constant of unfolding, k(u)(H2O), from ~1 × 10(-16) s(-1) to ~1 × 10(-4) s(-1) at 25 °C. This correlates with a decrease in the unfolding activation energy by 16.3 kcal mol(-1) in the apoprotein, extrapolated to zero SDS. These results suggest that changes in bR stability induced by retinal binding are mediated solely by changes in the activation barrier for unfolding. The results are consistent with a model in which bR is kinetically stabilized via a very slow rate of unfolding arising from protein

  11. In vivo and in vitro covalent binding of chlorobenzene to nucleic acids.

    PubMed

    Grilli, S; Arfellini, G; Colacci, A; Mazzullo, M; Prodi, G

    1985-08-01

    At 22 hr after ip injection into male Wistar rats and BALB/c mice, chlorobenzene was covalently bound to DNA, RNA and proteins of the liver, kidney and lung, as has been found with various weak carcinogens. A microsome-mediated interaction with DNA occurred in vitro. The interaction was enhanced by pretreatment in vivo with phenobarbitone but was suppressed by addition of 2-diethylaminoethyl-2,2-diphenylvalerate HC1 in vitro. These results indicate the involvement of cytochrome P-450. Liver microsomes were efficient bioactivators, whereas cytosol was ineffective. The extent of in vitro interaction of chlorobenzene with synthetic polyribonucleotides was of the same order as that with DNA. Finally, ultraviolet irradiation (lambda = 254 nm or lambda max = 365 nm) activated this environmental contaminant to forms capable of interacting with DNA. The results represent evidence for genotoxicity of chlorobenzene. PMID:3930452

  12. Possible contribution of cell-wall-bound ferulic acid in drought resistance and recovery in triticale seedlings.

    PubMed

    Hura, T; Hura, K; Grzesiak, S

    2009-11-01

    Studies were undertaken to estimate whether the presence of free and cell-wall-bound ferulic acid in leaf tissues can support drought resistance and its recovery under rehydration. An experiment was carried out on two genotypes of winter triticale: Lamberto and Ticino, at the propagation phase. Lamberto exhibited high content of ferulic acid bound with carbohydrates of the cell-wall under drought and rehydration. The markedly better parameters of chlorophyll fluorescence for this variety under both treatments correlated strongly and positively with the high contents of cell-wall-bound ferulic acid. The photosynthetic apparatus of Lamberto, in relation to Ticino, proved to be the more efficient after 4 weeks of drought treatment. The after-effects of soil drought better elicited the function disturbances of the photosynthetic apparatus in Ticino, which did not fully recover in comparison to Lamberto. Ferulic acid covalently bound to carbohydrates of the cell wall may act as a light filter limiting mesophyll penetration under drought conditions and can also support drought adaptation by down-regulation of leaf growth. The observed increase in the content of cell-wall-bound ferulic acid, as a response to water deficit in the leaf, could be one of the protective mechanisms induced by drought conditions. The ability to accumulate phenolic compounds in dehydrated leaves might be an additional and reliable biochemical parameter indicating the resistance of plants to drought stress. PMID:19464752

  13. Synthesis and immunological properties of conjugates composed of group B streptococcus type III capsular polysaccharide covalently bound to tetanus toxoid.

    PubMed

    Lagergard, T; Shiloach, J; Robbins, J B; Schneerson, R

    1990-03-01

    A synthetic scheme for covalently binding group B streptococcus type III to tetanus toxoid (TT), using adipic acid dihydrazide as a spacer, is described. Type III alone or as a conjugate with TT was injected subcutaneously into laboratory mice, and the type-specific and TT antibody responses elicited by these immunogens were assayed. Type III-TT elicited significantly higher levels of type-specific antibodies after each immunization than did the type III alone. These levels were related to the dosage of the conjugate, enhanced by Freund adjuvant, and exhibited booster responses. Type III alone elicited only immunoglobulin M (IgM) antibodies in Swiss albino mice and mostly IgM and low levels of IgG antibodies of the IgG3 subclass in BALB/c mice. Type III-TT conjugates, in contrast, elicited mostly IgG antibodies in both strains of mice. IgA type III antibodies were not detected. The first two immunizations with the conjugates elicited type III antibodies in the IgG1 and in the IgG3 subclasses. Low levels of IgG2a type III antibodies were detected after a third injection of type III-TT. Conjugate-induced antibodies facilitated opsonization of group B streptococcus type III organisms and did not react with the structurally related pneumococcus type 14. TT alone or as a component of type III-TT induced mostly antibodies of the IgG class: IgG1 levels were the highest of the four subclasses. No IgA TT antibodies were detected. The conjugation procedure, therefore, enhanced the immunogenicity of and conferred T-cell dependent properties to the type III while preserving the immunogenicity of the TT component. The T-cell dependent properties of the conjugates were responsible for stimulating IgG type III antibodies which could be boosted. Evaluation of type III-TT conjugates in antibody-negative women of child-bearing age is planned. PMID:2407652

  14. Targeted covalent inactivation of protein kinases by resorcylic acid lactone polyketides

    PubMed Central

    Schirmer, Andreas; Kennedy, Jonathan; Murli, Sumati; Reid, Ralph; Santi, Daniel V.

    2006-01-01

    Resorcylic acid lactones containing a cis-enone are susceptible to Michael addition reactions and are potent inhibitors of several protein kinases. A structural-bioinformatics analysis identified a conserved Cys residue in the ATP-binding site of the kinases reported to be inhibited by cis-enone resorcylic acid lactones but absent in those that are not. Mining of the kinome database revealed that a subset of some 46 kinases contained this Cys residue. Screening a panel of 124 kinases with the resorcylic acid lactone hypothemycin showed that 18 of 19 targets containing the conserved Cys were inhibited. Kinetic analyses showed time-dependent inhibition, a hallmark of covalent inactivation, and biochemical studies of the interaction of extracellular signal-regulated kinase (ERK)2 with hypothemycin confirmed covalent adduct formation. Resorcylic acid lactones are unique among kinase inhibitors in that they target mitogen-activated protein (MAP) kinase pathways at four levels: mitogen receptors, MAP kinase kinase (MEK)1/2 and ERK1/2, and certain downstream ERK substrates. Cell lines dependent on the activation of Tyr kinase mitogen receptor targets of the resorcylic acid lactones were unusually sensitive toward hypothemycin and showed the expected inhibition of kinase phosphorylation due to inhibition of the mitogen receptors and/or MEK1/2 and ERK1/2. Among cells without mitogen receptor targets, those harboring an ERK pathway-activating B-RAF V600E mutation were selectively and potently inhibited by hypothemycin. Hypothemycin also prevented stimulated activation of the p38 cascade through inhibition of the Cys-containing targets MEK3/6 and TGF-β-activated kinase 1 and of the JNK/SAPK (c-Jun N-terminal kinase/stress-activated protein kinase) cascade through inhibition of MEK4/7. PMID:16537514

  15. Covalent protein crosslinks: general detection, quantitation, and characterization via modification with diphenylborinic acid.

    PubMed

    Graham, L; Gallop, P M

    1994-03-01

    Progressive crosslinking of proteins appears to be a general phenomenon in aging cells and tissues. Crosslinked proteins can form insoluble aggregates which become increasingly resistant to proteolysis as more crosslinks form. However, most evidence for progressive crosslinking with age is indirect, and little is known about the chemical mechanisms involved. We have therefore developed a method for detection and isolation of any type of stable covalent crosslink from protein hydrolysates which requires no prior knowledge of the molecular structure of whatever crosslink(s) may be present. It utilizes the specificity of the diphenylborinic acid reagent for alpha-amino acid groups and the chromatographic properties and uv absorbance of the crosslink derivatives. The method is demonstrated using eight different crosslinks from collagen and fibrin, and a general procedure is given for detection of any type of crosslink in a protein hydrolysate. PMID:8203759

  16. Photogeneration of singlet oxygen by the phenothiazine derivatives covalently bound to the surface-modified glassy carbon

    NASA Astrophysics Data System (ADS)

    Blacha-Grzechnik, Agata; Piwowar, Katarzyna; Krukiewicz, Katarzyna; Koscielniak, Piotr; Szuber, Jacek; Zak, Jerzy K.

    2016-05-01

    The selected group of four amine-derivatives of phenothiazine was covalently grafted to the glassy carbon surface in the four-step procedure consisting of the electrochemical reduction of the diazonium salt followed by the electrochemical and chemical post-modification steps. The proposed strategy involves the bonding of linker molecule to which the photosensitizer is attached. The synthesized organic layers were characterized by means of cyclic voltammetry, XPS and Raman Spectroscopy. It was shown that the phenothiazines immobilized via proposed strategy retain their photochemical properties and are able to generate 1O2 when activated by the laser radiation. The effectiveness of in situ singlet oxygen generation by those new solid photoactive materials was determined by means of UVVis spectroscopy. The reported, covalently modified solid surfaces may find their application as the singlet oxygen photogenerators in the fine chemicals' synthesis or in the wastewater treatment.

  17. Modeling the reactions of 1-naphthylamine and 4-methylaniline with humic acids: Spectroscopic investigations of the covalent linkages

    SciTech Connect

    Ononye, A.I. . Dept. of Chemistry); Graveel, J.G. . Dept. of Agronomy)

    1994-04-01

    The covalent binding of two aromatic amines, 1-naphthylamine and 4-methylaniline, to substituted quinones in aqueous solutions has been studied as a model for the covalent binding of amines to humic acids. An imine compound was the only type of product identified for the reactions between 1-naphthylamine and selected quinones. Reactions of 4-methylaniline with the same quinones gave a 1,4- addition product in addition to the imine compound, although only the imine produce was formed when the quinone had bulky substituents at the 2- and 6- positions. These results provide additional insight into the reactions of aromatic amines with humic acid quinones.

  18. Synthesis of Photoactivatable Analogues of Lysophosphatidic Acid and Covalent Labeling of Plasma Proteins

    PubMed Central

    Li, Zaiguo; Baker, Daniel L.; Tigyi, Gabor; Bittman, Robert

    2008-01-01

    Lysophosphatidic acid bearing a benzophenone group in either the sn-1 or sn-2 chain of an oleoyl-type ester or oleyl-type ether chain and 32P in the phosphate group were synthesized. The benzophenone moiety was introduced by selective hydroboration of the double bond of enyne 11 at low temperature, followed by Suzuki reaction with 4-bromobenzophenone. The key intermediates for the preparation of ester-linked LPA 1 and 3 were obtained in one pot by a modified DIBAL-H reduction of orthoformate intermediate 22. These probes were shown to covalently modify a single protein target in rat plasma containing albumin and several protein targets in rat plasma containing a low level of albumin. PMID:16408973

  19. Fluorometric assay for quantitation of biotin covalently attached to proteins and nucleic acids.

    PubMed

    Batchelor, Robert H; Sarkez, Adam; Cox, W Gregory; Johnson, Iain

    2007-10-01

    As a component of the (strept)avidin affinity system, biotin is often covalently linked to proteins or nucleic acids. We describe here a microplate-based high-throughput fluorometric assay for biotin linked to either proteins or nucleic acids based on fluorescence resonance energy transfer (FRET). This assay utilizes a complex of Alexa Fluoro 488 dye-labeled avidin with a quencher dye, 2-(4'-hydroxyazobenzene) benzoic acid (HABA), occupying the biotin binding sites of the avidin. In the absence of biotin, HABA quenches the fluorescence emission of the Alexa Fluor 488 dyes via FRET HABA is displaced when biotin binds to the Alexa Fluor 488 dye-labeled avidin, resulting in decreased FRET efficiency. This mechanism results in an increase in fluorescence intensity directly related to the amount of biotin present in the sample. The assay is able to detect as little as 4 pmol biotin in a 0.1 mL volume within 15 min of adding sample to the reagent, with a Z-factor > 0.9. PMID:18019342

  20. Affinity isolation of cultured tumor cells by means of drugs and hormones covalently bound to glass and Sepharose beads.

    PubMed Central

    Venter, B R; Venter, J C; Kaplan, N O

    1976-01-01

    Isoproterenol, corticotropin (ACTH), and triodothyronine immobilized on glass and Sepharose beads by diazotization procedures have been shown to interact with cultured tumor cells of "target tissue" origin. Cells used were rat glioma cells (C6), rat adrenal tumor cells (Y-1), and rat pituitary tumor cells (GH3). The rat glioma cells bound principally to immobilized isoproterenol, whereas the rat adrenal tumor cells bound to immobilized corticotropin, and rat pituitary tumor cells bound to immobilized triiodothyronine. Binding was inhibited by preincubation of the cells in soluble drug or hormone. With C6 cells there was a positive correlation between adenylate cyclase [ATP pyrophosphate-lyase (cyclizing, EC 4.6.1.1] stimulation and the degree of binding to the immobilized isoproterenol. Norepinephrine, bound through the ethanolamine side chain via an amide linkage, did not bind cells, demonstrating specific structural requirements for drug-cell interactions. HeLa cells were shown to bind tightly to diphtheria toxin coupled to Sepharose beads via an amide bond. This binding was inhibited by prior incubation of the Sepharose toxin with purified antitoxin. Toxin bound to Sepharose via an azo bond did not bind cells. These data suggest that the cell affinities are due to cell surface receptors interacting with the immobilized drugs and hormones, and that the observed affinities possibly reflect the relative receptor complement of these cells. Images PMID:180534

  1. Non-covalent bonded 2D-3D supramolecular architectures based on 4-dimethylaminopyridine and organic acids

    NASA Astrophysics Data System (ADS)

    Zhang, Huan; Jin, Shouwen; Wen, Xianhong; Liu, Bin; Fang, Yang; Zhang, Yani; Wang, Daqi

    2015-07-01

    Studies concentrating on non-covalent weak interactions between the organic base of 4-dimethylaminopyridine, and acidic derivatives have led to an increased understanding of the role 4-dimethylaminopyridine has in binding with the organic acid derivatives. Here anhydrous and hydrous multicomponent organic acid-base adducts of 4-dimethylaminopyridine have been prepared with organic acids such as 1,3-benzodioxole-5-carboxylic acid, p-aminobenzoic acid, 2,4-dihydroxybenzoic acid, 3,5-dihydroxybenzoic acid, 5-chlorosalicylic acid, 5-bromosalicylic acid, 5-nitrosalicylic acid, and 5-sulfosalicylic acid. The 4-dimethylaminopyridine is only monoprotonated. All compounds are organic salts with the 1:1 ratio of the cation and the anion. For the 5-sulfosalicylic acid only one H is ionized to exhibit the valence number of -1. The eight crystalline complexes were characterized by X-ray diffraction analysis, IR, mp, and elemental analysis. These structures adopted the hetero supramolecular synthons. Analysis of the crystal packing of 1-8 suggests that there are Nsbnd H⋯O, Osbnd H⋯O, and Osbnd H⋯S hydrogen bonds (charge assisted or neutral) between the organic acid and the 4-dimethylaminopyridine moieties in the studied compounds. Except the classical hydrogen bonding interactions, the secondary propagating interactions also play important roles in structure extension. For the synergistic effect of the various non-covalent interactions, the complexes displayed 2D-3D framework structures.

  2. Triple-helical collagen hydrogels via covalent aromatic functionalization with 1,3-Phenylenediacetic acid

    PubMed Central

    Tronci, Giuseppe; Doyle, Amanda; Russell, Stephen J.; Wood, David J.

    2016-01-01

    Chemical crosslinking of collagen is a general strategy to reproduce macroscale tissue properties in physiological environment. However, simultaneous control of protein conformation, material properties and biofunctionality is highly challenging with current synthetic strategies. Consequently, the potentially-diverse clinical applications of collagen-based biomaterials cannot be fully realised. In order to establish defined biomacromolecular systems for mineralised tissue applications, type I collagen was functionalised with 1,3-Phenylenediacetic acid (Ph) and investigated at the molecular, macroscopic and functional levels. Preserved triple helix conformation was observed in obtained covalent networks via ATR-FTIR (AIII/A1450 ~ 1) and WAXS, while network crosslinking degree (C: 87-99 mol.-%) could be adjusted based on specific reaction conditions. Decreased swelling ratio (SR: 823-1285 wt.-%) and increased thermo-mechanical (Td: 80-88 °C; E: 28-35 kPa; σmax: 6-8 kPa; εb: 53-58 %) properties were observed compared to state-of-the-art carbodiimide (EDC)-crosslinked collagen controls, likely related to the intermolecular covalent incorporation of the aromatic segment. Ph-crosslinked hydrogels displayed nearly intact material integrity and only a slight mass decrease (MR: 5-11 wt. %) following 1-week incubation in either PBS or simulated body fluid (SBF), in contrast to EDC-crosslinked collagen (MR: 33-58 wt. %). Furthermore, FTIR, SEM and EDS revealed deposition of a calcium-phosphate phase on SBF-retrieved samples, whereby an increased calcium phosphate ratio (Ca/P: 0.84-1.41) was observed in hydrogels with higher Ph content. 72-hour material extracts were well tolerated by L929 mouse fibroblasts, whereby cell confluence and metabolic activity (MTS assay) were comparable to those of cells cultured in cell culture medium (positive control). In light of their controlled structure-function properties, these biocompatible collagen hydrogels represent attractive

  3. Transfer of a weakly bound electron in collisions of Rydberg atoms with neutral particles. I. Long-range interaction effects in the ionic-covalent coupling

    SciTech Connect

    Lebedev, V. S. Narits, A. A.

    2013-10-15

    Ion-pair formation processes are studied in collisions of Rydberg atoms with neutral particles possessing small electron affinities. Nonadiabatic transitions from a Rydberg covalent term to an ionic term of a quasi-molecule are considered using the modified Landau-Zener theory supplemented with calculation of survival factors of an anion decaying in the Coulomb field of a positive ion core. Using the technique of irreducible tensor operators and the momentum representation of the wavefunction of a highly excited atom, exact expressions are obtained for transition matrix elements and the ionic-covalent coupling parameter. The approach developed in the paper provides the description beyond the scope of a conventional assumption about a small variation of the wavefunction of the Rydberg atom on the range of electron coordinates determined by the characteristic radius of the wavefunction of the anion. This allows one to correctly consider long-range effects of the interaction between a weakly bound electron and the neutral core of a negative ion in processes under study. It is shown by the example of thermal collisions of Xe(nf) atoms with CH{sub 3}CN molecules that this is very important for a reliable quantitative description of anion formation with a low binding energy. The results are compared with experiments and calculations performed within the framework of a number of approximate methods.

  4. Building, characterising and catalytic activity testing of Co-C-protected amino acid complexes covalently grafted onto chloropropylated silica gel

    NASA Astrophysics Data System (ADS)

    Varga, G.; Timár, Z.; Csendes, Z.; Bajnóczi, É. G.; Carlson, S.; Canton, S. E.; Bagi, L.; Sipos, P.; Pálinkó, I.

    2015-06-01

    Co-C-protected amino acid (C-protected L-histidine, L-tyrosine, L-cysteine and L-cystine) complexes were covalently grafted onto chloropropylated silica gel, and the materials thus obtained were structurally characterised by mid/far IR and X-ray absorption spectroscopies. The superoxide dismutase-like activities of the substances were determined via the Beauchamp-Fridovich test reaction. It was found that covalent grafting and the preparation of the anchored complexes were successful in most cases. The coordinating groups varied upon changing the conditions of the syntheses. All materials displayed catalytic activity, although catalytic activities differed widely.

  5. Gambogic acid deactivates cytosolic and mitochondrial thioredoxins by covalent binding to the functional domain.

    PubMed

    Yang, Jing; Li, Chenglin; Ding, Li; Guo, Qinglong; You, Qidong; Jin, Shaohong

    2012-06-22

    Gambogic acid (1) is a cytotoxic caged xanthone derived from the resin of Garcinia hanburyi. Compound 1 selectively induces apoptosis in cancer cells, at least partially, by targeting the stress response to reactive oxygen species (ROS). However, the molecular mechanism of ROS toxicity stimulated by 1 remains poorly understood. In this study, mass spectrometric and biochemical pharmacological approaches were used that resulted in the identification of both cytosolic thioredoxin (TRX-1) and mitochondrial thioredoxin (TRX-2) as the molecular targets of 1. The results obtained showed that 1 deactivates TRX-1/2 proteins by covalent binding to the active cysteine residues in the functional domain via Michael addition reactions. Since both TRX-1 and TRX-2 play key roles in regulating the redox signaling of cancer cells, the present findings may shed light on the relationship between protein binding and cellular ROS accumulation induced by 1. This provides support for the current clinical trials of gambogic acid (1) being conducted alone or in combination with other agents that appear to increase ROS generation in order to selectively kill cancer cells. PMID:22663155

  6. Covalent grafting of copper amino acid complexes onto chloropropylated silica gel—an FT-IR study

    NASA Astrophysics Data System (ADS)

    Jakab, N. I.; Hernadi, K.; Kiss, J. T.; Pálinkó, I.

    2005-06-01

    Cu(amino acid) complexes were immobilised on silica gel by covalent anchoring. The amino acids were L-histidine and L-tyrosine and their BOC—( tert-butoxycarbonyl) or methyl ester protected derivatives. To gain control over the synthesis the appropriately protected amino acid was reacted with chloropropylated silica gel first. This modified material as is, or after deprotecting the anchored amino acids, was used in further steps of building the immobilised Cu(II) complex. The covalently grafted complexes were studied by FT-IR spectroscopy and computer modelling. Materials containing protected histidine ligands showed catalase activity (decomposition of H 2O 2), those containing protected or unprotected tyrosine ligands displayed tyrosinase activity (the decomposed H 2O 2 oxidised the tyrosine skeleton to a quinoidal structure).

  7. Loss of covalently linked lipid as the mechanism for radiation-induced release of membrane-bound polysaccharide and exonuclease from Micrococcus radiodurans. [/sup 60/CO

    SciTech Connect

    Mitchel, R.E.J.

    1981-08-01

    The mechanism of ..gamma..-radiation-induced release of polysaccharide and exonuclease from the midwall membrane of Micrococcus radiodurans has been examined. These two components appear to be released independently, but by very similar processes. Direct analysis of radiation-released polysaccharide indicated the absence of an alkali-labile neutral lipid normally present in the native material. Radiation-induced release therefore probably results from the radiolytic cleavage of a covalently linked lipid which normally serves to anchor these substances to the membrane. The absence of a natural membrane-bound carotenoid had no effect on the rate of release of these components. Likewise, the absence of exonuclease in an exonuclease minus mutant did not influence the release of polysaccharide. It is suggested that the major pathway of radical transfer from the initiating .OH and culminating in the cleavage of the neutral lipid anchor may not be via the membrane.

  8. Rapid acquisition of multidimensional solid-state NMR spectra of proteins facilitated by covalently bound paramagnetic tags.

    PubMed

    Nadaud, Philippe S; Helmus, Jonathan J; Sengupta, Ishita; Jaroniec, Christopher P

    2010-07-21

    We describe a condensed data collection approach that facilitates rapid acquisition of multidimensional magic-angle spinning solid-state nuclear magnetic resonance (SSNMR) spectra of proteins by combining rapid sample spinning, optimized low-power radio frequency pulse schemes and covalently attached paramagnetic tags to enhance protein (1)H spin-lattice relaxation. Using EDTA-Cu(2+)-modified K28C and N8C mutants of the B1 immunoglobulin binding domain of protein G as models, we demonstrate that high resolution and sensitivity 2D and 3D SSNMR chemical shift correlation spectra can be recorded in as little as several minutes and several hours, respectively, for samples containing approximately 0.1-0.2 micromol of (13)C,(15)N- or (2)H,(13)C,(15)N-labeled protein. This mode of data acquisition is naturally suited toward the structural SSNMR studies of paramagnetic proteins, for which the typical (1)H longitudinal relaxation time constants are inherently a factor of at least approximately 3-4 lower relative to their diamagnetic counterparts. To illustrate this, we demonstrate the rapid site-specific determination of backbone amide (15)N longitudinal paramagnetic relaxation enhancements using a pseudo-3D SSNMR experiment based on (15)N-(13)C correlation spectroscopy, and we show that such measurements yield valuable long-range (15)N-Cu(2+) distance restraints which report on the three-dimensional protein fold. PMID:20583834

  9. Antioxidant and antiradical SiO2 nanoparticles covalently functionalized with gallic acid.

    PubMed

    Deligiannakis, Yiannis; Sotiriou, Georgios A; Pratsinis, Sotiris E

    2012-12-01

    Gallic acid (GA) and its derivatives are natural polyphenolic substances widely used as antioxidants in nutrients, medicine and polymers. Here, nanoantioxidant materials are engineered by covalently grafting GA on SiO(2) nanoparticles (NPs). A proof-of-concept is provided herein, using four types of well-characterized SiO(2) NPs of specific surface area (SSA) 96-352 m(2)/g. All such hybrid SiO(2)-GA NPs had the same surface density of GA molecules (~1 GA per nm(2)). The radical-scavenging capacity (RSC) of the SiO(2)-GA NPs was quantified in comparison with pure GA based on the 2,2-diphenyl-1-picrylhydrazyl (DPPH(•)) radical method, using electron paramagnetic resonance (EPR) and UV-vis spectroscopy. The scavenging of DPPH radicals by these nanoantioxidant SiO(2)-GA NPs showed mixed-phase kinetics: An initial fast-phase (t(1/2) <1 min) corresponding to a H-Atom Transfer (HAT) mechanism, followed by a slow-phase attributed to secondary radical-radical reactions. The slow-reactions resulted in radical-induced NP agglomeration, that was more prominent for high-SSA NPs. After their interaction with DPPH radicals, the nanoantioxidant particles can be reused by simple washing with no impairment of their RSC. PMID:23121088

  10. Bioavailability of Chlorocatechols in Naturally Contaminated Sediment Samples and of Chloroguaiacols Covalently Bound to C2-Guaiacyl Residues

    PubMed Central

    Allard, Ann-Sofie; Hynning, Per-Åke; Remberger, Mikael; Neilson, Alasdair H.

    1994-01-01

    Bacteria in anaerobic enrichment cultures that dechlorinated a range of chlorocatechols were used to examine the stability of endogenous chlorocatechols in a contaminated sediment sample and in interstitial water prepared from it. During incubation of the sediment sample for 450 days with or without added cells, there was a decrease in the concentration of solvent-extractable chlorocatechols but not in that of the total chlorocatechols, including sediment-associated components. In the presence of azide, the decrease in the concentrations of the former was eliminated or substantially decreased. Control experiments in which 3,4,5-trichlorocatechol was added to the sediment suspensions after 130 days showed that its dechlorination was accomplished not only by the added cells but also by the endemic microbial flora. It was concluded that the endogenous chlorocatechols in the sediment were not accessible to microorganisms with dechlorinating activity. On the other hand, microorganisms were apparently responsible for decreasing the solvent extractability of the chlorocatechols, and this effect decreased with increasing length of exposure time. Similar experiments carried out for 70 days with the sediment interstitial water showed that the chlorocatechols that were known to be associated with organic matter were also inaccessible to microbial dechlorination. Experiments with model compounds in which 4,5,6-trichloroguaiacol and tetrachloroguaiacol were covalently linked to C2-guaiacyl residues showed that these compounds were resistant to O demethylation or dechlorination during incubation with a culture having these activities. The only effect of microbial action was the quantitative reduction in 12 days of the C′1 keto group to an alcohol which was stable against further transformation for up to 65 days. The results of these experiments are consistent with the existence of chlorocatechols and chloroguaiacols in contaminated sediments and illustrate the cardinal

  11. Hyaluronic acid and alginate covalent nanogels by template cross-linking in polyion complex micelle nanoreactors.

    PubMed

    De Santis, Serena; Diociaiuti, Marco; Cametti, Cesare; Masci, Giancarlo

    2014-01-30

    Hyaluronic acid (HA) and alginate (AL) covalent nanogels cross-linked with l-lysine ethyl ester were prepared by template chemical cross-linking of the polysaccharide in polyion complex micelle (PIC) nanoreactors. By using this method we were able to prepare HA and AL nanogels without organic solvents. PICs were prepared by using poly(ethylene oxide)-block-poly[(3-acrylamidopropyl)-trimethylammonium chloride] (PEO-b-PAMPTMA) or poly[(N-isopropylacrylamide)-block-PAMPTMA] (PNIPAAM-b-PAMPTMA). Only PNIPAAM-b-PAMPTMA block copolymers allowed to prepare PIC with small and controlled size. Short polysaccharide chains (Xn=50 and 63 for AL and HA, respectively, where Xn is the number of monosaccharidic units present in the polysaccharide) where used to optimize PIC formation. The remarkable difference in charge density and rigidity of HA and AL did not have a significant influence on the formation of PICs. PICs with small size (diameter of about 50-80 nm) and low polydispersity were obtained up to 5mg/mL of polymer. After cross-linking with l-lysine ethyl ester, the nanoreactors were dissociated by adding NaCl. The nanogels were easily purified and isolated by dialysis. The dissociation of the nanoreactors and the formation of the nanogels were confirmed by (1)H NMR, DLS, TEM and ζ-potential measurements. The size of the smallest nanogels in solution in the swollen state was 50-70 nm in presence of salt and 80-100 nm in water. PMID:24299754

  12. Conformational Analysis of Free and Bound Retinoic Acid

    PubMed Central

    Fu, Zheng; Li, Xue; Merz, Kenneth M.

    2012-01-01

    The conformational profiles of unbound all-trans and 9-cis retinoic acid (RA) have been determined using classical and quantum mechanical calculations. Sixty-six all-trans-RA (ATRA) and forty-eight 9-cis-RA energy minimum conformers were identified via HF/6-31G* geometry optimizations in vacuo. Their relative conformational energies were estimated utilizing the M06, M06-2x and MP2 methods combined with the 6-311+G(d,p), aug-cc-pVDZ and aug-cc-pVTZ basis sets, as well as complete basis set MP2 extrapolations using the latter two basis sets. Single-point energy calculations performed with the M06-2x density functional were found to yield similar results to MP2/CBS for the low-energy retinoic acid conformations. Not unexpectedly, the conformational propensities of retinoic acid were governed by the orientation and arrangement of the torsion angles associated with the polyene tail. We also used previously reported QM/MM X-ray refinement results on four ATRA-protein crystal structures plus one newly refined 9-cis-RA complex (PDB ID 1XDK) in order to investigate the conformational preferences of bound retinoic acid. In the re-refined RA conformers the conjugated double bonds are nearly coplanar, which is consistent with the global minimum identified by the Omega/QM method rather than the corresponding crystallographically determined conformations given in the PDB. Consequently, a 91.3% average reduction of the local strain energy in the gas phase, as well as 92.1% in PCM solvent, was observed using the QM/MM refined structures versus the PDB deposited RA conformations. These results thus demonstrate that our QM/MM X-ray refinement approach can significantly enhance the quality of X-ray crystal structures refined by conventional refinement protocols, thereby providing reliable drug-target structural information for use in structure-based drug discovery applications. PMID:22844234

  13. Hyaluronic Acid-Based Hydrogels Containing Covalently Integrated Drug Depots: Implication for Controlling Inflammation in Mechanically Stressed Tissues

    PubMed Central

    Xiao, Longxi; Tong, Zhixiang; Chen, Yingchao; Pochan, Darrin J.; Sabanayagam, Chandran R.; Jia, Xinqiao

    2013-01-01

    Synthetic hydrogels containing covalently-integrated soft and deformable drug depots capable of releasing therapeutic molecules in response to mechanical forces are attractive candidates for the treatment of degenerated tissues that are normally load bearing. Herein, radically crosslinkable block copolymer micelles (xBCM) assembled from an amphiphilic block copolymer consisting of hydrophilic poly(acrylic acid) (PAA) partially modified with 2-hydroxyethyl acrylate, and hydrophobic poly(n-butyl acryclate) (PnBA) were employed as the drug depots and the microscopic crosslinkers for the preparation of hyaluronic acid (HA)-based, hydrogels. HA hydrogels containing covalently integrated micelles (HAxBCM) were prepared by radical polymerization of glycidyl methacrylate (GMA)-modified HA (HAGMA) in the presence of xBCMs. When micelles prepared from the parent PAA-b-PnBA without any polymerizable double bonds were used, hydrogels containing physically entrapped micelles (HApBCM) were obtained. The addition of xBCMs to a HAGMA precursor solution accelerated the gelation kinetics and altered the hydrogel mechanical properties. The resultant HAxBCM gels exhibit an elastic modulus of 847 ± 43 Pa and a compressive modulus of 9.2 ± 0.7 kPa. Diffusion analysis of Nile Red (NR)-labeled xBCMs employing fluorescence correlation spectroscopy confirmed the covalent immobilization of xBCMs in HA networks. Covalent integration of dexamethasone (DEX)-loaded xBCMs in HA gels significantly reduced the initial burst release and provided sustained release over a prolonged period. Importantly, DEX release from HAxBCM gels was accelerated by intermittently-applied external compression in a strain-dependent manner. Culturing macrophages in the presence of DEX-releasing HAxBCM gels significantly reduced cellular production of inflammatory cytokines. Incorporating mechano-responsive modules in synthetic matrices offers a novel strategy to harvest mechanical stress present in the healing wounds

  14. Hyaluronic acid-based hydrogels containing covalently integrated drug depots: implication for controlling inflammation in mechanically stressed tissues.

    PubMed

    Xiao, Longxi; Tong, Zhixiang; Chen, Yingchao; Pochan, Darrin J; Sabanayagam, Chandran R; Jia, Xinqiao

    2013-11-11

    Synthetic hydrogels containing covalently integrated soft and deformable drug depots capable of releasing therapeutic molecules in response to mechanical forces are attractive candidates for the treatment of degenerated tissues that are normally load bearing. Herein, radically cross-linkable block copolymer micelles (xBCM) assembled from an amphiphilic block copolymer consisting of hydrophilic poly(acrylic acid) (PAA) partially modified with 2-hydroxyethyl acrylate, and hydrophobic poly(n-butyl acryclate) (PnBA) were employed as the drug depots and the microscopic cross-linkers for the preparation of hyaluronic acid (HA)-based, hydrogels. HA hydrogels containing covalently integrated micelles (HAxBCM) were prepared by radical polymerization of glycidyl methacrylate (GMA)-modified HA (HAGMA) in the presence of xBCMs. When micelles prepared from the parent PAA-b-PnBA without any polymerizable double bonds were used, hydrogels containing physically entrapped micelles (HApBCM) were obtained. The addition of xBCMs to a HAGMA precursor solution accelerated the gelation kinetics and altered the hydrogel mechanical properties. The resultant HAxBCM gels exhibit an elastic modulus of 847 ± 43 Pa and a compressive modulus of 9.2 ± 0.7 kPa. Diffusion analysis of Nile Red (NR)-labeled xBCMs employing fluorescence correlation spectroscopy confirmed the covalent immobilization of xBCMs in HA networks. Covalent integration of dexamethasone (DEX)-loaded xBCMs in HA gels significantly reduced the initial burst release and provided sustained release over a prolonged period. Importantly, DEX release from HAxBCM gels was accelerated by intermittently applied external compression in a strain-dependent manner. Culturing macrophages in the presence of DEX-releasing HAxBCM gels significantly reduced cellular production of inflammatory cytokines. Incorporating mechano-responsive modules in synthetic matrices offers a novel strategy to harvest mechanical stress present in the healing

  15. Crystal structure of the dithiol oxidase DsbA enzyme from proteus mirabilis bound non-covalently to an active site peptide ligand.

    PubMed

    Kurth, Fabian; Duprez, Wilko; Premkumar, Lakshmanane; Schembri, Mark A; Fairlie, David P; Martin, Jennifer L

    2014-07-11

    The disulfide bond forming DsbA enzymes and their DsbB interaction partners are attractive targets for development of antivirulence drugs because both are essential for virulence factor assembly in Gram-negative pathogens. Here we characterize PmDsbA from Proteus mirabilis, a bacterial pathogen increasingly associated with multidrug resistance. PmDsbA exhibits the characteristic properties of a DsbA, including an oxidizing potential, destabilizing disulfide, acidic active site cysteine, and dithiol oxidase catalytic activity. We evaluated a peptide, PWATCDS, derived from the partner protein DsbB and showed by thermal shift and isothermal titration calorimetry that it binds to PmDsbA. The crystal structures of PmDsbA, and the active site variant PmDsbAC30S were determined to high resolution. Analysis of these structures allows categorization of PmDsbA into the DsbA class exemplified by the archetypal Escherichia coli DsbA enzyme. We also present a crystal structure of PmDsbAC30S in complex with the peptide PWATCDS. The structure shows that the peptide binds non-covalently to the active site CXXC motif, the cis-Pro loop, and the hydrophobic groove adjacent to the active site of the enzyme. This high-resolution structural data provides a critical advance for future structure-based design of non-covalent peptidomimetic inhibitors. Such inhibitors would represent an entirely new antibacterial class that work by switching off the DSB virulence assembly machinery. PMID:24831013

  16. Crystal Structure of the Dithiol Oxidase DsbA Enzyme from Proteus Mirabilis Bound Non-covalently to an Active Site Peptide Ligand

    PubMed Central

    Kurth, Fabian; Duprez, Wilko; Premkumar, Lakshmanane; Schembri, Mark A.; Fairlie, David P.; Martin, Jennifer L.

    2014-01-01

    The disulfide bond forming DsbA enzymes and their DsbB interaction partners are attractive targets for development of antivirulence drugs because both are essential for virulence factor assembly in Gram-negative pathogens. Here we characterize PmDsbA from Proteus mirabilis, a bacterial pathogen increasingly associated with multidrug resistance. PmDsbA exhibits the characteristic properties of a DsbA, including an oxidizing potential, destabilizing disulfide, acidic active site cysteine, and dithiol oxidase catalytic activity. We evaluated a peptide, PWATCDS, derived from the partner protein DsbB and showed by thermal shift and isothermal titration calorimetry that it binds to PmDsbA. The crystal structures of PmDsbA, and the active site variant PmDsbAC30S were determined to high resolution. Analysis of these structures allows categorization of PmDsbA into the DsbA class exemplified by the archetypal Escherichia coli DsbA enzyme. We also present a crystal structure of PmDsbAC30S in complex with the peptide PWATCDS. The structure shows that the peptide binds non-covalently to the active site CXXC motif, the cis-Pro loop, and the hydrophobic groove adjacent to the active site of the enzyme. This high-resolution structural data provides a critical advance for future structure-based design of non-covalent peptidomimetic inhibitors. Such inhibitors would represent an entirely new antibacterial class that work by switching off the DSB virulence assembly machinery. PMID:24831013

  17. Direct electrical communication between chemically modified enzymes and metal electrodes. 1. Electron transfer from glucose oxidase to metal electrodes via electron relays, bound covalently to the enzyme

    SciTech Connect

    Degani, Y.; Heller, A.

    1987-03-12

    Glucose-reduced glucose oxidase does not directly transfer electrons to conventional electrodes because the distance between its redox centers and the electrode surface exceeds, even on closest approach, the distance across which electrons are transferred at sufficient rates. Therefore, electrical communication between the redox centers of this enzyme and electrodes required either the presence, and diffusion to and from the enzyme's redox center, of O/sub 2/ and H/sub 2/O/sub 2/, or the presence of members of a redox couple, or the use of special electrodes like TTF/TCNQ. They show here that direct electrical communication between the redox center of a large enzyme molecule and a simple metal electrode can be established through chemical modification of the enzyme. When a sufficient number of electron-relaying centers are attached through covalent bonding to the protein of glucose oxidase, electrons are transferred from the enzyme's redox centers to relays that are closer to the periphery of the enzyme. Because some of the relays are located sufficiently close to the enzyme's surface, electrons are transferred at practical rates to the electrode. As a result, a glucose-concentration-dependent current flows in an electrochemical cell made with conventional electrodes when the electrolytic solution contains the relay-modified enzyme. Such a current does not flow when the solution contains the natural enzyme. Specifically, electrical communication is established between the FAD/FADH/sub 2/ centers of glucose oxidase and gold, platinum, or carbon electrodes through the covalent bonding of an average of 12 molecules of ferrocenecarboxylic acid per glucose oxidase molecule.

  18. Structure of the red fluorescent protein from a lancelet (Branchiostoma lanceolatum): a novel GYG chromophore covalently bound to a nearby tyrosine

    PubMed Central

    Pletnev, Vladimir Z.; Pletneva, Nadya V.; Lukyanov, Konstantin A.; Souslova, Ekaterina A.; Fradkov, Arkady F.; Chudakov, Dmitry M.; Chepurnykh, Tatyana; Yampolsky, Ilia V.; Wlodawer, Alexander; Dauter, Zbigniew; Pletnev, Sergei

    2013-01-01

    A key property of proteins of the green fluorescent protein (GFP) family is their ability to form a chromophore group by post-translational modifications of internal amino acids, e.g. Ser65-Tyr66-Gly67 in GFP from the jellyfish Aequorea victoria (Cnidaria). Numerous structural studies have demonstrated that the green GFP-like chromophore represents the ‘core’ structure, which can be extended in red-shifted proteins owing to modifications of the protein backbone at the first chromophore-forming position. Here, the three-dimensional structures of green laGFP (λex/λem = 502/511 nm) and red laRFP (λex/λem ≃ 521/592 nm), which are fluorescent proteins (FPs) from the lancelet Branchiostoma lanceolatum (Chordata), were determined together with the structure of a red variant laRFP-ΔS83 (deletion of Ser83) with improved folding. Lancelet FPs are evolutionarily distant and share only ∼20% sequence identity with cnidarian FPs, which have been extensively characterized and widely used as genetically encoded probes. The structure of red-emitting laRFP revealed three exceptional features that have not been observed in wild-type fluorescent proteins from Cnidaria reported to date: (i) an unusual chromophore-forming sequence Gly58-Tyr59-Gly60, (ii) the presence of Gln211 at the position of the conserved catalytic Glu (Glu222 in Aequorea GFP), which proved to be crucial for chromophore formation, and (iii) the absence of modifications typical of known red chromophores and the presence of an extremely unusual covalent bond between the Tyr59 Cβ atom and the hydroxyl of the proximal Tyr62. The impact of this covalent bond on the red emission and the large Stokes shift (∼70 nm) of laRFP was verified by extensive structure-based site-directed mutagenesis. PMID:23999308

  19. Structure of the red fluorescent protein from a lancelet (Branchiostoma lanceolatum): a novel GYG chromophore covalently bound to a nearby tyrosine.

    PubMed

    Pletnev, Vladimir Z; Pletneva, Nadya V; Lukyanov, Konstantin A; Souslova, Ekaterina A; Fradkov, Arkady F; Chudakov, Dmitry M; Chepurnykh, Tatyana; Yampolsky, Ilia V; Wlodawer, Alexander; Dauter, Zbigniew; Pletnev, Sergei

    2013-09-01

    A key property of proteins of the green fluorescent protein (GFP) family is their ability to form a chromophore group by post-translational modifications of internal amino acids, e.g. Ser65-Tyr66-Gly67 in GFP from the jellyfish Aequorea victoria (Cnidaria). Numerous structural studies have demonstrated that the green GFP-like chromophore represents the `core' structure, which can be extended in red-shifted proteins owing to modifications of the protein backbone at the first chromophore-forming position. Here, the three-dimensional structures of green laGFP (λex/λem = 502/511 nm) and red laRFP (λex/λem ≃ 521/592 nm), which are fluorescent proteins (FPs) from the lancelet Branchiostoma lanceolatum (Chordata), were determined together with the structure of a red variant laRFP-ΔS83 (deletion of Ser83) with improved folding. Lancelet FPs are evolutionarily distant and share only ∼20% sequence identity with cnidarian FPs, which have been extensively characterized and widely used as genetically encoded probes. The structure of red-emitting laRFP revealed three exceptional features that have not been observed in wild-type fluorescent proteins from Cnidaria reported to date: (i) an unusual chromophore-forming sequence Gly58-Tyr59-Gly60, (ii) the presence of Gln211 at the position of the conserved catalytic Glu (Glu222 in Aequorea GFP), which proved to be crucial for chromophore formation, and (iii) the absence of modifications typical of known red chromophores and the presence of an extremely unusual covalent bond between the Tyr59 C(β) atom and the hydroxyl of the proximal Tyr62. The impact of this covalent bond on the red emission and the large Stokes shift (∼70 nm) of laRFP was verified by extensive structure-based site-directed mutagenesis. PMID:23999308

  20. Effect of recombinant bovine granulocyte colony-stimulating factor covalently bound to polyethylene glycol injection on neutrophil number and function in periparturient dairy cows.

    PubMed

    Kimura, Kayoko; Goff, Jesse P; Canning, Peter; Wang, Chong; Roth, James A

    2014-01-01

    Dairy cows often experience decreased immune function around the time of calving, typified by impaired polymorphonuclear neutrophil (PMN) function and a transient neutropenia. This is associated with increased disease incidence, including mastitis, retained placenta, and metritis. In an attempt to improve PMN functional capacity during the periparturient period, we injected cows with recombinant bovine granulocyte colony-stimulating factor covalently bound to polyethylene glycol (PEG rbG-CSF) twice subcutaneously, about 6d before calving and within 24h after calving. Twenty-one cows in their second pregnancy were enrolled in this study and divided into 2 groups: PEG rbG-CSF treated (n=11) and saline-treated controls (n=10). The PMN numbers quickly and dramatically increased after PEG rbG-CSF administration and remained elevated through the end of the experiment (13d after calving). Exocytosis of myeloperoxidase by stimulated PMN, which is generally decreased in periparturient cows, was markedly increased by PEG rbG-CSF after injection. Higher myeloperoxidase exocytosis persisted for at least 10d after calving. The PMN superoxide anion release and phagocytosis activity did not differ between groups. Injection of PEG rbG-CSF was safe for cows, with no significant negative effects observed. The greatest single effect of PEG rbG-CSF administration was a dramatic increase in circulating numbers of PMN. The increased numbers of PMN ready to move to a site of infection early in the course of an infection may improve the ability of the cow to ward off clinical disease in the periparturient period. PMID:24881799

  1. Label-free fluorescent molecular beacon based on a small fluorescent molecule non-covalently bound to the intentional gap site in the stem moiety.

    PubMed

    Gao, Qiang; Lin, Kai; Zhang, Hongge; Qi, Honglan; Zhang, Chengxiao

    2010-12-15

    A label-free fluorescent molecular beacon (MB) based on a fluorescent molecule, 5,6,7-trimethyl-1,8-naphthyridin-2-ylamine (ATMND) which is non-covalently bound to the intentional gap site in the stem moiety of the label-free MB, was developed. In the absence of a cDNA, ATMND fluorescence is significantly quenched because it binds to the unpaired cytosine at the gap site by hydrogen bonding. As a result, the label-free MB shows almost no fluorescence. Upon hybridization with cDNA, the label-free MB undergoes a conformational change to destroy the gap site. This results in an effective fluorescent enhancement because of the release of the ATMND from the gap site to the solution. Fluorescence titration shows that ATMND strongly binds to the cytosine at the gap site (K(11)>10(6)). Circular-dichroism spectroscopy indicates that the binding of ATMND at the gap site of the stem moiety does not induce a significant conformational change to the hairpin DNA. Under optimal conditions, the fluorescent intensity of the label-free MB increases with an increase in cDNA concentration from 50 nM to 1.5 μM. A detection limit of 20 nM cDNA was achieved. A single mismatched target ss-DNA can be effectively discriminated from cDNA. The advantage of the label-free MB is that both its ends can be left free to introduce other useful functionalities. In addition, the label-free MB synthesis introduced in this paper is relatively simple and inexpensive because no label is required. PMID:21111170

  2. Synthesis of a Sulfonated Two-Dimensional Covalent Organic Framework as an Efficient Solid Acid Catalyst for Biobased Chemical Conversion.

    PubMed

    Peng, Yongwu; Hu, Zhigang; Gao, Yongjun; Yuan, Daqiang; Kang, Zixi; Qian, Yuhong; Yan, Ning; Zhao, Dan

    2015-10-12

    Because of limited framework stability tolerance, de novo synthesis of sulfonated covalent organic frameworks (COFs) remains challenging and unexplored. Herein, a sulfonated two-dimensional crystalline COF, termed TFP-DABA, was synthesized directly from 1,3,5-triformylphloroglucinol and 2,5-diaminobenzenesulfonic acid through a previously reported Schiff base condensation reaction, followed by irreversible enol-to-keto tautomerization, which strengthened its structural stability. TFP-DABA is a highly efficient solid acid catalyst for fructose conversion with remarkable yields (97 % for 5-hydroxymethylfurfural and 65 % for 2,5-diformylfuran), good chemoselectivity, and good recyclability. The present study sheds light on the de novo synthesis of sulfonated COFs as novel solid acid catalysts for biobased chemical conversion. PMID:26448524

  3. Poly(adenylic acid) in small amounts, free or covalently linked to substrate, protects RNA from hydrolysis by ribonuclease.

    PubMed Central

    Karpetsky, T P; Shriver, K K; Levy, C C

    1981-01-01

    Short lengths (18 residues) of poly(A), covalently linked to the 3'-termini of Escherichia coli 5 S rRNA, induce powerful inhibitions (38-87%) of the activities of RNAases (ribonucleases) from Citrobacter sp., Enterobacter sp., bovine pancreas, human spleen and human plasma. As the polypurine chain length is extended, enzyme activity declines. Furthermore, poly(A) sequences, present only on a small subpopulation of RNA, and accounting for less than 1% of total RNA, serve to protect all RNA, polyadenylated or not, from enzyme-catalysed degradation. The quantity of 3'-terminal adenylic acid residues, relative to the amount of substrate, determines enzyme activity. The exact distribution of a fixed amount of poly(A) residues on the 3'-termini of substrate molecules is unimportant in this respect. Comparison of the efficacies of inhibition of RNAase activity, by using linked poly(A) and similar quantities of free poly(A), revealed that although the free polypurine inhibits RNAase activity, covalent linkage of poly(A) to RNA is more advantageous to the stability of an RNA substrate. However, the ratio of inhibited activities obtained by using linked or free poly(A) may change considerably with alterations in either substrate concentration or polyadenylic acid segment length. PMID:6171250

  4. A thiamin-bound, pre-decarboxylation reaction intermediate analogue in the pyruvate dehydrogenase E1 subunit induces large scale disorder-to-order transformations in the enzyme and reveals novel structural features in the covalently bound adduct.

    PubMed

    Arjunan, Palaniappa; Sax, Martin; Brunskill, Andrew; Chandrasekhar, Krishnamoorthy; Nemeria, Natalia; Zhang, Sheng; Jordan, Frank; Furey, William

    2006-06-01

    The crystal structure of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) has been determined with phosphonolactylthiamin diphosphate (PLThDP) in its active site. PLThDP serves as a structural and electrostatic analogue of the natural intermediate alpha-lactylthiamin diphosphate (LThDP), in which the carboxylate from the natural substrate pyruvate is replaced by a phosphonate group. This represents the first example of an experimentally determined, three-dimensional structure of a thiamin diphosphate (ThDP)-dependent enzyme containing a covalently bound, pre-decarboxylation reaction intermediate analogue and should serve as a model for the corresponding intermediates in other ThDP-dependent decarboxylases. Regarding the PDHc-specific reaction, the presence of PLThDP induces large scale conformational changes in the enzyme. In conjunction with the E1-PLThDP and E1-ThDP structures, analysis of a H407A E1-PLThDP variant structure shows that an interaction between His-407 and PLThDP is essential for stabilization of two loop regions in the active site that are otherwise disordered in the absence of intermediate analogue. This ordering completes formation of the active site and creates a new ordered surface likely involved in interactions with the lipoyl domains of E2s within the PDHc complex. The tetrahedral intermediate analogue is tightly held in the active site through direct hydrogen bonds to residues His-407, Tyr-599, and His-640 and reveals a new, enzyme-induced, strain-related feature that appears to aid in the decarboxylation process. This feature is almost certainly present in all ThDP-dependent decarboxylases; thus its inclusion in our understanding of general thiamin catalysis is important. PMID:16531404

  5. 15N NMR investigation of the covalent binding of reduced TNT amines to soil humic acid, model compounds, and lignocellulose.

    PubMed

    Thorn, K A; Kennedy, K R

    2002-09-01

    The five major reductive degradation products of TNT-4ADNT (4-amino-2,6-dinitrotoluene), 2ADNT (2-amino-4,6-dinitrotoluene), 2,4DANT (2,4-diamino-6-nitrotoluene), 2,6DANT (2,6-diamino-4-nitrotoluene), and TAT (2,4,6-triaminotoluene)-labeled with 15N in the amine positions, were reacted with the IHSS soil humic acid and analyzed by 15N NMR spectrometry. In the absence of catalysts, all five amines underwent nucleophilic addition reactions with quinone and other carbonyl groups in the soil humic acid to form both heterocyclic and nonheterocyclic condensation products. Imine formation via 1,2-addition of the amines to quinone groups in the soil humic acid was significant with the diamines and TAT but not the monoamines. Horseradish peroxidase (HRP) catalyzed an increase in the incorporation of all five amines into the humic acid. In the case of the diamines and TAT, HRP also shifted the binding away from heterocyclic condensation product toward imine formation. A comparison of quantitative liquid phase with solid-state CP/MAS 15N NMR indicated that the CP experiment underestimated imine and heterocyclic nitrogens in humic acid, even with contact times optimal for observation of these nitrogens. Covalent binding of the mono- and diamines to 4-methylcatechol, the HRP catalyzed condensation of 4ADNT and 2,4DANT to coniferyl alcohol, and the binding of 2,4DANT to lignocellulose with and without birnessite were also examined. PMID:12322752

  6. 15N NMR investigation of the covalent binding of reduced TNT amines to soil humic acid, model compounds, and lignocellulose

    USGS Publications Warehouse

    Thorn, K.A.; Kennedy, K.R.

    2002-01-01

    The five major reductive degradation products of TNT-4ADNT (4-amino-2,6-dinitrotoluene), 2ADNT (2-amino-4,6-dinitrotoluene), 2,4DANT (2,4-diamino-6-nitrotoluene), 2,6DANT (2,6-diamino-4-nitrotoluene), and TAT (2,4,6-triaminotoluene)-labeled with 15N in the amine positions, were reacted with the IHSS soil humic acid and analyzed by 15N NMR spectrometry. In the absence of catalysts, all five amines underwent nucleophilic addition reactions with quinone and other carbonyl groups in the soil humic acid to form both heterocyclic and nonheterocyclic condensation products. Imine formation via 1,2-addition of the amines to quinone groups in the soil humic acid was significant with the diamines and TAT but not the monoamines. Horseradish peroxidase (HRP) catalyzed an increase in the incorporation of all five amines into the humic acid. In the case of the diamines and TAT, HRP also shifted the binding away from heterocyclic condensation product toward imine formation. A comparison of quantitative liquid phase with solid-state CP/MAS 15N NMR indicated that the CP experiment underestimated imine and heterocyclic nitrogens in humic acid, even with contact times optimal for observation of these nitrogens. Covalent binding of the mono- and diamines to 4-methylcatechol, the HRP catalyzed condensation of 4ADNT and 2,4DANT to coniferyl alcohol, and the binding of 2,4DANT to lignocellulose with and without birnessite were also examined.

  7. Covalent immobilization of redox enzyme on electrospun nonwoven poly(acrylonitrile-co-acrylic acid) nanofiber mesh filled with carbon nanotubes: a comprehensive study.

    PubMed

    Wang, Zhen-Gang; Ke, Bei-Bei; Xu, Zhi-Kang

    2007-07-01

    In this work, novel conductive composite nanofiber mesh possessing reactive groups was electrospun from solutions containing poly(acrylonitrile-co-acrylic acid) (PANCAA) and multi-walled carbon nanotubes (MWCNTs) for redoxase immobilization, assuming that the incorporated MWCNTs could behave as electrons transferor during enzyme catalysis. The covalent immobilization of catalase from bovine liver on the neat PANCAA nanofiber mesh or the composite one was processed in the presence of EDC/NHS. Results indicated that both the amount and activity retention of bound catalase on the composite nanofiber mesh were higher than those on the neat PANCAA nanofiber mesh, and the activity increased up to 42%. Kinetic parameters, K(m) and V(max), for the catalases immobilized on the composite nanofiber mesh were lower and higher than those on the neat one, respectively. This enhanced activity might be ascribed to either promoted electron transfer through charge-transfer complexes and the pi system of carbon nanotubes or rendered biocompatibility by modified MWCNTs. Furthermore, the immobilized catalases revealed much more stability after MWCNTs were incorporated into the polymer nanofiber mesh. However, there was no significant difference in optimum pH value and temperature, thermal stability and operational stability between these two immobilized preparations, while the two ones appeared more advantageous than the free in these properties. The effect of MWCNTs incorporation on another redox enzyme, peroxidase, was also studied and it was found that the activity increased by 68% in comparison of composite one with neat preparation. PMID:17171660

  8. Breaking the dogma: PCB-derived semiquinone free radicals do not form covalent adducts with DNA, GSH, and amino acids.

    PubMed

    Wangpradit, Orarat; Rahaman, Asif; Mariappan, S V Santhana; Buettner, Garry R; Robertson, Larry W; Luthe, Gregor

    2016-02-01

    Covalent bond formations of free radical metabolites with biomolecules like DNA and proteins are thought to constitute a major mechanism of toxicity and carcinogenesis. Glutathione (GSH) is generally accepted as a radical scavenger protecting the cell. In the present study, we investigated a semiquinone radical (SQ(●-)) metabolite of the semivolatile 4-chlorobiphenyl, using electron paramagnetic resonance spectroscopy, and oxygen consumption. Proton nuclear magnetic resonance ((1)H NMR) and liquid chromatography-mass spectrometry (LC-MS) were also employed to elucidate the radical interaction with DNA, amino acids, and GSH. We found that DNA and oligonucleotides stabilized SQ(●-) by electron delocalization in the π-stacking system, resulting in persistent radical intercalated, rather than forming a covalent bond with SQ(●-). This finding was strongly supported by the semiempirical calculation of the semioccupied molecular orbital and the linear combination of the atomic orbitals, indicating 9.8 kcal mol(-1) energy gain. The insertion of SQ(●-) into the DNA strand may result in DNA strand breaks and interruption of DNA replication process or even activate radical mediated secondary reactions. The presence of amino acids resulted in a decrease of the electron paramagnetic resonance (EPR) signal of SQ(●-) and correlated with their isoelectric points. The pH shifts the equilibrium of the dianions of hydroquinone and influenced indirectly the formation of SQ(●-). Similar findings were observed with GSH and Cys. GSH and Cys functioned as indirect radical scavengers; their activities depend on their chemical equilibria with the corresponding quinones, and their further reaction via Michael addition. The generally accepted role of GSH as radical scavenger in biological systems should be reconsidered based upon these findings, questioning the generally accepted view of radical interaction of semiquinones with biologically active compounds, like DNA, amino acids

  9. Nucleic acid-binding molecules with high affinity and base sequence specificity: intercalating agents covalently linked to oligodeoxynucleotides.

    PubMed Central

    Asseline, U; Delarue, M; Lancelot, G; Toulmé, F; Thuong, N T; Montenay-Garestier, T; Hélène, C

    1984-01-01

    Oligodeoxyribonucleotides covalently linked to an intercalating agent via a polymethylene linker were synthesized. Oligothymidylates attached to an acridine dye (Acr) through the 3'-phosphate group [(Tp)n(CH2) mAcr ] specifically interact with the complementary sequence. The interaction is strongly stabilized by the intercalating agent. By using absorption and fluorescence spectroscopies, it is shown that complex formation between (Tp)n(CH2) mAcr and poly(rA) involves the formation of n A X T base pairs, where n is the number of thymines in the oligonucleotide. The acridine ring intercalates between A X T base pairs. Fluorescence excitation spectra reveal the existence of two environments for the acridine ring, whose relative contributions depend on the linker length (m). The binding of (Tp)4(CH2) mAcr to poly(rA) is analyzed in terms of site binding and cooperative interactions between oligonucleotides along the polynucleotide lattice. Thermodynamic parameters show that the covalent attachment of the acridine ring strongly stabilizes the binding of the oligonucleotide to its complementary sequence. The stabilization depends on the linker length; the compound with m = 5 gives a more stable complex than that with m = 3. These results open the way to the synthesis of a family of molecules exhibiting both high-affinity and high-specificity for a nucleic acid base sequence. PMID:6587350

  10. The Fungal Product Terreic Acid is a Covalent Inhibitor of the Bacterial Cell Wall Biosynthetic Enzyme UDP-N-acetylglucosamine 1-carboxyvinyltransferase (MurA)†

    PubMed Central

    Han, Huijong; Yang, Yan; Olesen, Sanne H.; Becker, Andreas; Betzi, Stephane; Schönbrunn, Ernst

    2010-01-01

    Terreic acid is a metabolite with antibiotic properties produced by the fungus Aspergillus terreus. We found that terreic acid is a covalent inhibitor of the bacterial cell wall biosynthetic enzyme MurA from E. cloacae and E. coli in-vitro. The crystal structure of the MurA dead-end complex with terreic acid revealed that the quinine ring is covalently attached to the thiol group of Cys115, the molecular target of the antibiotic fosfomycin. Kinetic characterization established that the inactivation requires the presence of substrate UNAG (UDP-N-acetylglucosamine), proceeding with an inactivation rate constant of kinact = 130 M−1s−1. Although the mechanisms of inactivation are similar, fosfomycin is approximately 50 times more potent than terreic acid, and the structural consequences of covalent modification by these two inhibitors are fundamentally different. The MurA-fosfomycin complex exists in the closed enzyme conformation, with the Cys115-fosfomycin adduct buried in the active site. In contrast, the dead-end complex with terreic acid is open, free of UNAG, and has the Cys115-terreic acid adduct solvent–exposed. It appears that terreic acid reacts with Cys115 in the closed, binary state of the enzyme, but that the resulting Cys115-terreic acid adduct imposes steric clashes in the active site. As a consequence, the loop containing Cys115 rearranges, the enzyme opens and UNAG is released. The differential kinetic and structural characteristics of MurA inactivation by terreic acid and fosfomycin reflect the importance of non-covalent binding potential, even for covalent inhibitors, to ensure inactivation efficiency and specificity. PMID:20392080

  11. Toward Self-Adjuvanting Subunit Vaccines: Model Peptide and Protein Antigens Incorporating Covalently Bound Toll-Like Receptor-7 Agonistic Imidazoquinolines

    PubMed Central

    Shukla, Nikunj M.; Lewis, Tyler C.; Day, Timothy P.; Mutz, Cole A.; Ukani, Rehman; Hamilton, Chase D.; Balakrishna, Rajalakshmi

    2011-01-01

    Toll-like receptor (TLR)-7 agonists show prominent Th1-biased immunostimulatory activities. A TLR7-active N1-(4-aminomethyl)benzyl substituted imidazoquinoline 1 served as a convenient precursor for the syntheses of isothiocyanate and maleimide derivatives for covalent attachment to free amine and thiol groups of peptides and proteins. 1 was also amenable to direct reductive amination with maltoheptaose without significant loss of activity. Covalent conjugation of the isothiocyanate derivative 2 to α-lactalbumin could be achieved under mild, non-denaturing conditions, in a controlled manner and with full preservation of antigenicity. The self-adjuvanting α-lactalbumin construct induced robust, high-affinity immunoglobulin titers in murine models. The premise of covalently decorating protein antigens with adjuvants offers the possibility of drastically reducing systemic exposure of the adjuvant, and yet eliciting strong, Th1-biased immune responses. PMID:21549593

  12. Structures of the Michaelis Complex (1.2A) and the Covalent Acyl Intermediate (2.0A ) of Cefamandole Bound in the Active Sites of the Mycobacterium tuberculosis beta-Lactamase K72A and E166A Mutants

    SciTech Connect

    L Tremblay; h Xu; J Blanchard

    2011-12-31

    The genome of Mycobacterium tuberculosis (TB) contains a gene that encodes a highly active {beta}-lactamase, BlaC, that imparts TB with resistance to {beta}-lactam chemotherapy. The structure of covalent BlaC-{beta}-lactam complexes suggests that active site residues K73 and E166 are essential for acylation and deacylation, respectively. We have prepared the K73A and E166A mutant forms of BlaC and have determined the structures of the Michaelis complex of cefamandole and the covalently bound acyl intermediate of cefamandole at resolutions of 1.2 and 2.0 {angstrom}, respectively. These structures provide insight into the details of the catalytic mechanism.

  13. 9-O-Acetylation of sialic acids is catalysed by CASD1 via a covalent acetyl-enzyme intermediate

    PubMed Central

    Baumann, Anna-Maria T.; Bakkers, Mark J. G.; Buettner, Falk F. R.; Hartmann, Maike; Grove, Melanie; Langereis, Martijn A.; de Groot, Raoul J.; Mühlenhoff, Martina

    2015-01-01

    Sialic acids, terminal sugars of glycoproteins and glycolipids, play important roles in development, cellular recognition processes and host–pathogen interactions. A common modification of sialic acids is 9-O-acetylation, which has been implicated in sialoglycan recognition, ganglioside biology, and the survival and drug resistance of acute lymphoblastic leukaemia cells. Despite many functional implications, the molecular basis of 9-O-acetylation has remained elusive thus far. Following cellular approaches, including selective gene knockout by CRISPR/Cas genome editing, we here show that CASD1—a previously identified human candidate gene—is essential for sialic acid 9-O-acetylation. In vitro assays with the purified N-terminal luminal domain of CASD1 demonstrate transfer of acetyl groups from acetyl-coenzyme A to CMP-activated sialic acid and formation of a covalent acetyl-enzyme intermediate. Our study provides direct evidence that CASD1 is a sialate O-acetyltransferase and serves as key enzyme in the biosynthesis of 9-O-acetylated sialoglycans. PMID:26169044

  14. 9-O-Acetylation of sialic acids is catalysed by CASD1 via a covalent acetyl-enzyme intermediate.

    PubMed

    Baumann, Anna-Maria T; Bakkers, Mark J G; Buettner, Falk F R; Hartmann, Maike; Grove, Melanie; Langereis, Martijn A; de Groot, Raoul J; Mühlenhoff, Martina

    2015-01-01

    Sialic acids, terminal sugars of glycoproteins and glycolipids, play important roles in development, cellular recognition processes and host-pathogen interactions. A common modification of sialic acids is 9-O-acetylation, which has been implicated in sialoglycan recognition, ganglioside biology, and the survival and drug resistance of acute lymphoblastic leukaemia cells. Despite many functional implications, the molecular basis of 9-O-acetylation has remained elusive thus far. Following cellular approaches, including selective gene knockout by CRISPR/Cas genome editing, we here show that CASD1--a previously identified human candidate gene--is essential for sialic acid 9-O-acetylation. In vitro assays with the purified N-terminal luminal domain of CASD1 demonstrate transfer of acetyl groups from acetyl-coenzyme A to CMP-activated sialic acid and formation of a covalent acetyl-enzyme intermediate. Our study provides direct evidence that CASD1 is a sialate O-acetyltransferase and serves as key enzyme in the biosynthesis of 9-O-acetylated sialoglycans. PMID:26169044

  15. Gas-phase ion/ion reactions of peptides and proteins: acid/base, redox, and covalent chemistries

    PubMed Central

    Prentice, Boone M.

    2013-01-01

    Gas-phase ion/ion reactions are emerging as useful and flexible means for the manipulation and characterization of peptide and protein biopolymers. Acid/base-like chemical reactions (i.e., proton transfer reactions) and reduction/oxidation (redox) reactions (i.e., electron transfer reactions) represent relatively mature classes of gas-phase chemical reactions. Even so, especially in regards to redox chemistry, the widespread utility of these two types of chemistries is undergoing rapid growth and development. Additionally, a relatively new class of gas-phase ion/ion transformations is emerging which involves the selective formation of functional-group-specific covalent bonds. This feature details our current work and perspective on the developments and current capabilities of these three areas of ion/ion chemistry with an eye towards possible future directions of the field. PMID:23257901

  16. Using spin labels to study molecular processes in soils: Covalent binding of aromatic amines to humic acids of soils

    NASA Astrophysics Data System (ADS)

    Aleksandrova, O. N.; Kholodov, V. A.; Perminova, I. V.

    2015-08-01

    Interactions of aliphatic and aromatic amines with soil and humic acids isolated from it are studied by means of spin labels and electron paramagnetic resonance (EPR) spectroscopy. Nitroxyl radicals containing amino groups are used as spin labels. It is found experimentally that aromatic amines are instantaneously converted to the bound state. It is shown that the microareas of their incorporation are characterized by a significant delay in the reduction of the nitroxyl fragment of spin-label molecules, indicating the formation of condensed structures typical of an oxidative binding mechanism. It is concluded that aliphatic amines do not bind to humic acids. It is noted that the studied process allows elucidating the formation of bound xenobiotic residues in soils.

  17. Covalently linked organic networks

    NASA Astrophysics Data System (ADS)

    Tsotsalas, Manuel; Addicoat, Matthew

    2015-02-01

    In this review, we intend to give an overview of the synthesis of well-defined covalently-bound organic network materials such as covalent organic frameworks (COFs), conjugated microporous frameworks (CMPs) and other “ideal polymer networks” and discuss the different approaches in their synthesis and their potential applications. In addition we will describe the common computational approaches and highlight recent achievements in the computational study of their structure and properties. For further information the interested reader is referred to several excellent and more detailed reviews dealing with the synthesis [Dawson 2012; Ding 2013; Feng 2012] and computational aspects [Han 2009; Colón 2014] of the materials presented here.

  18. Fabricating an Amperometric Cholesterol Biosensor by a Covalent Linkage between Poly(3-thiopheneacetic acid) and Cholesterol Oxidase

    PubMed Central

    Nien, Po-Chin; Chen, Po-Yen; Ho, Kuo-Chuan

    2009-01-01

    In this study, use of the covalent enzyme immobilization method was proposed to attach cholesterol oxidase (ChO) on a conducting polymer, poly(3-thiopheneacetic acid), [poly(3-TPAA)]. Three red-orange poly(3-TPAA) films, named electrodes A, B and C, were electropolymerized on a platinum electrode by applying a constant current of 1.5 mA, for 5, 20 and 100 s, respectively. Further, 1-ethyl-3-(3-dimethylamiopropyl)carbodiimide hydrochloride (EDC · HCl) and N-hydroxysuccinimide (NHS) were used to activate the free carboxylic groups of the conducting polymer. Afterwards, the amino groups of the cholesterol oxidase were linked on the activated groups to form peptide bonds. The best sensitivity obtained for electrode B is 4.49 mA M−1 cm−2, with a linear concentration ranging from 0 to 8 mM, which is suitable for the analysis of cholesterol in humans. The response time (t95) is between 70 and 90 s and the limit of detection is 0.42 mM, based on the signal to noise ratio equal to 3. The interference of species such as ascorbic acid and uric acid increased to 5.2 and 10.3% of the original current response, respectively, based on the current response of cholesterol (100%). With respect to the long-term stability, the sensing response retains 88% of the original current after 13 days. PMID:22573987

  19. Kinetics of rapid covalent bond formation of aniline with humic acid: ESR investigations with nitroxide spin labels

    NASA Astrophysics Data System (ADS)

    Glinka, Kevin; Matthies, Michael; Theiling, Marius; Hideg, Kalman; Steinhoff, Heinz-Jürgen

    2016-04-01

    Sulfonamide antibiotics used in livestock farming are distributed to farmland by application of slurry as fertilizer. Previous work suggests rapid covalent binding of the aniline moiety to humic acids found in soil. In the current work, kinetics of this binding were measured in X-band EPR spectroscopy by incubating Leonardite humic acid (LHA) with a paramagnetic aniline spin label (anilino-NO (2,5,5-Trimethyl-2-(3-aminophenyl)pyrrolidin-1-oxyl)). Binding was detected by a pronounced broadening of the spectral lines after incubation of LHA with anilino-NO. The time evolution of the amplitude of this feature was used for determining the reaction kinetics. Single- and double-exponential models were fitted to the data obtained for modelling one or two first-order reactions. Reaction rates of 0.16 min-1 and 0.012 min-1, were found respectively. Addition of laccase peroxidase did not change the kinetics but significantly enhanced the reacting fraction of anilino-NO. This EPR-based method provides a technically simple and effective method for following rapid binding processes of a xenobiotic substance to humic acids.

  20. X-ray Crystallographic Analysis of α-Ketoheterocycle Inhibitors Bound to a Humanized Variant of Fatty Acid Amide Hydrolase

    PubMed Central

    Mileni, Mauro; Garfunkle, Joie; Ezzili, Cyrine; Kimball, F. Scott; Cravatt, Benjamin F.; Stevens, Raymond C.; Boger, Dale L.

    2009-01-01

    Three cocrystal X-ray structures of the α-ketoheterocycle inhibitors 3–5 bound to a humanized variant of fatty acid amide hydrolase (FAAH) are disclosed and comparatively discussed alongside those of 1 (OL-135) and its isomer 2. These five X-ray structures systematically probe each of the three active site regions key to substrate or inhibitor binding: (1) the conformationally mobile acyl chain-binding pocket and membrane access channel responsible for fatty acid amide substrate and inhibitor acyl chain binding, (2) the atypical active site catalytic residues and surrounding oxyanion hole that covalently binds the core of the α-ketoheterocycle inhibitors captured as deprotonated hemiketals mimicking the tetrahedral intermediate of the enzyme catalyzed reaction, and (3) the cytosolic port and its uniquely important imbedded ordered water molecules and a newly identified anion binding site. The detailed analysis of their key active site interactions and their implications on the interpretation of the available structure–activity relationships are discussed providing important insights for future design. PMID:19924997

  1. X-ray Crystallographic Analysis of [alpha]-Ketoheterocycle Inhibitors Bound to a Humanized Variant of Fatty Acid Amide Hydrolase

    SciTech Connect

    Mileni, Mauro; Garfunkle, Joie; Ezzili, Cyrine; Kimball, F.Scott; Cravatt, Benjamin F.; Stevens, Raymond C.; Boger, Dale L.

    2010-11-03

    Three cocrystal X-ray structures of the {alpha}-ketoheterocycle inhibitors 3-5 bound to a humanized variant of fatty acid amide hydrolase (FAAH) are disclosed and comparatively discussed alongside those of 1 (OL-135) and its isomer 2. These five X-ray structures systematically probe each of the three active site regions key to substrate or inhibitor binding: (1) the conformationally mobile acyl chain-binding pocket and membrane access channel responsible for fatty acid amide substrate and inhibitor acyl chain binding, (2) the atypical active site catalytic residues and surrounding oxyanion hole that covalently binds the core of the {alpha}-ketoheterocycle inhibitors captured as deprotonated hemiketals mimicking the tetrahedral intermediate of the enzyme-catalyzed reaction, and (3) the cytosolic port and its uniquely important imbedded ordered water molecules and a newly identified anion binding site. The detailed analysis of their key active site interactions and their implications on the interpretation of the available structure-activity relationships are discussed providing important insights for future design.

  2. Conversion of covalently mercurated nucleic acids to tritiated and halogenated derivatives.

    PubMed Central

    Dale, R M; Ward, D C; Livingston, D C; Martin, E

    1975-01-01

    Mercurated nucleic acids are converted to the corresponding tritiated, brominated, and iodinated derivatives by treatment with sodium borotritiide, N-bromosuccinimide, and elemental iodine, respectively. All three reactions occur under mild conditions in neutral aqueous solutions. Mercury-halogen conversions are essentially quantitative at both the mono- and polynucleotide levels. Tritiation reactions also proceed efficiently with mononucleotides, although polymers undergo incomplete demercuration. In spite of the latter limitation , these reactions provide novel and efficient synthetic routes to radiolabeled nucleic acid derivatives. PMID:1144066

  3. Cells labeled with multiple fluorophores bound to a nucleic acid carrier

    SciTech Connect

    Dattagupta, N.; Kamarch, M.E.

    1989-04-25

    In passing labeled cells through a cell sorter, the improvement which comprises employing a labeled cell comprising a cell, an antibody specific to and bound to such cell, a nucleic acid fragment joined to the antibody, and a plurality of labels on the nucleic acid fragment. Because of the presence of multiple labels, the sensitivity of the separation of labeled cells in increased.

  4. Hierarchically structured, hyaluronic acid-based hydrogel matrices via the covalent integration of microgels into macroscopic networks$

    PubMed Central

    Jha, Amit K.; Malik, Manisha S.; Farach-Carson, Mary C.; Duncan, Randall L.; Jia, Xinqiao

    2010-01-01

    We aimed to develop biomimetic hydrogel matrices that not only exhibit structural hierarchy and mechanical integrity, but also present biological cues in a controlled fashion. To this end, photocrosslinkable, hyaluronic acid (HA)-based hydrogel particles (HGPs) were synthesized via an inverse emulsion crosslinking process followed by chemical modification with glycidyl methacrylate (GMA). HA modified with GMA (HA-GMA) was employed as the soluble macromer. Macroscopic hydrogels containing covalently integrated hydrogel particles (HA-c-HGP) were prepared by radical polymerization of HA-GMA in the presence of crosslinkable HGPs. The covalent linkages between the hydrogel particles and the secondary HA matrix resulted in the formation of a diffuse, fibrilar interface around the particles. Compared to the traditional bulk gels synthesized by photocrosslinking of HA-GMA, these hydrogels exhibited a reduced sol fraction and a lower equilibrium swelling ratio. When tested under uniaxial compression, the HA-c-HGP gels were more pliable than the HA-p-HGP gels and fractured at higher strain than the HA-GMA gels. Primary bovine chondrocytes were photoencapsulated in the HA matrices with minimal cell damage. The 3D microenvironment created by HA-GMA and HA HGPs not only maintained the chondrocyte phenotype but also fostered the production of cartilage specific extracellular matrix. To further improve the biological activities of the HA-c-HGP gels, bone morphogenetic protein 2 (BMP-2) was loaded into the immobilized HGPs. BMP-2 was released from the HA-c-HGP gels in a controlled manner with reduced initial burst over prolonged periods of time. The HA-c-HGP gels are promising candidates for use as bioactive matrices for cartilage tissue engineering. PMID:20936090

  5. Direct demonstration of insulin receptor internalization. A quantitative electron microscopic study of covalently bound /sup 125/I-photoreactive insulin incubated with isolated hepatocytes

    SciTech Connect

    Gorden, P.; Carpentier, J.L.; Moule, M.L.; Yip, C.C.; Orci, L.

    1982-07-01

    When /sup 125/I-insulin is incubated with isolated rodent hepatocytes at 37 degrees C, the ligand initially binds to the plasma membrane of the cell and is subsequently internalized by adsorptive endocytosis. To confirm directly that the insulin receptor is internalized with the ligand, we covalently linked photoreactive /sup 125/I-N sigma B29 (azidobenzoyl) insulin to its specific hepatocyte receptor and followed its fate by quantitative electron microscopic autoradiography. We found that the covalently linked photoreactive insulin is internalized by the cell in fashion analogous to the internalization of ordinary /sup 125/I-insulin, indicating that, at least under these conditions, the insulin receptor is internalized with the ligand.

  6. Dynamic nuclear polarization of nucleic acid with endogenously bound manganese.

    PubMed

    Wenk, Patricia; Kaushik, Monu; Richter, Diane; Vogel, Marc; Suess, Beatrix; Corzilius, Björn

    2015-09-01

    We report the direct dynamic nuclear polarization (DNP) of (13)C nuclei of a uniformly [(13)C,(15)N]-labeled, paramagnetic full-length hammerhead ribozyme (HHRz) complex with Mn(2+) where the enhanced polarization is fully provided by the endogenously bound metal ion and no exogenous polarizing agent is added. A (13)C enhancement factor of ε = 8 was observed by intra-complex DNP at 9.4 T. In contrast, "conventional" indirect and direct DNP experiments were performed using AMUPol as polarizing agent where we obtained a (1)H enhancement factor of ε ≈ 250. Comparison with the diamagnetic (Mg(2+)) HHRz complex shows that the presence of Mn(2+) only marginally influences the (DNP-enhanced) NMR properties of the RNA. Furthermore two-dimensional correlation spectra ((15)N-(13)C and (13)C-(13)C) reveal structural inhomogeneity in the frozen, amorphous state indicating the coexistence of several conformational states. These demonstrations of intra-complex DNP using an endogenous metal ion as well as DNP-enhanced MAS NMR of RNA in general yield important information for the development of new methods in structural biology. PMID:26219517

  7. Irreversible inactivation of snake venom l-amino acid oxidase by covalent modification during catalysis of l-propargylglycine.

    PubMed

    Mitra, Jyotirmoy; Bhattacharyya, Debasish

    2013-01-01

    Snake venom l-amino acid oxidase (SV-LAAO, a flavor-enzyme) has attracted considerable attention due to its multifunctional nature, which is manifest in diverse clinical and biological effects such as inhibition of platelet aggregation, induction of cell apoptosis and cytotoxicity against various cells. The majority of these effects are mediated by H2O2 generated during the catalytic conversion of l-amino acids. The substrate analog l-propargylglycine (LPG) irreversibly inhibited the enzyme from Crotalus adamanteus and Crotalus atrox in a dose- and time-dependent manner. Inactivation was irreversible which was significantly protected by the substrate l-phenylalanine. A Kitz-Wilson replot of the inhibition kinetics suggested formation of reversible enzyme-LPG complex, which occurred prior to modification and inactivation of the enzyme. UV-visible and fluorescence spectra of the enzyme and the cofactor strongly suggested formation of covalent adduct between LPG and an active site residue of the enzyme. A molecular modeling study revealed that the FAD-binding, substrate-binding and the helical domains are conserved in SV-LAAOs and both His223 and Arg322 are the important active site residues that are likely to get modified by LPG. Chymotrypsin digest of the LPG inactivated enzyme followed by RP-HPLC and MALDI mass analysis identified His223 as the site of modification. The findings reported here contribute towards complete inactivation of SV-LAAO as a part of snake envenomation management. PMID:23772385

  8. ABS polymer electroless plating through a one-step poly(acrylic acid) covalent grafting.

    PubMed

    Garcia, Alexandre; Berthelot, Thomas; Viel, Pascal; Mesnage, Alice; Jégou, Pascale; Nekelson, Fabien; Roussel, Sébastien; Palacin, Serge

    2010-04-01

    A new, efficient, palladium- and chromium-free process for the electroless plating of acrylonitrile-butadiene-styrene (ABS) polymers has been developed. The process is based on the ion-exchange properties of poly(acrylic acid) (PAA) chemically grafted onto ABS via a simple and one-step method that prevents using classical surface conditioning. Hence, ABS electroless plating can be obtained in three steps, namely: (i) the grafting of PAA onto ABS, (ii) the copper Cu(0) seeding of the ABS surface, and (iii) the nickel or copper metallization using commercial-like electroless plating bath. IR, XPS, and SEM were used to characterize each step of the process, and the Cu loading was quantified by atomic absorption spectroscopy. This process successfully compares with the commercial one based on chromic acid etching and palladium-based seed layer, because the final metallic layer showed excellent adhesion with the ABS substrate. PMID:20361751

  9. Free and bound cinnamic acid derivatives in corsica sweet blond oranges.

    PubMed

    Carrera, Eric; El Kebir, Mohamed Vall Ould; Jacquemond, Camille; Luro, François; Lozano, Yves; Gaydou, Emile M

    2010-03-01

    Total determination of cinnamic acids (CA), including hydroxycinnamic acid derivatives is generally not accurate since, during hydrolysis, a possible degradation of dihydroxy CA such as caffeic acid could occur. Evaluations of CA (ferulic, p-coumaric, sinapic, cinnamic and caffeic acids) before and after hydrolysis have been undertaken using standards and either with or without addition of ascorbic acid and EDTA. The method was then applied to the determination of free and bound CA in five blond cultivars (Navelina, Washington navel, Pera, Salustiana and Valencia late) of sweet oranges [Citrus sinensis (L.) Osb.]. Four parts of the fruits (peel juice, flavedo, albedo and juice) have been investigated. Results show that CA are mainly bound (86% up to 92%) in the four fruit parts. The mean of total CA contents was found to be higher in peel juice (1.5 g kg(-1)) in comparison with flavedo (0.7 g kg(-1)), albedo (0.1 g kg(-1)) and juice (0.6 g kg(-1)). Free and bound ferulic acid represented 55-70% of CA in juices, followed by p-coumaric acid (20%), sinapic acid (10%) and caffeic acid (9%). Total contents of each CA in the four fruit parts are discussed and show the potential interest in orange peel wastes. PMID:20420324

  10. Rational design of resorcylic acid lactone analogues as covalent MNK1/2 kinase inhibitors by tuning the reactivity of an enamide Michael acceptor.

    PubMed

    Xu, Jin; Chen, Anqi; Joy, Joma; Xavier, Vanessa Joanne; Ong, Esther H Q; Hill, Jeffrey; Chai, Christina L L

    2013-09-01

    Recent biological and computational advances in drug design have led to renewed interest in targeted covalent inhibition as an efficient and practical approach for the development of new drugs. As part of our continuing efforts in the exploration of the therapeutic potential of resorcylic acid lactones (RALs), we report herein the design, synthesis, and biological evaluation of conveniently accessible RAL enamide analogues as novel covalent inhibitors of MAP kinase interacting kinases (MNKs). In this study, we have successfully demonstrated that the covalent binding ability of RAL enamides can be tuned by attaching an electron-withdrawing motif, such as an acyl group, to enhance its reactivity toward the cysteine residues at the MNK1/2 binding sites. We have also shown that ¹H NMR spectroscopy is a convenient and effective tool for screening the covalent binding activities of enamides using cysteamine as a mimic of the key cysteine residue in the enzyme, whereas mass spectrometric analysis confirms covalent modification of the kinases. Preliminary optimization of the initial hit led to the discovery of enamides with low micromolar activity in MNK assays. Cancer cell line assays have identified RAL enamides that inhibit the growth of cancer cells with similar potency to the natural product L-783,277. PMID:23929665

  11. Physicochemical characterisation of β-carotene emulsion stabilised by covalent complexes of α-lactalbumin with (-)-epigallocatechin gallate or chlorogenic acid.

    PubMed

    Wang, Xiaoya; Liu, Fuguo; Liu, Lei; Wei, Zihao; Yuan, Fang; Gao, Yanxiang

    2015-04-15

    In this study the impact of covalent complexes of α-lactalbumin (α-La) with (-)-epigallocatechin gallate (EGCG) or chlorogenic acid (CA) was investigated on the physicochemical properties of β-carotene oil-in-water emulsions. EGCG, or CA, was covalently linked to α-La at pH 8.0, as evidenced by increased total phenolic content and declined fluorescence intensity. Compared with those stabilised by α-La alone and α-La-CA or EGCG mixture, the emulsion stabilised by the α-La-EGCG covalent complex exhibited the least changes in particle size and transmission profiles, using a novel centrifugal sedimentation technique, indicating an improvement in the physical stability. The least degradation of β-carotene occurred in the emulsion stabilised with the α-La-EGCG covalent complex when stored at 25 °C. These results implied that protein-polyphenol covalent complexes were able to enhance the physical stability of β-carotene emulsion and inhibit the degradation of β-carotene in oil-in-water emulsion, and the effect was influenced by the types of the phenolic compounds. PMID:25466060

  12. Crystal Structure of Antagonist Bound Human Lysophosphatidic Acid Receptor 1

    PubMed Central

    Chrencik, Jill E.; Roth, Christopher B.; Terakado, Masahiko; Kurata, Haruto; Omi, Rie; Kihara, Yasuyuki; Warshaviak, Dora; Nakade, Shinji; Asmar-Rovira, Guillermo; Mileni, Mauro; Mizuno, Hirotaka; Griffith, Mark T.; Rodgers, Caroline; Han, Gye Won; Velasquez, Jeffrey; Chun, Jerold; Stevens, Raymond C.

    2015-01-01

    Summary Lipid biology continues to emerge as an area of significant therapeutic interest, particularly as the result of an enhanced understanding of the wealth of signaling molecules with diverse physiological properties. This growth in knowledge is epitomized by lysophosphatidic acid (LPA), which functions through interactions with six cognate G protein-coupled receptors. Herein we present three crystal structures of LPA1 in complex with antagonist tool compounds selected and designed through structural and stability analysis. Structural analysis combined with molecular dynamics identified a basis for ligand access to the LPA1 binding pocket from the extracellular space contrasting with the proposed access for the sphingosine 1-phosphate receptor. Characteristics of the LPA1 binding pocket raise the possibility of promiscuous ligand recognition of phosphorylated endocannabinoids. Cell-based assays confirmed this hypothesis, linking the distinct receptor systems through metabolically related ligands with potential functional and therapeutic implications for treatment of disease. PMID:26091040

  13. Evidence that the reaction of the UDP-N-acetylglucosamine 1-carboxyvinyltransferase proceeds through the O-phosphothioketal of pyruvic acid bound to Cys115 of the enzyme.

    PubMed

    Wanke, C; Amrhein, N

    1993-12-15

    The enzyme UDP-N-acetylglucosamine 1-carboxyvinyltransferase (enolpyruvyltransferase, EC 2.5.1.7) catalyses the transfer of the intact 1-carboxyvinyl moiety of phosphoenolpyruvate to the 3'-hydroxyl group of the glucosamine moiety of UDP-(2')-N-acetylglucosamine with the concomitant release of inorganic phosphate, the first committed step in the biosynthesis of the bacterial cell wall peptidoglycan. Overexpression of the enzyme from Enterobacter cloacae in Escherichia coli allowed the isolation of large amounts of purified enzyme (approx. 900 mg/20 g fresh mass bacteria). By incubating the enzyme with 14C-labelled phosphoenolpyruvate, 32P-labelled orthophosphate and unlabelled UDP-(2')-N-acetyl-(3')-1-carboxyvinylglucosamine, we were able to isolate and characterise a reaction intermediate, covalently bound to the protein. It contains stoichiometric quantities of the C3 moiety (0.98 mol/mol) as well as of the phosphate moiety (0.95 mol/mol) of phosphoenolpyruvate relative to protein. The rapid turnover of this protein-bound intermediate in the presence of UDP-(2')-N-acetylglucosamine towards the product UDP-(2')-N-acetyl-(3')-1-carboxyvinylglucosamine suggests that the intermediate is kinetically competent. We also present evidence that the intermediate is bound as the O-phosphothioketal of pyruvic acid to Cys115 of the enzyme. This is the same Cys residue to which phosphomycin, an irreversible inhibitor of the UDP-GlcNAc carboxyvinyltransferase, binds covalently. Exchange of Cys115 for a Ser residue resulted in an inactive enzyme, demonstrating the essential role of Cys115 for the reaction. The only other enzyme known to catalyse the transfer of the intact 1-carboxyvinyl moiety of phosphoenolpyruvate to a substrate is the 3-phosphoshikimate 1-carboxyvinyltransferase (EC 2.5.1.19), the sixth enzyme of the shikimate pathway. The reaction of this synthase is known to proceed through a single, tightly but not covalently bound, tetrahedral intermediate. Even though the

  14. A GC-ECD method for estimation of free and bound amino acids, gamma-aminobutyric acid, salicylic acid, and acetyl salicylic acid from Solanum lycopersicum (L.).

    PubMed

    Meher, Hari Charan; Gajbhiye, Vijay T; Singh, Ghanendra

    2011-01-01

    A gas chromatograph with electron capture detection method for estimation of selected metabolites--amino acids (free and bound), gamma-aminobutyric acid (GABA), salicylic acid (SA), and acetyl salicylic acid (ASA) from tomato--is reported. The method is based on nitrophenylation of the metabolites by 1-fluoro-2, 4-dinitrobenzene under aqueous alkaline conditions to form dinitophenyl derivatives. The derivatives were stable under the operating conditions of GC. Analysis of bound amino acids comprised perchloric acid precipitation of protein, alkylation (carboxymethylation) with iodoacetic acid, vapor-phase hydrolysis, and derivatization with 1-fluoro-2,4-dinitrobenzene in that order. The metabolites were resolved in 35 min, using a temperature-programmed run. The method is rapid, sensitive, and precise. It easily measured the typical amino acids (aspartate, asparagine, glutamate, glutamine, alanine, leucine, lysine, and phenylalanine) used for identification and quantification of a protein, resolved amino acids of the same mass (leucine and isoleucine), satisfactorily measured sulfur amino acid (methionine, cystine, and cysteine), and quantified GABA, SA, and ASA, as well. The developed method was validated for specificity, linearity, and precision. It has been applied and recommended for estimation of 25 metabolites from Solanum lycopersicum (L.). PMID:21391500

  15. Covalent immobilization of Candida rugosa lipase on aldehyde functionalized hydrophobic support and the application for synthesis of oleic acid ester.

    PubMed

    Temoçin, Zülfikar

    2013-01-01

    This study focuses on Candida rugosa lipase (CRL) immobilization by covalent attachment on poly(ethylene terephthalate)-grafted glycidyl methacrylate (PET-g-GMA) fiber. The immobilization yielded a protein loading of 2.38 mg g(-1) of PET-g-GMA fiber. The performances of the immobilized and free CRLs were evaluated with regard to hydrolysis of olive oil and esterification of oleic acid. The optimum activity pH of the CRL was changed by immobilization to neutral range. The maximum activity of the free and immobilized CRLs occurred at 40 and 45 °C respectively. The immobilized lipase retained 65% of its original activity at 50 °C for 2 h. It was found that the immobilized lipase stored at 4 °C retained 90% of its original activity after 35 days, whereas the free lipase stored at 4 °C retained 69% of its original activity after the same period. In the esterification experiments, the immobilized CRL could maintain a high activity at a water content range from 1.5 to 6% (v/v), while the activity of free CRL showed a clear dependence on water content and decreased rapidly at above 3% (v/v) water content. In addition, after five reuses, the esterification percent yield of the immobilized CRL slightly decreased from 29 to 27%. PMID:23574345

  16. Dynamic Covalent Chemistry-based Sensing: Pyrenyl Derivatives of Phenylboronic Acid for Saccharide and Formaldehyde.

    PubMed

    Chang, Xingmao; Fan, Jiayun; Wang, Min; Wang, Zhaolong; Peng, Haonan; He, Gang; Fang, Yu

    2016-01-01

    We synthesized two specially designed pyrenyl (Py) derivatives of phenylboronic acid, PSNB1 and PSNB2, of which PSNB2 self-assemble to form dynamic aggregate in methanol-water mixture (1:99, v/v) via intermolecular H-bonding and pi-pi stacking. Interestingly, the dynamic aggregate shows smart response to presence of fructose (F) as evidenced by fluorescence color change from green to blue. More interestingly, the fluorescence emission of the resulted PSNB2-F changes from blue to green with the addition of formaldehyde (FA). The reason behind is formation of a PSNB2-F dimer via FA cross-linking. Based upon the reactions as found, sensitive and fast sensing of F and FA in water was realized, of which the experimental DLs could be significantly lower than 10 μM for both analytes, and the response times are less than 1 min. It is believed that not only the materials as created may have the potential to find real-life applications but also the strategy as developed can be adopted to develop other dynamic materials. PMID:27498703

  17. Glucose-responsive insulin activity by covalent modification with aliphatic phenylboronic acid conjugates.

    PubMed

    Chou, Danny Hung-Chieh; Webber, Matthew J; Tang, Benjamin C; Lin, Amy B; Thapa, Lavanya S; Deng, David; Truong, Jonathan V; Cortinas, Abel B; Langer, Robert; Anderson, Daniel G

    2015-02-24

    Since its discovery and isolation, exogenous insulin has dramatically changed the outlook for patients with diabetes. However, even when patients strictly follow an insulin regimen, serious complications can result as patients experience both hyperglycemic and hypoglycemic states. Several chemically or genetically modified insulins have been developed that tune the pharmacokinetics of insulin activity for personalized therapy. Here, we demonstrate a strategy for the chemical modification of insulin intended to promote both long-lasting and glucose-responsive activity through the incorporation of an aliphatic domain to facilitate hydrophobic interactions, as well as a phenylboronic acid for glucose sensing. These synthetic insulin derivatives enable rapid reversal of blood glucose in a diabetic mouse model following glucose challenge, with some derivatives responding to repeated glucose challenges over a 13-h period. The best-performing insulin derivative provides glucose control that is superior to native insulin, with responsiveness to glucose challenge improved over a clinically used long-acting insulin derivative. Moreover, continuous glucose monitoring reveals responsiveness matching that of a healthy pancreas. This synthetic approach to insulin modification could afford both long-term and glucose-mediated insulin activity, thereby reducing the number of administrations and improving the fidelity of glycemic control for insulin therapy. The described work is to our knowledge the first demonstration of a glucose-binding modified insulin molecule with glucose-responsive activity verified in vivo. PMID:25675515

  18. Glucose-responsive insulin activity by covalent modification with aliphatic phenylboronic acid conjugates

    PubMed Central

    Chou, Danny Hung-Chieh; Webber, Matthew J.; Tang, Benjamin C.; Lin, Amy B.; Thapa, Lavanya S.; Deng, David; Truong, Jonathan V.; Cortinas, Abel B.; Langer, Robert; Anderson, Daniel G.

    2015-01-01

    Since its discovery and isolation, exogenous insulin has dramatically changed the outlook for patients with diabetes. However, even when patients strictly follow an insulin regimen, serious complications can result as patients experience both hyperglycemic and hypoglycemic states. Several chemically or genetically modified insulins have been developed that tune the pharmacokinetics of insulin activity for personalized therapy. Here, we demonstrate a strategy for the chemical modification of insulin intended to promote both long-lasting and glucose-responsive activity through the incorporation of an aliphatic domain to facilitate hydrophobic interactions, as well as a phenylboronic acid for glucose sensing. These synthetic insulin derivatives enable rapid reversal of blood glucose in a diabetic mouse model following glucose challenge, with some derivatives responding to repeated glucose challenges over a 13-h period. The best-performing insulin derivative provides glucose control that is superior to native insulin, with responsiveness to glucose challenge improved over a clinically used long-acting insulin derivative. Moreover, continuous glucose monitoring reveals responsiveness matching that of a healthy pancreas. This synthetic approach to insulin modification could afford both long-term and glucose-mediated insulin activity, thereby reducing the number of administrations and improving the fidelity of glycemic control for insulin therapy. The described work is to our knowledge the first demonstration of a glucose-binding modified insulin molecule with glucose-responsive activity verified in vivo. PMID:25675515

  19. Dynamic Covalent Chemistry-based Sensing: Pyrenyl Derivatives of Phenylboronic Acid for Saccharide and Formaldehyde

    NASA Astrophysics Data System (ADS)

    Chang, Xingmao; Fan, Jiayun; Wang, Min; Wang, Zhaolong; Peng, Haonan; He, Gang; Fang, Yu

    2016-08-01

    We synthesized two specially designed pyrenyl (Py) derivatives of phenylboronic acid, PSNB1 and PSNB2, of which PSNB2 self-assemble to form dynamic aggregate in methanol-water mixture (1:99, v/v) via intermolecular H-bonding and pi-pi stacking. Interestingly, the dynamic aggregate shows smart response to presence of fructose (F) as evidenced by fluorescence color change from green to blue. More interestingly, the fluorescence emission of the resulted PSNB2-F changes from blue to green with the addition of formaldehyde (FA). The reason behind is formation of a PSNB2-F dimer via FA cross-linking. Based upon the reactions as found, sensitive and fast sensing of F and FA in water was realized, of which the experimental DLs could be significantly lower than 10 μM for both analytes, and the response times are less than 1 min. It is believed that not only the materials as created may have the potential to find real-life applications but also the strategy as developed can be adopted to develop other dynamic materials.

  20. Dynamic Covalent Chemistry-based Sensing: Pyrenyl Derivatives of Phenylboronic Acid for Saccharide and Formaldehyde

    PubMed Central

    Chang, Xingmao; Fan, Jiayun; Wang, Min; Wang, Zhaolong; Peng, Haonan; He, Gang; Fang, Yu

    2016-01-01

    We synthesized two specially designed pyrenyl (Py) derivatives of phenylboronic acid, PSNB1 and PSNB2, of which PSNB2 self-assemble to form dynamic aggregate in methanol-water mixture (1:99, v/v) via intermolecular H-bonding and pi-pi stacking. Interestingly, the dynamic aggregate shows smart response to presence of fructose (F) as evidenced by fluorescence color change from green to blue. More interestingly, the fluorescence emission of the resulted PSNB2-F changes from blue to green with the addition of formaldehyde (FA). The reason behind is formation of a PSNB2-F dimer via FA cross-linking. Based upon the reactions as found, sensitive and fast sensing of F and FA in water was realized, of which the experimental DLs could be significantly lower than 10 μM for both analytes, and the response times are less than 1 min. It is believed that not only the materials as created may have the potential to find real-life applications but also the strategy as developed can be adopted to develop other dynamic materials. PMID:27498703

  1. Seven supramolecular frameworks constructed from combination of hydrogen-bonds and other non-covalent associations between organic acids and bis-imidazoles

    NASA Astrophysics Data System (ADS)

    Jin, Shouwen; Zhang, Huan; Zhao, Ying; Jin, Li; Ye, Xianghang; Liu, Hui; Wang, Daqi

    2015-11-01

    Seven crystalline organic acid-base adducts derived from bis(N-imidazolyl) and organic acids (2,4,6-trinitrophenol, p-nitrobenzoic acid, 3,5-dinitrobenzoic acid, oxalic acid, m-phthalic acid, and 1,5-naphthalenedisulfonic acid) were prepared and characterized by X-ray diffraction analysis, IR, mp, and elemental analysis. The seven compounds are all organic salts. In salts 1, and 3, the L1 are monoprotonated, while in 4 and 6 the L1 are diprotonated. All supramolecular architectures involve extensive classical hydrogen bonds and C-H⋯O interactions. The role of weak and strong non-covalent interactions in the crystal packing is analyzed. The complexes displayed 2D-3D framework structures for the synergistic effect of the various non-covalent interactions. The results presented herein indicate that the strength and directionality of the N-H⋯N, N-H⋯O, O-H⋯O, O-H⋯N, N-H⋯S, and O-H⋯S hydrogen bonds between the organic acids and the ditopic imidazoles are sufficient to bring about the formation of binary organic salts.

  2. Covalently bonded sulfonic acid magnetic graphene oxide: Fe3O4@GO-Pr-SO3H as a powerful hybrid catalyst for synthesis of indazolophthalazinetriones.

    PubMed

    Doustkhah, Esmail; Rostamnia, Sadegh

    2016-09-15

    Multistep synthesis of covalently sulfonated magnetic graphene oxide was achieved by starting from Hummer's method to produce graphene oxide (GO) from chemical oxidation of graphite. Then, GO nanosheets were applied to support Fe3O4 nanoparticles (Fe3O4@GO) using co-precipitation method in the presence of GO sheets. This strategy led to formation of uniform particles of Fe3O4 on the surface of GO sheets. Then, it was sulfonated (Fe3O4@GO-Pr-SO3H) through modification with 3-mercaptopropyltrimethoxysilane (MPTMS) and subsequent oxidation with hydrogen peroxide (H2O2). In comparison, the covalently bonded propyl sulfonic acid groups were more prevailing rather to sulfonic acids of GO itself. The proposed catalyst was more active and recyclable at least for 11 runs. PMID:27309948

  3. Non-covalent complexes of folic acid and oleic acid conjugated polyethylenimine: An efficient vehicle for antisense oligonucleotide delivery

    PubMed Central

    Yang, Shuang; Yang, Xuewei; Liu, Yan; Zheng, Bin; Meng, Lingjun; Lee, Robert J.; Xie, Jing; Teng, Lesheng

    2016-01-01

    Polyethylenimine (PEI) was conjugated to oleic acid (PEI-OA) and evaluated as a delivery agent for LOR-2501, an antisense oligonucleotide against ribonucleotide reductase R1 subunit. PEI-OA/LOR-2501 complexes were further coated with folic acid (FA/PEI-OA/LOR-2501) and evaluated in tumor cells. The level of cellular uptake of FA/PEI-OA/LOR-2501 was more than double that of PEI/LOR-2501 complexes, and was not affected by the expression level of folate receptor (FR) on the cell surface. Efficient delivery was seen in several cell lines. Furthermore, pathway specific cellular internalization inhibitors and markers were used to reveal the principal mechanism of cellular uptake. FA/PEI-OA/LOR-2501 significantly induced the downregulation of R1 mRNA and R1 protein. This novel formulation of FA/PEI-OA provides a reliable and highly efficient method for delivery of oligonucleotide and warrants further investigation. PMID:26263216

  4. Modulation of phenytoin teratogenicity and embryonic covalent binding by acetylsalicylic acid, caffeic acid, and alpha-phenyl-N-t-butylnitrone: implications for bioactivation by prostaglandin synthetase

    SciTech Connect

    Wells, P.G.; Zubovits, J.T.; Wong, S.T.; Molinari, L.M.; Ali, S.

    1989-02-01

    Teratogenicity of the anticonvulsant drug phenytoin is thought to involve its bioactivation by cytochromes P-450 to a reactive arene oxide intermediate. We hypothesized that phenytoin also may be bioactivated to a teratogenic free radical intermediate by another enzymatic system, prostaglandin synthetase. To evaluate the teratogenic contribution of this latter pathway, an irreversible inhibitor of prostaglandin synthetase, acetylsalicylic acid (ASA), 10 mg/kg intraperitoneally (ip), was administered to pregnant CD-1 mice at 9:00 AM on Gestational Days 12 and 13, 2 hr before phenytoin, 65 mg/kg ip. Other groups were pretreated 2 hr prior to phenytoin administration with either the antioxidant caffeic acid or the free radical spin trapping agent alpha-phenyl-N-t-butylnitrone (PBN). Caffeic acid and PBN were given ip in doses that respectively were up to 1.0 to 0.05 molar equivalents to the dose of phenytoin. Dams were killed on Day 19 and the fetuses were assessed for teratologic anomalies. A similar study evaluated the effect of ASA on the in vivo covalent binding of radiolabeled phenytoin administered on Day 12, in which case dams were killed 24 hr later on Day 13. ASA pretreatment produced a 50% reduction in the incidence of fetal cleft palates induced by phenytoin (p less than 0.05), without significantly altering the incidence of resorptions or mean fetal body weight. Pretreatment with either caffeic acid or PBN resulted in dose-related decreases in the incidence of fetal cleft palates produced by phenytoin, with maximal respective reductions of 71 and 82% at the highest doses of caffeic acid and PBN (p less than 0.05).

  5. Hydroxycinnamic acid bound arabinoxylans from millet brans-structural features and antioxidant activity.

    PubMed

    Bijalwan, Vandana; Ali, Usman; Kesarwani, Atul Kumar; Yadav, Kamalendra; Mazumder, Koushik

    2016-07-01

    Hydroxycinnamic acid bound arabinoxylans (HCA-AXs) were extracted from brans of five Indian millet varieties and response surface methodology was used to optimize the extraction conditions. The optimal condition to obtain highest yield of millet HCA-AXs was determined as follows: time 61min, temperature 66°C, ratio of solvent to sample 12ml/g. Linkage analysis indicated that hydroxycinnamic acid bound arabinoxylan from kodo millet (KM-HCA-AX) contained comparatively low branched arabinoxylan consisting of 14.6% mono-substituted, 1.2% di-substituted and 41.2% un-substituted Xylp residues. The HPLC analysis of millet HCA-AXs showed significant variation in the content of three major bound hydroxycinnamic acids (caffeic, p-coumaric and ferulic acid). The antioxidant activity of millet HCA-AXs were evaluated using three in vitro assay methods (DPPH, FRAP and β-carotene linoleate emulsion assays) which suggested both phenolic acid composition and structural characteristics of arabinoxylans could be correlated to their antioxidant potential, the detailed structural analysis revealed that low substituted KM-HCA-AX exhibited relatively higher antioxidant activity compared to other medium and highly substituted HCA-AXs from finger (FM), proso (PM), barnyard (BM) and foxtail (FOXM) millet. PMID:27050114

  6. Seasonal and spatial changes of free and bound organic acids in total suspended particles in Guangzhou, China

    NASA Astrophysics Data System (ADS)

    Ma, Shexia; Peng, Ping'an; Song, Jianzhong; Bi, Xinhui; Zhao, Jinping; He, Lulu; Sheng, Guoying; Fu, Jiamo

    2010-12-01

    The concentrations and compositions of free and bound organic acids in total suspended particles from typical urban, suburban and forest park sites of Guangzhou were determined in this study. The free form of organic acids (solvent extractable) in aerosols in Guangzhou varied with site and season. The suburban samples contained the highest contents of alkanoic, alkenoic and dicarboxylic acids. These findings were consistent with a higher supply of hydrocarbons and NOx in the suburban area. However, concentrations of aromatic acids were similar in the urban, suburban and forest park sites. Generally, winter season samples of the acids from anthropogenic sources contained more organic acids than summer season samples due to stronger removal by wet deposition in the summer. For the acids from botanic sources, the summer season samples were higher. In addition to the free acids, bound acids (solvent non-extractable) mainly formed by esterification of free acids were also found in the samples. In general, bound acids were higher than free acids. Esterification is mainly controlled by the pKa of organic acids and the atmospheric pH value. This explains why aromatic and dicarboxylic acids occur mainly as bound forms and why the samples from urban sites contained high levels of bound acids as the pH of rain water can reach 4.53. Concentrations of alkanoic and alkenoic acids in the aerosols of Guangzhou were much higher than those in the other areas studied.

  7. Reduction of the uptake by a macrophagic cell line of nanoparticles bearing heparin or dextran covalently bound to poly(methyl methacrylate).

    PubMed

    Jaulin, N; Appel, M; Passirani, C; Barratt, G; Labarre, D

    2000-01-01

    Amphiphilic and fluorescent covalently labelled core-shell nanoparticles based on poly(methyl methacrylate) (PMMA), were prepared by random copolymerisation of N-Vinyl carbazole (NVC) with MMA, initiated on polysaccharidic radicals, yielding diblock copolymers of either dextran-P(MMA-NVC) (Nanodex* particles), or heparin-P(MMA-NVC) (Nanohep* particles). Nanoparticles made from random copolymers of P(MMA-NVC) (PMMA*) were used as controls. The interactions between particles and a J774A1 murine macrophage-like cell line were quantified by direct measurement of the cell-associated fluorescence. The association with the cells occurred within 30 min. Nanodex* and Nanohep* showed considerably less association than the control PMMA* particles. Some of the particle uptake could be attributed to phagocytosis, but more than 50% of the cell-associated fluorescence persisted at low temperature or in the presence of cytochalasin B. The results suggest that both the adsorption and the internalisation processes can be inhibited by the presence of the polysaccharide chains. In conclusion, these results confirm that nanoparticles prepared with heparin or dextran chains on their surface, probably in a brush-like configuration, show "stealth" properties in vitro as had previously been observed in vivo. If this biomimetic approach can also be applied to biodegradable polymers, these systems would provide at least an alternative to PEG-modified particles as long-circulating drug carriers systems or imaging agents. PMID:10938526

  8. A FRET-based probe for epidermal growth factor receptor bound non-covalently to a pair of synthetic amphipathic helixes

    SciTech Connect

    Itoh, Reina E.; Kurokawa, Kazuo; Fujioka, Aki; Sharma, Alok; Mayer, Bruce J.; Matsuda, Michiyuki . E-mail: matsudam@biken.osaka-u.ac.jp

    2005-07-01

    Epidermal growth factor (EGF) receptor plays a pivotal role in a variety of cellular functions, such as proliferation, differentiation, and migration. To monitor the EGF receptor (EGFR) activity in living cells, we developed a probe for EGFR activity based on the principle of fluorescence resonance energy transfer (FRET). Previously, we developed a probe designated as Picchu (Phosphorylation indicator of the CrkII chimeric unit), which detects the tyrosine phosphorylation of the CrkII adaptor protein. We used a pair of synthetic amphipathic helixes, WinZipA2 and WinZipB1, to bind Picchu non-covalently to the carboxyl-terminus of the EGFR. Using this modified probe named Picchu-Z, the activity of EGFR was followed in EGF-stimulated Cos7 cells. We found that a high level of tyrosine phosphorylation of Picchu-Z probe remained after endocytosis until the point when the EGFR was translocated to the perinuclear region. These findings are in agreement with the previously reported 'signaling endosome' model. Furthermore, by pulse stimulation with EGF and by acute ablation of EGFR activity with AG1478, it was suggested that the phosphorylation of Picchu-Z probe, and probably the phosphorylation of EGFR also, underwent a rapid equilibrium ({tau} {sub 1/2} < 2 min) between the phosphorylated and dephosphorylated states in the presence of EGF.

  9. Charge Photoinjection in Intercalated and Covalently Bound [Re(CO)3(dppz)(py)]+-DNA Constructs Monitored by Time Resolved Visible and Infrared Spectroscopy

    PubMed Central

    Olmon, Eric D.; Sontz, Pamela A.; Blanco-Rodríguez, Ana María; Towrie, Michael; Clark, Ian P.; Vlček, Antonín; Barton, Jacqueline K.

    2011-01-01

    The complex [Re(CO)3(dppz)(py′-OR)]+ (dppz = dipyrido[3,2-a:2′,3′-c]phenazine; py′-OR = 4-functionalized pyridine) offers IR sensitivity and can oxidize DNA directly from the excited state, making it a promising probe for the study of DNA-mediated charge transport (CT). The behavior of several covalent and noncovalent Re-DNA constructs was monitored by time-resolved IR (TRIR) and UV/visible spectroscopies, as well as biochemical methods, confirming the long-range oxidation of DNA by the excited complex. Optical excitation of the complex leads to population of MLCT and at least two distinct intraligand states. Experimental observations that are consistent with charge injection from these excited states include similarity between long-time TRIR spectra and the reduced state spectrum observed by spectroelectrochemistry, the appearance of a guanine radical signal in TRIR spectra, and the eventual formation of permanent guanine oxidation products. The majority of reactivity occurs on the ultrafast timescale, although processes dependent on slower conformational motions of DNA, such as the accumulation of oxidative damage at guanine, are also observed. The ability to measure events on such disparate timescales, its superior selectivity in comparison to other spectroscopic techniques, and the ability to simultaneously monitor carbonyl ligand and DNA IR absorption bands makes TRIR a valuable tool for the study of CT in DNA. PMID:21827149

  10. Extractive and oxidative removal of copper bound to humic acid in soil.

    PubMed

    Hwang, Bo-Ram; Kim, Eun-Jung; Yang, Jung-Seok; Baek, Kitae

    2015-04-01

    Copper (Cu) is often found strongly bound to natural organic matter (NOM) in soil through the formation of strong Cu-NOM complexes. Therefore, in order to successfully remediate Cu-contaminated soils, effective removal of Cu bound to soil organic matter should be considered. In this study, we investigated soil washing methods for Cu removal from a synthetic Cu-contaminated model silica soil coated with humic acid (HA) and from field contaminated soil. Various reagents were studied to extract Cu bound to NOM, which included oxidant (H2O2), base (NaOH), and chelating agents (citric acid and ethylenediaminetetraacetic acid (EDTA)). Among the wash reagents, EDTA extracted Cu most effectively since EDTA formed very strong complexes with Cu, and Cu-HA complexes were transformed into Cu-EDTA complexes. NaOH extracted slightly less Cu compared to EDTA. HA was effectively extracted from the model soil under strongly alkaline conditions with NaOH, which seemed to concurrently release Cu bound to HA. However, chemical oxidation with H2O2 was not effective at destroying Cu-HA complexes. Fourier transform infrared spectroscopy and elemental analysis revealed that chelating agents such as citrate and EDTA were adsorbed onto the model soil via possible complexation between HA and extraction agents. The extraction of Cu from a field contaminated soil sample was effective with chelating agents, while oxidative removal with H2O2 and extractive removal with NaOH separated negligible amounts of Cu from the soil. Based on these results, Cu bound to organic matter in soil could be effectively removed by chelating agents, although remnant agents may remain in the soil. PMID:25388560

  11. Hydrogels with covalent and noncovalent crosslinks

    NASA Technical Reports Server (NTRS)

    Kilck, Kristi L. (Inventor); Yamaguchi, Nori (Inventor)

    2013-01-01

    A method for targeted delivery of therapeutic compounds from hydrogels is presented. The method involves administering to a cell a hydrogel in which a therapeutic compound is noncovalently bound to heparin. The hydrogel may contain covalent and non-covalent crosslinks.

  12. Quantification of total content of non-esterified fatty acids bound to human serum albumin.

    PubMed

    Pavićević, Ivan D; Jovanović, Vesna B; Takić, Marija M; Aćimović, Jelena M; Penezić, Ana Z; Mandić, Ljuba M

    2016-09-10

    Non-esterified fatty acids bound to the human serum albumin (HSA) contribute to several HSAs properties of special concern in pathologies, for instance to the reactivity of the free HSA-Cys34 thiol group (important antioxidative thiol pool in plasma), and to the affinity for binding of molecules and ions (for example cobalt as a prominent biomarker in heart ischemia). Therefore, the method for determination of FAs bound to HSA was developed. FAs were released from HSA (previously isolated from serum by ammonium sulfate precipitation) using acidic copper(II) sulfate in phosphoric acid, extracted by n-heptane-chloroform (4:1, v/v) mixture, spotted on TL silica-gel and then developed with n-heptane-chloroform-acetic acid (5:3:0.3, v/v/v). Common office flatbed scanner and software solution for densitometric image analysis, developed in R, were used. The linearity of calibration curve in concentration range from 0.1 to 5.0mmol/L stearic acid was achieved. The method was proved to be precise (with RSD of 1.4-4.7%) and accurate. Accuracy was examined by standard addition method (recoveries 97.2-102.5%) and by comparison to results of GC. The method is sample saving, technically less demanding, and cheap, and therefore suitable for determination of FAs/HSA ratio when elevated concentrations of free FAs are reliable diagnostic/risk parameter of pathological states. PMID:27394177

  13. Prediction of ultra-high ON/OFF ratio nanoelectromechanical switches from covalently bound C60 chains: An ab initio study

    NASA Astrophysics Data System (ADS)

    Kim, Han; Kim, Yong-Hoon

    2013-03-01

    Applying a first-principles computational approach combining density-functional theory and matrix Green's function calculations, we analyze the microscopic origin of the switching behavior experimentally observed for the fullerene C60 chains oligomerized via [2 +2] cycloaddition and propose a scheme to significantly improve the device performance. Considering infinite C60 chains, we first confirm that bound C60 chains with significant orbital hybridizations and band formation should in principle induce a higher conductance state. However, we find that large metal-C60 distances adopted in the scanning tunneling microscope (STM) setup can result in the experimentally observed opposite switching state assignment. The switching ordering and ratio is in fact found to sensitively depend on the STM tip metal species and the associated band bending direction in the C60-STM tip vacuum gap. We demonstrate that a junction configuration in which the C60-STM tip distance is maintained at short distances via nanoelectromechanical tip movement can achieve a metal-independent and drastically improved switching performance based on the intrinsically better electronic connectivity in the oligomerized C60 chains. This work was supported by Basic Science Research Grant (No. 2012R1A1A2044793) and EDISON Program (No. 2012M3C1A6035684) of the NRF of Korea.

  14. The in situ distribution of glycoprotein-bound 4-O-Acetylated sialic acids in vertebrates.

    PubMed

    Aamelfot, Maria; Dale, Ole Bendik; Weli, Simon Chioma; Koppang, Erling Olaf; Falk, Knut

    2014-05-01

    Sialic acids are located at the terminal branches of the cell glycocalyx and secreted glycan molecules. O-Acetylation is an important modification of the sialic acids, however very few studies have demonstrated the in situ distribution of the O-Acetylated sialic acids. Here the distribution of glycoprotein bound 4-O-Acetylated sialic acids (4-O-Ac sias) in vertebrates was determined using a novel virus histochemistry assay. The 4-O-Ac sias were found in the circulatory system, i.e. on the surface of endothelial cells and RBCs, of several vertebrate species, though most frequently in the cartilaginous fish (class Chondrichthyes) and the bony fish (class Osteichthyes). The O-Acetylated sialic acid was detected in 64 % of the examined fish species. Even though the sialic acid was found less commonly in higher vertebrates, it was found at the same location in the positive species. The general significance of this endothelial labelling pattern distribution is discussed. The seemingly conserved local position through the evolution of the vertebrates, suggests an evolutionary advantage of this sialic acid modification. PMID:24833039

  15. Chiral stationary phase covalently bound with a chiral pseudo-18-crown-6 ether for enantiomer separation of amino compounds using a normal mobile phase.

    PubMed

    Hirose, Keiji; Yongzhu, Jin; Nakamura, Takashi; Nishioka, Ryota; Ueshige, Tetsuro; Tobe, Yoshito

    2005-03-01

    In order to apply the excellent chiral recognition ability of chiral pseudo-18-crown-6 ethers that we developed to chiral separation, we prepared a chiral stationary phase (CSP) by immobilizing a chiral pseudo-18-crown-6-type host on 3-aminopropyl silica gel. A chiral column was prepared by the slurry-packing method in a stainless steel HPLC column. A liquid chromatography system using this CSP combined with the detection by mass spectrometry was used for enantiomer separation of amino compounds. A normal mobile phase can be used on this CSP as opposed to conventional dynamic coating-type CSPs. Enantiomers of 18 common natural amino acids were efficiently separated. The chiral separation observed for amino acid methyl esters, amino alcohols, and lipophilic amines was fair using this HPLC system. In view of the correlation between the enantiomer selectivity observed in chromatography and the complexion in solution, the chiral recognition in host-guest interactions might contribute to this enantiomer separation. PMID:15704196

  16. "Stereoscopic" 2D super-microporous phosphazene-based covalent organic framework: Design, synthesis and selective sorption towards uranium at high acidic condition.

    PubMed

    Zhang, Shuang; Zhao, Xiaosheng; Li, Bo; Bai, Chiyao; Li, Yang; Wang, Lei; Wen, Rui; Zhang, Meicheng; Ma, Lijian; Li, Shoujian

    2016-08-15

    So far, only five primary elements (C, H, O, N and B) and two types of spatial configuration (C2-C4, C6 and Td) are reported to build the monomers for synthesis of covalent organic frameworks (COFs), which have partially limited the route selection for accessing COFs with new topological structure and novel properties. Here, we reported the design and synthesis of a new "stereoscopic" 2D super-microporous phosphazene-based covalent organic framework (MPCOF) by using hexachorocyclotriphosphazene (a P-containing monomer in a C3-like spatial configuration) and p-phenylenediamine (a linker). The as-synthesized MPCOF shows high crystallinity, relatively high heat and acid stability and distinctive super-microporous structure with narrow pore-size distributions ranging from 1.0-2.1nm. The results of batch sorption experiments with a multi-ion solution containing 12 co-existing cations show that in the pH range of 1-2.5, MPCOF exhibits excellent separation efficiency for uranium with adsorption capacity more than 71mg/g and selectivity up to record-breaking 92%, and furthermore, an unreported sorption capacity (>50mg/g) and selectivity (>60%) were obtained under strong acidic condition (1M HNO3). Studies on sorption mechanism indicate that the uranium separation by MPCOF in acidic solution is realized mainly through both intra-particle diffusion and size-sieving effect. PMID:27107239

  17. Sequential photochemical and microbial degradation of organic molecules bound to humic acid

    SciTech Connect

    Amador, J.A.; Zika, R.G. ); Alexander, M. )

    1989-11-01

    We studied the effects of photochemical processes on the mineralization by soil microorganisms of (2-{sup 14}C)glycine bound to soil humic acid. Microbial mineralization of these complexes in the dark increased inversely with the molecular weight of the complex molecules. Sunlight irradiation of glycine-humic acid complexes resulted in loss of absorbance in the UV range and an increase in the amount of {sup 14}C-labeled low-molecular-weight photoproducts and the rate and extent of mineralization. More than half of the radioactivity in the low-molecular-weight photoproducts appears to be associated with carboxylic acids. Microbial mineralization of the organic carbon increased with solar flux and was proportional to the loss of A{sub 330}. Mineralization was proportional to the percentage of the original complex that was converted to low-molecular-weight photoproducts. Only light at wavelengths below 380 nm had an effect on the molecular weight distribution of the products formed from the glycine-humic acid complexes and on the subsequent microbial mineralization. Our results indicate that photochemical processes generate low-molecular-weight, readily biodegradable molecules from high-molecular-weight complexes of glycine with humic acid.

  18. BMAA detected as neither free nor protein bound amino acid in blue mussels.

    PubMed

    Rosén, Johan; Westerberg, Erik; Schmiedt, Sebastian; Hellenäs, Karl-Erik

    2016-01-01

    The results of this study imply that β-methylamino-alanine (BMAA) obtained from extracts of blue mussels from the Swedish west coast is neither free nor protein bound. The results were obtained by separation (precipitation and ultrafiltration) of low and high molecular weight compounds from neutral extracts of blue mussels, and treatment of these extracts with low and high concentrations of acids, varying time and temperature. The main portion of BMAA was obtained from the low molecular weight fraction, released or formed at 95 °C in dilute acids. The measured amount of BMAA did not increase by strong acid treatment. Lysine was used as reference and was only released at significant amounts when treating the high molecular weight fraction with concentrated acid. The results also indicated that breakage of peptide bonds was not involved in the formation/release of BMAA in these extracts unless any BMAA peptide bond would be significantly more susceptible to dilute acid than e.g. the monitored lysine peptide bond. BMAA was measured using isotope dilution and detection of the underivatized compound by HILIC-UHPLC-MS/MS (Hydrophilic Interaction Liquid Chromatography, Ultra-High Performance Liquid Chromatography, tandem Mass Spectrometry). The findings might add to the understanding of conflicting data in the literature regarding the occurrence of BMAA, and have implications for studies on possible biomagnification of BMAA in the food chain and bioavailability from food. PMID:26577502

  19. Sequential photochemical and microbial degradation of organic molecules bound to humic Acid.

    PubMed

    Amador, J A; Alexander, M; Zika, R G

    1989-11-01

    We studied the effects of photochemical processes on the mineralization by soil microorganisms of [2-C]glycine bound to soil humic acid. Microbial mineralization of these complexes in the dark increased inversely with the molecular weight of the complex molecules. Sunlight irradiation of glycine-humic acid complexes resulted in loss of absorbance in the UV range and an increase in the amount of C-labeled low-molecular-weight photoproducts and the rate and extent of mineralization. More than half of the radioactivity in the low-molecular-weight photoproducts appears to be associated with carboxylic acids. Microbial mineralization of the organic carbon increased with solar flux and was proportional to the loss of A(330). Mineralization was proportional to the percentage of the original complex that was converted to low-molecular-weight photoproducts. Only light at wavelengths below 380 nm had an effect on the molecular weight distribution of the products formed from the glycine-humic acid complexes and on the subsequent microbial mineralization. Our results indicate that photochemical processes generate low-molecular-weight, readily biodegradable molecules from high-molecular-weight complexes of glycine with humic acid. PMID:16348046

  20. Sequential Photochemical and Microbial Degradation of Organic Molecules Bound to Humic Acid

    PubMed Central

    Amador, José A.; Alexander, Martin; Zika, Rod G.

    1989-01-01

    We studied the effects of photochemical processes on the mineralization by soil microorganisms of [2-14C]glycine bound to soil humic acid. Microbial mineralization of these complexes in the dark increased inversely with the molecular weight of the complex molecules. Sunlight irradiation of glycine-humic acid complexes resulted in loss of absorbance in the UV range and an increase in the amount of 14C-labeled low-molecular-weight photoproducts and the rate and extent of mineralization. More than half of the radioactivity in the low-molecular-weight photoproducts appears to be associated with carboxylic acids. Microbial mineralization of the organic carbon increased with solar flux and was proportional to the loss of A330. Mineralization was proportional to the percentage of the original complex that was converted to low-molecular-weight photoproducts. Only light at wavelengths below 380 nm had an effect on the molecular weight distribution of the products formed from the glycine-humic acid complexes and on the subsequent microbial mineralization. Our results indicate that photochemical processes generate low-molecular-weight, readily biodegradable molecules from high-molecular-weight complexes of glycine with humic acid. PMID:16348046

  1. Crystal structure of a membrane-bound l-amino acid deaminase from Proteus vulgaris.

    PubMed

    Ju, Yingchen; Tong, Shuilong; Gao, Yongxiang; Zhao, Wei; Liu, Qi; Gu, Qiong; Xu, Jun; Niu, Liwen; Teng, Maikun; Zhou, Huihao

    2016-09-01

    l-amino acid oxidases/deaminases (LAAOs/LAADs) are a class of oxidoreductases catalyzing the oxidative deamination of l-amino acids to α-keto acids. They are widely distributed in eukaryotic and prokaryotic organisms, and exhibit diverse substrate specificity, post-translational modifications and cellular localization. While LAAOs isolated from snake venom have been extensively characterized, the structures and functions of LAAOs from other species are largely unknown. Here, we reported crystal structure of a bacterial membrane-bound LAAD from Proteus vulgaris (pvLAAD) in complex with flavin adenine dinucleotide (FAD). We found that the overall fold of pvLAAD does not resemble typical LAAOs. Instead it, is similar to d-amino acid oxidases (DAAOs) with an additional hydrophobic insertion module on protein surface. Structural analysis and liposome-binding assays suggested that the hydrophobic module serves as an extra membrane-binding site for LAADs. Bacteria from genera Proteus and Providencia were found to encode two classes of membrane-bound LAADs. Based on our structure, the key roles of residues Q278 and L317 in substrate selectivity were proposed and biochemically analyzed. While LAADs on the membrane were proposed to transfer electrons to respiratory chain for FAD re-oxidization, we observed that the purified pvLAAD could generate a significant amount of hydrogen peroxide in vitro, suggesting it could use dioxygen to directly re-oxidize FADH2 as what typical LAAOs usually do. These findings provide a novel insights for a better understanding this class of enzymes and will help developing biocatalysts for industrial applications. PMID:27422658

  2. Sialic Acid Is Required for Neuronal Inhibition by Soluble MAG but not for Membrane Bound MAG.

    PubMed

    Al-Bashir, Najat; Mellado, Wilfredo; Filbin, Marie T

    2016-01-01

    Myelin-Associated Glycoprotein (MAG), a major inhibitor of axonal growth, is a member of the immunoglobulin (Ig) super-family. Importantly, MAG (also known as Siglec-4) is a member of the Siglec family of proteins (sialic acid-binding, immunoglobulin-like lectins), MAG binds to complex gangliosides, specifically GD1a and/or GT1b. Therefore, it has been proposed as neuronal receptors for MAG inhibitory effect of axonal growth. Previously, we showed that MAG binds sialic acid through domain 1 at Arg118 and is able to inhibit axonal growth through domain 5. We developed a neurite outgrowth (NOG) assay, in which both wild type MAG and mutated MAG (MAG Arg118) are expressed on cells. In addition we also developed a soluble form NOG in which we utilized soluble MAG-Fc and mutated MAG (Arg118-Fc). Only MAG-Fc is able to inhibit NOG, but not mutated MAG (Arg118)-Fc that has been mutated at its sialic acid binding site. However, both forms of membrane bound MAG- and MAG (Arg118)- expressing cells still inhibit NOG. Here, we review various results from different groups regarding MAG's inhibition of axonal growth. Also, we propose a model in which the sialic acid binding is not necessary for the inhibition induced by the membrane form of MAG, but it is necessary for the soluble form of MAG. This finding highlights the importance of understanding the different mechanisms by which MAG inhibits NOG in both the soluble fragmented form and the membrane-bound form in myelin debris following CNS damage. PMID:27065798

  3. Sialic Acid Is Required for Neuronal Inhibition by Soluble MAG but not for Membrane Bound MAG

    PubMed Central

    Al-Bashir, Najat; Mellado, Wilfredo; Filbin, Marie T.

    2016-01-01

    Myelin-Associated Glycoprotein (MAG), a major inhibitor of axonal growth, is a member of the immunoglobulin (Ig) super-family. Importantly, MAG (also known as Siglec-4) is a member of the Siglec family of proteins (sialic acid-binding, immunoglobulin-like lectins), MAG binds to complex gangliosides, specifically GD1a and/or GT1b. Therefore, it has been proposed as neuronal receptors for MAG inhibitory effect of axonal growth. Previously, we showed that MAG binds sialic acid through domain 1 at Arg118 and is able to inhibit axonal growth through domain 5. We developed a neurite outgrowth (NOG) assay, in which both wild type MAG and mutated MAG (MAG Arg118) are expressed on cells. In addition we also developed a soluble form NOG in which we utilized soluble MAG-Fc and mutated MAG (Arg118-Fc). Only MAG-Fc is able to inhibit NOG, but not mutated MAG (Arg118)-Fc that has been mutated at its sialic acid binding site. However, both forms of membrane bound MAG- and MAG (Arg118)- expressing cells still inhibit NOG. Here, we review various results from different groups regarding MAG’s inhibition of axonal growth. Also, we propose a model in which the sialic acid binding is not necessary for the inhibition induced by the membrane form of MAG, but it is necessary for the soluble form of MAG. This finding highlights the importance of understanding the different mechanisms by which MAG inhibits NOG in both the soluble fragmented form and the membrane-bound form in myelin debris following CNS damage. PMID:27065798

  4. Dynamic Covalent Nanoparticle Building Blocks.

    PubMed

    Kay, Euan R

    2016-07-25

    Rational and generalisable methods for engineering surface functionality will be crucial to realising the technological potential of nanomaterials. Nanoparticle-bound dynamic covalent exchange combines the error-correcting and environment-responsive features of equilibrium processes with the stability, structural precision, and vast diversity of covalent chemistry, defining a new and powerful approach for manipulating structure, function and properties at nanomaterial surfaces. Dynamic covalent nanoparticle (DCNP) building blocks thus present a whole host of possibilities for constructing adaptive systems, devices and materials that incorporate both nanoscale and molecular functional components. At the same time, DCNPs have the potential to reveal fundamental insights regarding dynamic and complex chemical systems confined to nanoscale interfaces. PMID:27312526

  5. Construction of crystalline 2D covalent organic frameworks with remarkable chemical (acid/base) stability via a combined reversible and irreversible route.

    PubMed

    Kandambeth, Sharath; Mallick, Arijit; Lukose, Binit; Mane, Manoj V; Heine, Thomas; Banerjee, Rahul

    2012-12-01

    Two new chemically stable [acid and base] 2D crystalline covalent organic frameworks (COFs) (TpPa-1 and TpPa-2) were synthesized using combined reversible and irreversible organic reactions. Syntheses of these COFs were done by the Schiff base reactions of 1,3,5-triformylphloroglucinol (Tp) with p-phenylenediamine (Pa-1) and 2,5-dimethyl-p-phenylenediamine (Pa-2), respectively, in 1:1 mesitylene/dioxane. The expected enol-imine (OH) form underwent irreversible proton tautomerism, and only the keto-enamine form was observed. Because of the irreversible nature of the total reaction and the absence of an imine bond in the system, TpPa-1 and TpPa-2 showed strong resistance toward acid (9 N HCl) and boiling water. Moreover, TpPa-2 showed exceptional stability in base (9 N NaOH) as well. PMID:23153356

  6. Relevant insight of surface characterization techniques to study covalent grafting of a biopolymer to titanium implant and its acidic resistance

    NASA Astrophysics Data System (ADS)

    D'Almeida, Mélanie; Amalric, Julien; Brunon, Céline; Grosgogeat, Brigitte; Toury, Bérangère

    2015-02-01

    Peri-implant bacterial infections are the main cause of complications in dentistry. Our group has previously proposed the attachment of chitosan on titanium implants via a covalent bond to improve its antibacterial properties while maintaining its biocompatibility. A better knowledge of the coating preparation process allows a better understanding of the bioactive coating in biological conditions. In this work, several relevant characterization techniques were used to assess an implant device during its production phase and its resistance in natural media at different pH. The titanium surface was functionalized with 3-aminopropyltriethoxysilane (APTES) followed by grafting of an organic coupling agent; succinic anhydride, able to form two covalent links, with the substrate through a Ti-O-Si bond and the biopolymer through a peptide bond. Each step of the coating synthesis as well as the presence confirmation of the biopolymer on titanium after saliva immersion was followed by FTIR-ATR, SEM, EDS, 3D profilometry, XPS and ToF-SIMS analyses. Results allowed to highlight the efficiency of each step of the process, and to propose a mechanism occurring during the chitosan coating degradation in saliva media at pH 5 and at pH 3.

  7. 3-Nitropropionic Acid is a Suicide Inhibitor of MitochondrialRespiration that, Upon Oxidation by Complex II, Forms a Covalent AdductWith a Catalytic Base Arginine in the Active Site of the Enzyme

    SciTech Connect

    Huang, Li-shar; Sun, Gang; Cobessi, David; Wang, Andy C.; Shen,John T.; Tung, Eric Y.; Anderson, Vernon E.; Berry, Edward A.

    2005-12-01

    We report three new structures of mitochondrial respiratory Complex II (succinate ubiquinone oxidoreductase, E.C. 1.3.5.1) at up to 2.1 {angstrom} resolution, with various inhibitors. The structures define the conformation of the bound inhibitors and suggest the residues involved in substrate binding and catalysis at the dicarboxylate site. In particular they support the role of Arg297 as a general base catalyst accepting a proton in the dehydrogenation of succinate. The dicarboxylate ligand in oxaloacetate-containing crystals appears to be the same as that reported for Shewanella flavocytochrome c treated with fumarate. The plant and fungal toxin 3-nitropropionic acid, an irreversible inactivator of succinate dehydrogenase, forms a covalent adduct with the side chain of Arg297. The modification eliminates a trypsin cleavage site in the flavoprotein, and tandem mass spectroscopic analysis of the new fragment shows the mass of Arg 297 to be increased by 83 Da and to have potential of losing 44 Da, consistent with decarboxylation, during fragmentation.

  8. Specific covalent immobilization of proteins through dityrosine cross-links.

    PubMed

    Endrizzi, Betsy J; Huang, Gang; Kiser, Patrick F; Stewart, Russell J

    2006-12-19

    Dityrosine cross-links are widely observed in nature in structural proteins such as elastin and silk. Natural oxidative cross-linking between tyrosine residues is catalyzed by a diverse group of metalloenzymes. Dityrosine formation is also catalyzed in vitro by metal-peptide complexes such as Gly-Gly-His-Ni(II). On the basis of these observations, a system was developed to specifically and covalently surface immobilize proteins through dityrosine cross-links. Methacrylate monomers of the catalytic peptide Gly-Gly-His-Tyr-OH (GGHY) and the Ni(II)-chelating group nitrilotriacetic acid (NTA) were copolymerized with acrylamide into microbeads. Green fluorescent protein (GFP), as a model protein, was genetically tagged with a tyrosine-modified His6 peptide on its carboxy terminus. GFP-YGH6, specifically associated with the NTA-Ni(II) groups, was covalently coupled to the bead surface through dityrosine bond formation catalyzed by the colocalized GGHY-Ni(II) complex. After extensive washing with EDTA to disrupt metal coordination bonds, we observed that up to 75% of the initially bound GFP-YGH6 remained covalently bound to the bead while retaining its structure and activity. Dityrosine cross-linking was confirmed by quenching the reaction with free tyrosine. The method may find particular utility in the construction and optimization of protein microarrays. PMID:17154619

  9. Non-covalent probes for the investigation of structure and dynamics of protein-nucleic acid assemblies: the case of NC-mediated dimerization of genomic RNA in HIV-1

    PubMed Central

    Turner, Kevin B.; Kohlway, Andrew S.; Hagan, Nathan A.; Fabris, Daniele

    2009-01-01

    The nature of specific RNA-RNA and protein-RNA interactions involved in the process of genome dimerization and isomerization in HIV-1, which is mediated in vitro by the stemloop 1 (SL1) of the packaging signal and by the nucleocapsid (NC) domain of the viral Gag polyprotein, was investigated by using archetypical nucleic acid ligands as non-covalent probes. Small-molecule ligands make contact with their target substrates through complex combinations of H-bonds, salt bridges, and hydrophobic interactions. Therefore, their binding patterns assessed by electrospray ionization (ESI) mass spectrometry can provide valuable insights into the factors determining specific recognition between species involved in biopolymer assemblies. In the case of SL1, dimerization and isomerization create unique structural features capable of sustaining stable interactions with classic nucleic acid ligands. The binding modes exhibited by intercalators and minor groove binders were adversely affected by the significant distortion of the duplex formed by palindrome annealing in the kissing-loop (KL) dimer, whereas the modes observed for the corresponding extended duplex (ED) confirmed a more regular helical structure. Consistent with the ability to establish electrostatic interactions with highly negative pockets typical of helix anomalies, polycationic aminoglycosides bound to the stem-bulge motif conserved in all SL1 conformers, to the unpaired nucleotides located at the hinge between kissing hairpins in KL, and to the exposed bases flanking the palindrome duplex in ED. The patterns afforded by intercalators and minor groove binders did not display detectable variations when the corresponding NC-SL1 complexes were submitted to probing. In contrast, aminoglycosides displayed the ability to compete with the protein for overlapping sites, producing opposite effects on the isomerization process. Indeed, displacing NC from the stem-bulges of the KL dimer induced inhibition of stem melting and

  10. Enzymatic amplification-free nucleic acid hybridisation sensing on nanostructured thick-film electrodes by using covalently attached methylene blue.

    PubMed

    García-González, Raquel; Costa-García, Agustín; Fernández-Abedul, M Teresa

    2015-09-01

    Amplification-free (referring to enzymatic amplification step) detection methodologies are increasing in biosensor development due to the need of faster and simpler protocols. However, for maintaining sensitivity without this step, highly detectable molecules or very sensitive detection techniques are required. The nanostructuration of transducer surfaces with carbon nanotubes (CNTs), gold nanoparticles (AuNPs) or both in nanohybrid configurations has been employed in this work for DNA hybridisation sensing purposes. Methylene blue (MB), covalently attached to single stranded DNA, (ssDNA) was incubated with a complementary sequence immobilized on nanostructured screen-printed electrodes (AuSPEs). Although CNTs can increase notoriously the signal of the marker, adsorptive properties should also be considered when bioassays are performed because non-specific adsorption (NSA) phenomena are magnified. In this work, strategies for decreasing NSA were thoroughly evaluated for the detection of Mycoplasma pneumoniae (MP) on CNTs-nanostructured screen-printed electrodes. Among them, the employ of UV-radiation or long incubation times (72h) allowed obtaining higher signals for the complementary strand with respect to the non-complementary one. The use of CNTs/AuNPs nanohybrids, together with the use of streptavidin-biotin (ST-B) interaction allows the higher differentiation (with a 3.5 ratio) in the genosensing of M. pneumoniae. PMID:26003686

  11. On the role of mercury in the non-covalent stabilisation of consecutive U-Hg(II)-U metal-mediated nucleic acid base pairs: metallophilic attraction enters the world of nucleic acids.

    PubMed

    Benda, Ladislav; Straka, Michal; Tanaka, Yoshiyuki; Sychrovský, Vladimír

    2011-01-01

    Metal atoms with a closed-shell electronic structure and positive charge as for example the Au(I), Pt(II), Ag(I), Tl(I) or Hg(II) atoms do not in some compounds repel each other due to the so-called metallophilic attraction (P. Pyykkö, Chem. Rev., 1997, 97, 597-636). Here we highlight the role of the Hg(II)Hg(II) metallophilic attraction between the consecutive metal-mediated mismatched base pairs of nucleic acids. Usually, the base stacking dominates the non-covalent interactions between steps of native nucleic acids. In the presence of metal-mediated base pairs these non-covalent interactions are enriched by the metal-base interactions and the metallophilic attraction. The two interactions arising due to the metal linkage of the mismatches were found in this study to have a stabilizing effect on nucleic acid structure. The calculated data are consistent with recent experimental observations. The stabilization due to the metallophilic attraction seems to be a generally important concept for the nucleic acids containing heavy metals with short contacts. PMID:21049099

  12. QM/MM Refinement and Analysis of Protein Bound Retinoic Acid

    PubMed Central

    Li, Xue; Fu, Zheng; Merz, Kenneth M.

    2011-01-01

    Retinoic acid (RA) is a vitamin A derivative, which modifies the appearance of fine wrinkles and roughness of facial skin and treats acne and activates gene transcription by binding to heterodimers of the retinoic acid receptor (RAR) and the retinoic X receptor (RXR). There are series of protein bound RA complexes available in the protein databank (PDB), which provides a broad range of information about the different bioactive conformations of RA. In order to gain further insights into the observed bioactive RA conformations we applied quantum mechanic (QM)/molecular mechanic (MM) approaches to re-refine the available RA protein-ligand complexes. MP2 complete basis set (CBS) extrapolations single energy calculations are also carried out for both the experimental conformations and QM optimized geometries of RA in the gas as well as solution phase. The results demonstrate that the re-refined structures show better geometries for RA than seen in the originally deposited PDB structures through the use of quantum mechanics for the ligand in the X-ray refinement procedure. QM/MM re-refined conformations also reduced the computed strain energies found in the deposited crystal conformations for RA. Finally, the dependence of ligand strain on resolution is analyzed. It is shown that ligand strain is not converged in our calculations and is likely an artifact of the typical resolutions employed to study protein-ligand complexes. PMID:22108894

  13. Rapid detection and isolation of covalent DNA/protein complexes: application to topoisomerase I and II.

    PubMed Central

    Trask, D K; DiDonato, J A; Muller, M T

    1984-01-01

    A rapid and simple method has been developed which allows detection and isolation of covalent DNA/protein adducts. The method is based upon the use of an ionic detergent, SDS, to neutralize cationic sites of weakly bound proteins thereby resulting in their dissociation off the helix. Proteins tightly or covalently bound to DNA that are not dissociable by SDS, result in the precipitation of the DNA fragment by the addition of KCl; however, free nucleic acid does not precipitate. The method is particularly useful as an analytical tool to titrate the binding of prototypic covalent binding proteins, topoisomerase I and II; thus, quantitation of topoisomerase activity is possible under defined conditions. As an analytical tool the method can be used as a general assay in the purification of as yet unidentified topoisomerases or other activities that bind DNA covalently. Moreover, the technology can be adapted for use in a preparative mode to separate covalent complexes from free DNA in a single step. Images Fig. 2. Fig. 4. PMID:6325181

  14. Novel characterisation of minor α-linolenic acid isomers in linseed oil by gas chromatography and covalent adduct chemical ionisation tandem mass spectrometry.

    PubMed

    Gómez-Cortés, P; Brenna, J T; Lawrence, P; de la Fuente, M A

    2016-06-01

    Discrimination between polyunsaturated fatty acid isomers with three double bonds is a great challenge, due to structural similarities and similar polarities. In this study, we report the identification of four minor geometrical isomers of α-linolenic acid (ALA) present in linseed oil samples: (9E,12Z,15E)-, (9Z,12Z,15E)-, (9Z,12E,15Z)- and (9E,12Z,15Z)-octadeca-9,12,15-trienoic acids, chromatographically resolved by gas chromatography (GC) using a new and highly polar ionic phase column (SLB-IL111). Gas chromatography-electron ionisation mass spectrometry (GC-EIMS) determined that the four unknown compounds were C18:3 n-3 isomers. The positional 9-12-15 C18:3 configuration was achieved by covalent adduct chemical ionisation tandem mass spectrometry (CACI-MS/MS) while geometrical configuration was established with analytical standards based on relative retention. We hypothesised that these isomers are formed during linseed oil deodorisation and postulate preferred and unfavoured isomerisation pathways of ALA. PMID:26830571

  15. Gi-mediated tyrosine phosphorylation of Grb2 (growth-factor-receptor-bound protein 2)-bound dynamin-II by lysophosphatidic acid.

    PubMed Central

    Kranenburg, O; Verlaan, I; Moolenaar, W H

    1999-01-01

    Lysophosphatidic acid (LPA) is the prototypic G-protein-coupled receptor agonist that activates the Ras-mitogen-activated protein (MAP) kinase cascade through pertussis toxin (PTX)-sensitive Gi and enhanced tyrosine kinase activity. We recently detected a 100 kDa protein (p100) that binds to the C-terminal SH3 domain of growth-factor-receptor-bound protein 2 (Grb2) and becomes tyrosine phosphorylated in a PTX-sensitive manner in LPA-treated Rat-1 cells [Kranenburg, Verlaan, Hordijk and Moolenaar (1997) EMBO J. 16, 3097-3105]. Through glutathione S-transferase-Grb2 affinity purification and microsequencing, we have now identified p100 as dynamin-II, a GTPase that regulates clathrin-mediated endocytosis. We show that in Rat-1 cells, Grb2-bound dynamin-II is rapidly tyrosine phosphorylated in response to LPA in a PTX-sensitive manner. Thus, tyrosine phosphorylation of Grb2-bound dynamin-II may be a critical event in Gi-mediated activation of the Ras-MAP kinase cascade in fibroblasts. PMID:10085221

  16. Bound phenolics in foods, a review.

    PubMed

    Acosta-Estrada, Beatriz A; Gutiérrez-Uribe, Janet A; Serna-Saldívar, Sergio O

    2014-01-01

    Among phytochemicals, phenolic compounds have been extensively researched due to their diverse health benefits. Phenolic compounds occur mostly as soluble conjugates and insoluble forms, covalently bound to sugar moieties or cell wall structural components. Absorption mechanisms for bound phenolic compounds in the gastrointestinal tract greatly depend on the liberation of sugar moieties. Food processes such as fermentation, malting, thermoplastic extrusion or enzymatic, alkaline and acid hydrolyses occasionally assisted with microwave or ultrasound have potential to release phenolics associated to cell walls. Different kinds of wet chemistry methodologies to release and detect bound phenolic have been developed. These include harsh heat treatments, chemical modifications or biocatalysis. New protocols for processing and determining phenolics in food matrices must be devised in order to release bound phenolics and for quality control in the growing functional food industry. PMID:24444905

  17. Stimulation of proximal tubular cell apoptosis by albumin-bound fatty acids mediated by peroxisome proliferator activated receptor-gamma.

    PubMed

    Arici, Mustafa; Chana, Ravinder; Lewington, Andrew; Brown, Jez; Brunskill, Nigel John

    2003-01-01

    In nephrotic syndrome, large quantities of albumin enter the kidney tubule. This albumin carries with it a heavy load of fatty acids to which the proximal tubule cells are exposed at high concentration. It is postulated that exposure to fatty acids in this way is injurious to proximal tubule cells. This study has examined the ability of fatty acids to interact with peroxisome proliferator-activated receptors (PPAR) in primary cultures of human proximal tubule cells. Luciferase reporter assays in transiently transfected human proximal tubule cells were used to show that albumin bound fatty acids and other agonists activate PPARgamma in a dose-dependent manner. One of the consequences of this activation is apoptosis of the cells as determined by changes in cell morphology, evidence of PARP cleavage, and appearance of DNA laddering. Overexpression of PPARgamma in these cells also results in enhanced apoptosis. Both fatty acid-induced PPAR activation and apoptosis in these cells can be blocked by PPAR response element decoy oligonucleotides. Activation of PPARgamma by the specific agonist PGJ(2) is associated with inhibition of cell proliferation, whereas activation by albumin bound fatty acids is accompanied by increased proliferation. However, the net balance of apoptosis/proliferation favors deletion of cells. These results implicate albumin-bound fatty acids as important mediators of tubular injury in nephrosis and provide fresh impetus for pursuit of lipid-lowering strategies in proteinuric renal disease. PMID:12506134

  18. Complete amino acid analysis of peptides and proteins after hydrolysis by a mixture of Sepharose-bound peptidases

    PubMed Central

    Bennett, H. P. J.; Elliott, D. F.; Evans, B. E.; Lowry, P. J.; McMartin, C.

    1972-01-01

    Incubation with a mixture of Sepharose-bound peptidases was shown to result in the quantitative release of amino acids from certain peptides and S-aminoethylated proteins. Subtraction of the low background values of amino acids generated by the enzymes enables amino acid ratios of corticotrophin-(1–24)-tetracosapeptide to be determined with a standard deviation on repeat digestions of 3–5%. Good values were obtained for amino acids that are completely or partially destroyed on acid hydrolysis, i.e. tryptophan, tyrosine, serine, asparagine and glutamine. Experiments with peptides containing d-amino acids showed that the enzyme mixture is stereospecific and could therefore be used to detect the presence of d-residues in peptides. The enzyme mixture completely hydrolyses peptide fragments obtained after Edman degradation and should therefore be useful for determining sequences of peptides containing acid-labile amino acid residues. The activities of the bound enzymes were unaltered over a period of 7 months and they provide a simple, reproducible procedure for the quantitative determination of amino acids in peptides and proteins containing l-amino acids. PMID:4349115

  19. A comparative study of serum protein-bound sialic acid in benign and malignant prostatic growth: possible role of oxidative stress in sialic acid homeostasis.

    PubMed

    Goswami, K; Nandeesha, H; Koner, B C; Nandakumar, D N

    2007-01-01

    Benign and malignant prostatic growths are associated with an increase in sialoconjugates (e.g. prostate-specific antigen (PSA)) in blood. Oxidative stress plays a crucial role in pathogenesis of various malignancies. The objective of this study was to evaluate oxidative stress parameters and protein-bound sialic acid level in sera of prostatic tumor cases and to asses for any association between them. Sera samples were collected and estimated for carbonylation of proteins, lipid peroxidation products, PSA and protein-bound sialic acid from 10 patients in each group with prostatic carcinoma (Ca prostate) and benign prostatic hyperplasia (BPH) along with 10 healthy male subjects of similar age group as control. In carcinoma prostate cases, lipid peroxides, protein carbonyls, protein-bound sialic acid and PSA were significantly increased compared to BPH and controls. There was significant association between oxidative stress parameters (lipid peroxide and protein carbonyl) and sialoconjugates (PSA and protein-bound sialic acid). In BPH cases, serum lipid peroxides and protein-bound sialic acid were significantly higher in comparison to controls and protein carbonyls were correlated with protein-bound sialic acid. ROC curve for sialic acid showed that it can be used as a marker to differentiate carcinoma prostate from benign growth of prostate at a cutoff level of 11.38 mug/mg protein with a sensitivity of 100% and specificity of 80%. We conclude that oxidative stress might be associated with the degree of sialylation of protein and graded changes in these parameters possibly unveil the pathogenic demarcation from benign to malignant condition of prostate. PMID:17404581

  20. Supramolecular architectures constructed by lanthanum, amino acids and 1,10-phenanthroline via non-covalent bond interactions

    NASA Astrophysics Data System (ADS)

    Zheng, Xiang-Jun; Jin, Lin-Pei

    2003-07-01

    Three supramolecular lanthanum coordination compounds of amino acids, with 1,10-phenanthroline (phen), [La 2(APA) 6(phen) 2(H 2O) 2](ClO 4) 6(phen) 4·2H 2O ( 1), [La 2(ABA) 6(phen) 2(H 2O) 2](ClO 4) 6 (phen) 6·4H 2O ( 2), and [La 2(AHA) 4(phen) 4](ClO 4) 6(phen) 4·2H 2O ( 3) (APA=3-aminopropionic acid; ABA=4-aminobutanoic acid; AHA=6-aminohexanoic acid) were synthesized and characterized by single crystal X-ray diffraction. The results show that the three coordination compounds are all composed of binuclear coordination cations built by metal-ligand coordination. Through hydrogen bonding and π-π stacking interactions, complex 1 forms a two-dimensional supramolecular sheet structure extending in the (001) plane, complex 2 forms a three-dimensional supramolecular network with many cavities occupied by ClO 4- and lattice H 2O molecules, and complex 3 forms a two-dimensional supramolecular lamellar structure in the (100) plane.

  1. A new maleimide-bound acid-cleavable solid-support reagent for profiling phosphorylation.

    PubMed

    Chowdhury, Saiful M; Munske, Gerhard R; Siems, William F; Bruce, James E

    2005-01-01

    A new chemical strategy for phosphopeptide profiling is reported in this study. Phosphorylation represents one of the most important classes of posttranslational modifications of proteins. Here we report a generalized strategy that employs solid-phase capture and mass-encoding steps to selectively enrich phosphopeptides from complex mixtures. This method exploits conversion of phosphates into thiols and reactive compounds to selectively isolate products of phosphorylation. Selective isolation of phosphopeptides is achieved with a simple, novel, acid-cleavable, solid-support-bound maleimide reagent. Our chemistry efforts have focused on minimization of linker size and simplification of reagent production with incorporation of common solid-phase peptide synthesis steps. Relative quantitation was demonstrated by modifying phosphopeptides with incorporation of ethanedithiol and propanedithiol. We observed that appropriate normalization is necessary to utilize mass tag strategies for relative quantitation of posttranslational modifications. The utility of solid-phase capture was determined with model phosphopeptides, and the method was demonstrated with enriching phosphopeptides from beta-casein, alpha-casein and ovalbumin. The solid-phase capture and release methods were also demonstrated with unfractionated whole histone protein mixtures to show this compound applicability in real biological samples. The new chemical strategy will ultimately be utilized for high-throughput profiling of phosphorylation and possibly other posttranslational modifications. PMID:15739244

  2. Dynamic covalent assembly and disassembly of nanoparticle aggregates.

    PubMed

    Borsley, Stefan; Kay, Euan R

    2016-07-12

    The quantitative assembly and disassembly of a new type of dynamic covalent nanoparticle (NP) building block is reported. In situ spectroscopic characterization reveals constitutionally adaptive NP-bound monolayers of boronate esters. Ditopic linker molecules are used to produce covalently connected AuNP assemblies, displaying open dendritic morphologies, and which, despite being linked by covalent bonds, can be fully disassembled on application of an appropriate chemical stimulus. PMID:27001937

  3. Extending the Diffuse Layer Model of Surface Acidity Behavior: III. Estimating Bound Site Activity Coefficients

    EPA Science Inventory

    Although detailed thermodynamic analyses of the 2-pK diffuse layer surface complexation model generally specify bound site activity coefficients for the purpose of accounting for those non-ideal excess free energies contributing to bound site electrochemical potentials, in applic...

  4. Locking GTPases covalently in their functional states

    NASA Astrophysics Data System (ADS)

    Wiegandt, David; Vieweg, Sophie; Hofmann, Frank; Koch, Daniel; Li, Fu; Wu, Yao-Wen; Itzen, Aymelt; Müller, Matthias P.; Goody, Roger S.

    2015-07-01

    GTPases act as key regulators of many cellular processes by switching between active (GTP-bound) and inactive (GDP-bound) states. In many cases, understanding their mode of action has been aided by artificially stabilizing one of these states either by designing mutant proteins or by complexation with non-hydrolysable GTP analogues. Because of inherent disadvantages in these approaches, we have developed acryl-bearing GTP and GDP derivatives that can be covalently linked with strategically placed cysteines within the GTPase of interest. Binding studies with GTPase-interacting proteins and X-ray crystallography analysis demonstrate that the molecular properties of the covalent GTPase-acryl-nucleotide adducts are a faithful reflection of those of the corresponding native states and are advantageously permanently locked in a defined nucleotide (that is active or inactive) state. In a first application, in vivo experiments using covalently locked Rab5 variants provide new insights into the mechanism of correct intracellular localization of Rab proteins.

  5. Composition of free and peptide-bound amino acids in beef chuck, loin, and round cuts.

    PubMed

    Wu, G; Cross, H R; Gehring, K B; Savell, J W; Arnold, A N; McNeill, S H

    2016-06-01

    Meat is a food for humans. However, beef consumption in the United States has steadily declined by >14% over the past decade due to a variety of factors, including insufficient knowledge of animal protein. This study quantified all proteinogenic AA as well as nutritionally and physiologically significant nonproteinogenic AA and small peptides in beef cuts from 3 subprimals (chuck, round, and loin). Beef carcasses ( = 10) were selected at 3 commercial packing plants in the United States. Retail-cut samples were analyzed for the nitrogenous substances after acid, alkaline, or enzymatic hydrolysis and after deproteinization. In these chuck, round, and loin cuts, total amounts of glutamate (free plus peptide bound) were the highest (69-75 mg/g dry weight) followed by lysine, leucine, arginine, and glutamine in descending order. This is the first study to determine aspartate, asparagine, glutamate, and glutamine in meat proteins of any animal species. In all the beef samples evaluated, glutamine was the most abundant free AA (4.0-5.7 mg/g dry weight) followed by taurine, alanine, glutamate, and β-alanine. Additionally, samples from all beef cuts had high concentrations of anserine, carnosine, and glutathione, which were 2.8 to 3.7, 15.2 to 24.2, and 0.68 to 0.79 mg/g dry weight, respectively. Beef top loin steaks appear to provide higher protein nutrition values than top round steaks and under blade roasts, but all are excellent sources of proteinogenic AA as well as antioxidant AA and peptides to improve human growth, development, and health. Our findings may help guide future decisions regarding human and animal nutrition. PMID:27285936

  6. Covalency in oxidized uranium

    NASA Astrophysics Data System (ADS)

    Tobin, J. G.; Yu, S.-W.; Qiao, R.; Yang, W. L.; Booth, C. H.; Shuh, D. K.; Duffin, A. M.; Sokaras, D.; Nordlund, D.; Weng, T.-C.

    2015-07-01

    Using x-ray emission spectroscopy and absorption spectroscopy, it has been possible to directly access the states in the unoccupied conduction bands that are involved with 5 f and 6 d covalency in oxidized uranium. By varying the oxidizing agent, the degree of 5 f covalency can be manipulated and monitored, clearly and irrevocably establishing the importance of 5 f covalency in the electronic structure of the key nuclear fuel, uranium dioxide.

  7. Development and Application of Pyrolysis Gas Chromatography/Mass Spectrometry for the Analysis of Bound Trinitrotoluene Residues in Soil

    USGS Publications Warehouse

    Weiss, J.M.; Mckay, A.J.; Derito, C.; Watanabe, C.; Thorn, K.A.; Madsen, E.L.

    2004-01-01

    TNT (trinitrotoluene) is a contaminant of global environmental significance, yet determining its environmental fate has posed longstanding challenges. To date, only differential extraction-based approaches have been able to determine the presence of covalently bound, reduced forms of TNT in field soils. Here, we employed thermal elution, pyrolysis, and gas chromatography/mass spectrometry (GC/MS) to distinguish between covalently bound and noncovalently bound reduced forms of TNT in soil. Model soil organic matter-based matrixes were used to develop an assay in which noncovalently bound (monomeric) aminodinitrotoluene (ADNT) and diaminonitrotoluene (DANT) were desorbed from the matrix and analyzed at a lower temperature than covalently bound forms of these same compounds. A thermal desorption technique, evolved gas analysis, was initially employed to differentiate between covalently bound and added 15N-labeled monomeric compounds. A refined thermal elution procedure, termed "double-shot analysis" (DSA), allowed a sample to be sequentially analyzed in two phases. In phase 1, all of an added 15N-labeled monomeric contaminant was eluted from the sample at relatively low temperature. In phase 2 during high-temperature pyrolysis, the remaining covalently bound contaminants were detected. DSA analysis of soil from the Louisiana Army Ammunition Plant (LAAP; ???5000 ppm TNT) revealed the presence of DANT, ADNT, and TNT. After scrutinizing the DSA data and comparing them to results from solvent-extracted and base/acid-hydrolyzed LAAP soil, we concluded that the TNT was a noncovalently bound "carryover" from phase 1. Thus, the pyrolysis-GC/MS technique successfully defined covalently bound pools of ADNT and DANT in the field soil sample.

  8. Spermatozoa bound to solid state hyaluronic acid show chromatin structure with high DNA chain integrity: an acridine orange fluorescence study.

    PubMed

    Yagci, Artay; Murk, William; Stronk, Jill; Huszar, Gabor

    2010-01-01

    During human spermiogenesis, the elongated spermatids undergo a plasma membrane remodeling step that facilitates formation of the zona pellucida and hyaluronic acid (HA) binding sites. Various biochemical sperm markers indicated that human sperm bound to HA exhibit attributes similar to that of zona pellucida-bound sperm, including minimal DNA fragmentation, normal shape, and low frequency of chromosomal aneuploidies. In this work, we tested the hypothesis that HA-bound sperm would be enhanced in sperm of high DNA chain integrity and green acridine orange fluorescence (AOF) compared with the original sperm in semen. Sperm DNA integrity in semen and in their respective HA-bound sperm fractions was studied in 50 men tested for fertility. In the semen samples, the proportions of sperm with green AOF (high DNA integrity) and red AOF (DNA breaks) were 54.9% ± 2.0% and 45.0% ± 1.9%, whereas in the HA-bound sperm fraction, the respective proportions were 99% and 1.0%, respectively. The data indeed demonstrated that HA shows a high degree of selectivity for sperm with high DNA integrity. These findings are important from the points of view of human sperm DNA integrity, sperm function, and the potential efficacy of HA-mediated sperm selection for intracytoplasmic sperm injection. PMID:20133967

  9. Comparative immunogenicity of conjugates composed of Escherichia coli O111 O-specific polysaccharide, prepared by treatment with acetic acid or hydrazine, bound to tetanus toxoid by two synthetic schemes.

    PubMed Central

    Gupta, R K; Egan, W; Bryla, D A; Robbins, J B; Szu, S C

    1995-01-01

    Escherichia coli O111, of various H types and virulence factors, causes enteritis throughout the world, especially in young children. This O type is found rarely in healthy individuals. Serum antibodies to the O-specific polysaccharide of O111 lipopolysaccharide (LPS) protect mice and dogs against infection with this E. coli serotype. The O111 O-specific polysaccharide is composed of a pentasaccharide repeat unit with two colitoses bound to the C-3 and C-6 of glucose in a trisaccharide backbone; this structure is identical to that of Salmonella adelaide (O35), another enteric pathogen. Nonpyrogenic O111 O-specific polysaccharide was prepared by treatment of its LPS with acetic acid (O-SP) or the organic base hydrazine (DeA-LPS). The O-SP had a reduced concentration of colitose. These products were derivatized with adipic acid dihydrazide (ADH) or thiolated with N-succinimidyl-3(2-pyridyldithio) propionate (SPDP). The four derivatives were covalently bound to tetanus toxoid (TT) by carbodiimide-mediated condensation or with SPDP to form conjugates. Immunization of BALB/c and general-purpose mice by a clinically acceptable route showed that DeA-LPS-TTADH, of the four conjugates, elicited the highest level of LPS antibodies. Possible reasons to explain this differential immunogenicity between the four conjugates are discussed. PMID:7542631

  10. Toward "stable-on-the-table" enzymes: improving key properties of catalase by covalent conjugation with poly(acrylic acid).

    PubMed

    Riccardi, Caterina M; Cole, Kyle S; Benson, Kyle R; Ward, Jessamyn R; Bassett, Kayla M; Zhang, Yiren; Zore, Omkar V; Stromer, Bobbi; Kasi, Rajeswari M; Kumar, Challa V

    2014-08-20

    Several key properties of catalase such as thermal stability, resistance to protease degradation, and resistance to ascorbate inhibition were improved, while retaining its structure and activity, by conjugation to poly(acrylic acid) (PAA, Mw 8000) via carbodiimide chemistry where the amine groups on the protein are appended to the carboxyl groups of the polymer. Catalase conjugation was examined at three different pH values (pH 5.0, 6.0, and 7.0) and at three distinct mole ratios (1:100, 1:500, and 1:1000) of catalase to PAA at each reaction pH. The corresponding products are labeled as Cat-PAA(x)-y, where x is the protein to polymer mole ratio and y is the pH used for the synthesis. The coupling reaction consumed about 60-70% of the primary amines on the catalase; all samples were completely water-soluble and formed nanogels, as evidenced by gel electrophoresis and electron microscopy. The UV circular dichroism (CD) spectra indicated substantial retention of protein secondary structure for all samples, which increased to 100% with increasing pH of the synthesis and polymer mole fraction. Soret CD bands of all samples indicated loss of ∼50% of band intensities, independent of the reaction pH. Catalytic activities of the conjugates increased with increasing synthesis pH, where 55-80% and 90-100% activity was retained for all samples synthesized at pH 5.0 and pH 7.0, respectively, and the Km or Vmax values of Cat-PAA(100)-7 did not differ significantly from those of the free enzyme. All conjugates synthesized at pH 7.0 were thermally stable even when heated to ∼85-90 °C, while native catalase denatured between 55 and 65 °C. All conjugates retained 40-90% of their original activities even after storing for 10 weeks at 8 °C, while unmodified catalase lost all of its activity within 2 weeks, under similar storage conditions. Interestingly, PAA surrounding catalase limited access to the enzyme from large molecules like proteases and significantly increased

  11. Covalent modification of proteins by cocaine

    NASA Astrophysics Data System (ADS)

    Deng, Shi-Xian; Bharat, Narine; Fischman, Marian C.; Landry, Donald W.

    2002-03-01

    Cocaine covalently modifies proteins through a reaction in which the methyl ester of cocaine acylates the -amino group of lysine residues. This reaction is highly specific in vitro, because no other amino acid reacts with cocaine, and only cocaine's methyl ester reacts with the lysine side chain. Covalently modified proteins were present in the plasma of rats and human subjects chronically exposed to cocaine. Modified endogenous proteins are immunogenic, and specific antibodies were elicited in mouse and detected in the plasma of human subjects. Covalent modification of proteins could explain cocaine's autoimmune effects and provide a new biochemical approach to cocaine's long-term actions.

  12. Gambogic acid covalently modifies IκB-kinase-β subunit to mediate suppression of lipopolysaccharide-induced activation of NF-κB in macrophages*

    PubMed Central

    Palempalli, Umamaheshwari D.; Gandhi, Ujjawal; Kalantari, Parisa; Vunta, Hema; Arner, Ryan J.; Narayan, Vivek; Ravindran, Anand; Prabhu, K. Sandeep

    2009-01-01

    Synopsis Gambogic acid (GA) is a polyprenylated xanthone abundant in the resin of Garcinia morella and G. hanburyi with a long history of use as a complementary and alternative medicine. The anti-tumor activity of GA has been well demonstrated and is thought to arise partly from the associated anti-inflammatory activity. Recent studies have indicated that the anti-tumor activity of GA is mediated by its ligation of the transferrin receptor TfR1. Since the cellular expression of TfR1 is down-regulated by lipopolysaccharide (LPS), we hypothesized that an alternative pathway exists in immune cells, such as macrophages, where GA could mitigate the expression of pro-inflammatory genes. Here we demonstrate that GA inhibits the LPS-dependent expression of nuclear factor-κB (NF-κB) target pro-inflammatory genes in macrophages. Western immunoblot, NF-κB luciferase reporter, and gel shift analyses revealed that GA strongly blocked the activation of NF-κB induced by LPS; while 9,10-dihydroGA that lacks the reactive α,β-unsaturated carbonyl group was ineffective. Moreover, GA was able to decrease nuclear p65 levels in RAW264.7 macrophages, where the expression of TfR1 was down-regulated by RNA interference. In-vitro kinase assays coupled with interaction studies using biotinylated GA as well as proteomic analysis demonstrated that IKKβ, a key kinase of the NF-κB signaling axis, was covalently modified by GA at Cys179 causing significant inhibition of its kinase activity. Taken together, these data demonstrate the potent anti-inflammatory activity of GA. PMID:19140805

  13. Gambogic acid covalently modifies IkappaB kinase-beta subunit to mediate suppression of lipopolysaccharide-induced activation of NF-kappaB in macrophages.

    PubMed

    Palempalli, Umamaheshwari D; Gandhi, Ujjawal; Kalantari, Parisa; Vunta, Hema; Arner, Ryan J; Narayan, Vivek; Ravindran, Anand; Prabhu, K Sandeep

    2009-04-15

    GA (gambogic acid) is a polyprenylated xanthone abundant in the resin of Garcinia morella and Garcinia hanburyi with a long history of use as a complementary and alternative medicine. The antitumour activity of GA has been well demonstrated and is thought to arise partly from the associated anti-inflammatory activity. Recent studies have indicated that the antitumour activity of GA is mediated by its ligation of TfR1 (transferrin receptor-1). Since the cellular expression of TfR1 is down-regulated by LPS (lipopolysaccharide), we hypothesized that an alternative pathway exists in immune cells, such as macrophages, where GA could mitigate the expression of pro-inflammatory genes. Here we demonstrate that GA inhibits the LPS-dependent expression of NF-kappaB (nuclear factor kappaB) target pro-inflammatory genes in macrophages. Western immunoblot, NF-kappaB-luciferase reporter and gel-shift analyses revealed that GA strongly blocked the activation of NF-kappaB induced by LPS, whereas 9,10-dihydro-GA, which lacks the reactive alpha,beta-unsaturated carbonyl group, was ineffective. Moreover, GA was able to decrease nuclear p65 levels in RAW264.7 macrophages, where the expression of TfR1 was down-regulated by RNA interference. in vitro kinase assays coupled with interaction studies using biotinylated GA as well as proteomic analysis demonstrated that IKKbeta [IkappaB (inhibitory kappaB) kinase-beta], a key kinase of the NF-kappaB signalling axis, was covalently modified by GA at Cys-179, causing significant inhibition of its kinase activity. Taken together, these results demonstrate the potent anti-inflammatory activity of GA. PMID:19140805

  14. Covalent Chemistry beyond Molecules.

    PubMed

    Jiang, Juncong; Zhao, Yingbo; Yaghi, Omar M

    2016-03-16

    Linking molecular building units by covalent bonds to make crystalline extended structures has given rise to metal-organic frameworks (MOFs) and covalent organic frameworks (COFs), thus bringing the precision and versatility of covalent chemistry beyond discrete molecules to extended structures. The key advance in this regard has been the development of strategies to overcome the "crystallization problem", which is usually encountered when attempting to link molecular building units into covalent solids. Currently, numerous MOFs and COFs are made as crystalline materials in which the large size of the constituent units provides for open frameworks. The molecular units thus reticulated become part of a new environment where they have (a) lower degrees of freedom because they are fixed into position within the framework; (b) well-defined spatial arrangements where their properties are influenced by the intricacies of the pores; and (c) ordered patterns onto which functional groups can be covalently attached to produce chemical complexity. The notion of covalent chemistry beyond molecules is further strengthened by the fact that covalent reactions can be carried out on such frameworks, with full retention of their crystallinity and porosity. MOFs are exemplars of how this chemistry has led to porosity with designed metrics and functionality, chemically-rich sequences of information within their frameworks, and well-defined mesoscopic constructs in which nanoMOFs enclose inorganic nanocrystals and give them new levels of spatial definition, stability, and functionality. PMID:26863450

  15. Identification of albumin-bound fatty acids as the major factor in serum-induced lipid accumulation by cultured cells.

    PubMed

    Mackenzie, C G; Mackenzie, J B; Reiss, O K; Wisneski, J A

    1970-11-01

    Factors responsible for the high lipogenic activity of rabbit serum were investigated using an assay procedure based on the gravimetric determination of the 24 hr increase in cell lipid. Cellular synthesis of fatty acids was inhibited by the presence of serum in the assay medium. Approximately 90% of the increase in cell lipid produced by serum fractions was due to triglyceride accumulation. Fractionation of rabbit serum by precipitation with ammonium sulfate or by ultracentrifugation in high density medium, both indicated that three-quarters of its lipogenic activity was associated with albumin. The lipoproteins prepared by ultracentrifugation also exhibited about one-half the activity of whole serum. The lipogenic activity of albumin was confirmed by the high potency of the albumin isolated in a nearly pure form from proteins of d>1.21 by precipitation with trichloroacetic acid and extraction with ethanol. As judged from chemical and isotopic analysis, neither the lipid content nor the lipid composition of the albumin was appreciably altered during its isolation. Of the albumin-bound lipids, only the free fatty acids, as determined by DEAE column chromatography, were present in an amount sufficient to account for the observed increase in cell triglycerides. In control experiments with horse serum of low lipogenic activity, the proteins of d>1.21 also possessed low activity in conjunction with a low content of free fatty acid. However, the albumin isolated from the latter preparation exhibited the high lipogenic activity of rabbit serum albumin. Chemical and isotopic analysis of the recovered horse serum albumin revealed that its free fatty acid content was the same as that of rabbit serum albumin. These results indicated that the isolation of horse serum albumin was attended by a substantial increase in its free fatty acid content. When the rabbit serum and horse serum content of media were adjusted to provide equivalent concentrations of albumin-bound fatty

  16. Acid-base properties of ionophore A23187 in methanol-water solutions and bound to unilamellar vesicles of dimyristoylphosphatidylcholine.

    PubMed

    Kauffman, R F; Taylor, R W; Pfeiffer, D R

    1982-05-11

    The acid-base properties of ionophore A23187 in methanol-water solutions (0--95% w/w) and bound to unilamellar vesicles of dimyristoylphosphatidylcholine were examined by ultraviolet and fluorescence spectroscopy, and the spectral properties for the acidic and basic forms were defined under these conditions. Standard mixed-solvent buffers were employed to calibrate pH measurement in the methanol-water solvents. In 65% methanol-water, two protonation equilibria were observed, the most basic of which displayed a value for the logarithm of the protonation constant (log KH) of 7.19 +/- 0.05 at 25 degrees C and 0.05 M ionic strength. Instability of A23187 was encountered below pH approximately 4; however, decomposition was slow enough to allow log KH for the more acidic equilibrium to be estimated as 1.28. Comparison of these results to those obtained with the methyl ester of A23187 (log KH = 1.32) and literature values for other model compounds allowed assignment of the more basic equilibrium to the carboxylic acid moiety and the more acidic one to the N-methylamino substituent of the benzoxazole ring. log KH of the carboxylic acid increased from 5.69 +/- 0.05 to 9.37 +/- 0.05 over the range of solvent polarity encompassed by water to 95% methanol-water. Values for the ground state (absorption) and first excited state (fluorescence) were equal within experimental error. The logarithm of the protonation constant for the membrane-bound ionophore, measured under conditions where the surface potential generated by ionization did not significantly alter the equilibrium, was found to be 7.85 +/- 0.05 at 25 degrees C and at ionic strength of 0.05 M in the aqueous phase. The value agrees with that observed in 80% methanol-water, as does the wavelength of maximum fluorescence emission for the membrane-bound free acid. An interfacial location for the monoprotonated form of the benzoxazolate moiety is proposed, both above and below the membrane phase transition temperature. The

  17. Hydrogen Isotopic Composition of Particulate-Bound Fatty Acids From the California Borderland Basins

    NASA Astrophysics Data System (ADS)

    Jones, A. A.; Sessions, A. L.; Campbell, B. J.; Valentine, D. L.

    2006-12-01

    We examined the hydrogen-isotopic composition of fatty acids associated with particulate organic matter (POM) from depth transects in three California Borderland stations. Our goals were to determine (1) the natural variability of δD values in POM-associated fatty acids and (2) the magnitude of isotopic fractionations associated with fatty acid degradation in the marine environment. Some differences in molecular abundance were observed between completely ventilated and occasionally suboxic sites, but no corresponding shifts in δD values were measured. Values of δD for specific fatty acids were generally consistent between stations. Saturated fatty acids (C14, C16, and C18) yielded δD values ranging from -230‰ to -132‰, with δD values generally decreasing with chain length. We found no evidence of extreme D-enrichment of the C18 fatty acid as has been observed in studies of isolated macroalgae (Chikaraishi, et al, 2004). The unsaturated C16 and C18 fatty acids showed a similar trend while the polyunsaturated fatty acid 22:6 was somewhat enriched in D (δD values ranging from -186‰ to -68‰) relative to 20:5 (-208‰ to -93‰). Unsaturated fatty acids tended to have more positive δD values than their saturated counterparts, opposite the trend observed in sediments from the same location. The bacterial fatty acid C15 showed even greater deuterium enrichment with δD values ranging from - 145‰ to -88‰. This offset can likely be attributed to differences in biosynthetic fractionation between bacteria and eukaryotes, to differences in hydrogen isotopic composition of the food sources of these organisms, or some combination of these two factors. Within the surface waters, fatty acids become enriched with depth by an average of 25‰. The C18:0 acid is a significant exception, becoming depleted by 48‰ over that same interval. Below 100 meters depth, all fatty acids tend to become slightly depleted in D with increasing depth. The difference in δD values

  18. Renoprotective effect of DPP-4 inhibitors against free fatty acid-bound albumin-induced renal proximal tubular cell injury.

    PubMed

    Tanaka, Yuki; Kume, Shinji; Chin-Kanasaki, Masami; Araki, Hisazumi; Araki, Shin-ichi; Ugi, Satoshi; Sugaya, Takeshi; Uzu, Takashi; Maegawa, Hiroshi

    2016-02-12

    Dipeptidyl peptidase (DPP)-4 inhibitors, a new class of antidiabetic agent, have recently been suggested to exert pleiotropic effects beyond glucose lowering. Renal prognosis in patients with diabetic nephropathy depends on the severity of tubulointerstitial injury induced by massive proteinuria. We thus examined the renoprotective effect of DPP-4 inhibitors on inflammation in cultured mouse proximal tubular cells stimulated with free fatty acid (FFA)-bound albumin. Linagliptin and higher concentrations of sitagliptin, vildagliptin, and alogliptin all inhibited FFA-bound albumin-induced increases in mRNA expression of MCP-1 in cultured mouse proximal tubular cells. Furthermore, linagliptin significantly inhibited tubulointerstitial injury induced by peritoneal injection of FFA-bound albumin, such as inflammation, fibrosis, and apoptosis, in mice without altering systemic characteristics including body weight, fasting blood glucose, and food intake. These results indicate that DPP-4 inhibitors pleiotropically exert a direct renoprotective effect, and may serve as an additional therapeutic strategy to protect proximal tubular cells against proteinuria in patients with diabetic nephropathy. PMID:26802469

  19. Ether- and Ester-Bound iso-Diabolic Acid and Other Lipids in Members of Acidobacteria Subdivision 4

    PubMed Central

    Rijpstra, W. Irene C.; Hopmans, Ellen C.; Foesel, Bärbel U.; Wüst, Pia K.; Overmann, Jörg; Tank, Marcus; Bryant, Donald A.; Dunfield, Peter F.; Houghton, Karen; Stott, Matthew B.

    2014-01-01

    Recently, iso-diabolic acid (13,16-dimethyl octacosanedioic acid) has been identified as a major membrane-spanning lipid of subdivisions 1 and 3 of the Acidobacteria, a highly diverse phylum within the Bacteria. This finding pointed to the Acidobacteria as a potential source for the bacterial glycerol dialkyl glycerol tetraethers that occur ubiquitously in peat, soil, lakes, and hot springs. Here, we examined the lipid composition of seven phylogenetically divergent strains of subdivision 4 of the Acidobacteria, a bacterial group that is commonly encountered in soil. Acid hydrolysis of total cell material released iso-diabolic acid derivatives in substantial quantities (11 to 48% of all fatty acids). In contrast to subdivisions 1 and 3 of the Acidobacteria, 6 out of the 7 species of subdivision 4 (excepting “Candidatus Chloracidobacterium thermophilum”) contained iso-diabolic acid ether bound to a glycerol in larger fractional abundance than iso-diabolic acid itself. This is in agreement with the analysis of intact polar lipids (IPLs) by high-performance liquid chromatography-mass spectrometry (HPLC-MS), which showed the dominance of mixed ether-ester glycerides. iso-Diabolic acid-containing IPLs were not identified, because these IPLs are not released with a Bligh-Dyer extraction, as observed before when studying lipid compositions of subdivisions 1 and 3 of the Acidobacteria. The presence of ether bonds in the membrane lipids does not seem to be an adaptation to temperature, because the five mesophilic isolates contained a larger amount of ether lipids than the thermophile “Ca. Chloracidobacterium thermophilum.” Furthermore, experiments with Pyrinomonas methylaliphatogenes did not reveal a major influence of growth temperature over the 50 to 69°C range. PMID:24928878

  20. A heteromeric membrane-bound prenyltransferase complex from hop catalyzes three sequential aromatic prenylations in the bitter acid pathway.

    PubMed

    Li, Haoxun; Ban, Zhaonan; Qin, Hao; Ma, Liya; King, Andrew J; Wang, Guodong

    2015-03-01

    Bitter acids (α and β types) account for more than 30% of the fresh weight of hop (Humulus lupulus) glandular trichomes and are well known for their contribution to the bitter taste of beer. These multiprenylated chemicals also show diverse biological activities, some of which have potential benefits to human health. The bitter acid biosynthetic pathway has been investigated extensively, and the genes for the early steps of bitter acid synthesis have been cloned and functionally characterized. However, little is known about the enzyme(s) that catalyze three sequential prenylation steps in the β-bitter acid pathway. Here, we employed a yeast (Saccharomyces cerevisiae) system for the functional identification of aromatic prenyltransferase (PT) genes. Two PT genes (HlPT1L and HlPT2) obtained from a hop trichome-specific complementary DNA library were functionally characterized using this yeast system. Coexpression of codon-optimized PT1L and PT2 in yeast, together with upstream genes, led to the production of bitter acids, but no bitter acids were detected when either of the PT genes was expressed by itself. Stepwise mutation of the aspartate-rich motifs in PT1L and PT2 further revealed the prenylation sequence of these two enzymes in β-bitter acid biosynthesis: PT1L catalyzed only the first prenylation step, and PT2 catalyzed the two subsequent prenylation steps. A metabolon formed through interactions between PT1L and PT2 was demonstrated using a yeast two-hybrid system, reciprocal coimmunoprecipitation, and in vitro biochemical assays. These results provide direct evidence of the involvement of a functional metabolon of membrane-bound prenyltransferases in bitter acid biosynthesis in hop. PMID:25564559

  1. Synergistic transcriptional enhancement does not depend on the number of acidic activation domains bound to the promoter.

    PubMed Central

    Oliviero, S; Struhl, K

    1991-01-01

    Many eukaryotic transcriptional activator proteins contain a DNA-binding domain that interacts with specific promoter sequences and an acidic activation region that is required to stimulate transcription. Transcriptional enhancement by such activator proteins is often synergistic and promiscuous; promoters containing multiple binding sites for an individual protein or even for unrelated proteins can be 10-100 times more active than promoters with single sites. It has been suggested that such synergy reflects a nonlinear response of the basic transcription machinery to the number and/or quality of acidic activation regions. Here, we determine the transcriptional activity of Jun-Fos heterodimers containing one or two GCN4 acidic activation regions on promoters containing one or two Ap-1 target sites. Surprisingly, heterodimers with one or two acidic regions activate transcription with similar efficiency and are equally synergistic (10- to 15-fold) on promoters containing two target sites. Thus, transcriptional synergy does not depend on the number of acidic activation regions but rather on the number of proteins bound to the promoter. This suggests that synergy is mediated either by cooperative DNA binding or by alternative mechanisms in which the DNA-binding domain plays a more direct role in transcription (e.g., changes in DNA structure, nucleosome displacement, or direct interactions with the transcriptional machinery). Images PMID:1898773

  2. Development and validation of an HPLC-method for determination of free and bound phenolic acids in cereals after solid-phase extraction.

    PubMed

    Irakli, Maria N; Samanidou, Victoria F; Biliaderis, Costas G; Papadoyannis, Ioannis N

    2012-10-01

    Whole cereal grains are a good source of phenolic acids associated with reduced risk of chronic diseases. This paper reports the development and validation of a high-performance liquid chromatography-diode array detection (HPLC-DAD) method for the determination of phenolic acids in cereals in either free or bound form. Extraction of free phenolic acids and clean-up was performed by an optimised solid-phase extraction (SPE) protocol on Oasis HLB cartridges using aqueous methanol as eluant. The mean recovery of analytes ranged between 84% and 106%. Bound phenolic acids were extracted using alkaline hydrolysis with mean recoveries of 80-95%, except for gallic acid, caffeic acid and protocatechuic acid. Both free and bound phenolic extracts were separated on a Nucleosil 100 C18 column, 5 μm (250 mm × 4.6 mm) thermostated at 30 °C, using a linear gradient elution system consisting of 1% (v/v) acetic acid in methanol. Method validation was performed by means of linearity, accuracy, intra-day and inter-day precision and sensitivity. Detection limits ranged between 0.13 and 0.18 μg/g. The method was applied to the analysis of free and bound phenolic acids contents in durum wheat, bread wheat, barley, oat, rice, rye, corn and triticale. PMID:25005991

  3. Identification/quantification of free and bound phenolic acids in peel and pulp of apples (Malus domestica) using high resolution mass spectrometry (HRMS).

    PubMed

    Lee, Jihyun; Chan, Bronte Lee Shan; Mitchell, Alyson E

    2017-01-15

    Free and bound phenolic acids were measured in the pulp and peel of four varieties of apples using high resolution mass spectrometry. Twenty-five phenolic acids were identified and included: 8 hydroxybenzoic acids, 11 hydroxycinnamic acids, 5 hydroxyphenylacetic acids, and 1 hydoxyphenylpropanoic acid. Several phenolics are tentatively identified for the first time in apples and include: methyl gallate, ethyl gallate, hydroxy phenyl acetic acid, three phenylacetic acid isomers, 3-(4-hydroxyphenyl)propionic acid, and homoveratric acid. With exception of chlorogenic and caffeic acid, most phenolic acids were quantified for the first time in apples. Significant varietal differences (p<0.05) were observed in both peel and pulp. The levels of total phenolic acids were higher in the pulp as compared to apple peel (dry weight) in all varieties. Coumaroylquinic, protocatechuic, 4-hydroxybenzoic, vanillic and t-ferulic acids were present in free forms. With exception of chlorogenic acid, all other phenolic acids were present only as bound forms. PMID:27542479

  4. Determination of seleno-amino acids bound to proteins in extra virgin olive oils.

    PubMed

    Torres, Sabier; Gil, Raul; Silva, María Fernanda; Pacheco, Pablo

    2016-04-15

    An analytical method has been developed to determine seleno-amino acids in proteins extracted from extra virgin olive oils (EVOOs). Different aqueous/organic solvents were tested to isolate proteins, an acetone:n-hexane combination being the best protein precipitant. In a first dimension chromatography, extracted proteins were analysed by size exclusion chromatography (SEC) coupled to inductively coupled plasma mass spectrometry (ICP-MS) to identify S and Se associations as proteins marker. Two fractions of 66 kDa (A) and 443 kDa (B) were identified. These fractions were submitted to microwave-assisted acid hydrolysis (MAAH) to release seleno-amino acids. In a second dimension chromatography seleno-amino acids were determined by reversed-phase chromatography (RPC) coupled to ICP-MS. Seleno-methylselenocysteine was determined with values ranging from 1.03-2.03±0.2 μg kg(-1) and selenocysteine at a concentration of 1.47±0.1 μg kg(-1). Variations of protein and seleno-amino acid concentrations were observed between EVOO varieties, contributing to EVOO cultivar differentiation. PMID:26616967

  5. Amino acids form strongly bound anions when substituted with superhalogen ligands

    NASA Astrophysics Data System (ADS)

    Sieradzan, Iwona; Anusiewicz, Iwona

    2013-04-01

    The properties of AA-Y- anions (where AA = cysteine, aspartic acid, lysine; Y = BF3, PF5) were investigated at the ab initio Outer Valence Green's Function (OVGF)/6-311++G(3df,3pd)//MP2/6-311++G(d,p) level of theory. It is shown that introducing a superhalogen-like substituent to an amino acid (i.e., Cys, Asp, and Lys) results in obtaining molecules that bind an excess electron relatively strongly. The electronic stabilities of such resulting daughter anions are predicted to be substantial (5.3-6.9 eV).

  6. Binding of the Covalent Flavin Assembly Factor to the Flavoprotein Subunit of Complex II.

    PubMed

    Maklashina, Elena; Rajagukguk, Sany; Starbird, Chrystal A; McDonald, W Hayes; Koganitsky, Anna; Eisenbach, Michael; Iverson, Tina M; Cecchini, Gary

    2016-02-01

    Escherichia coli harbors two highly conserved homologs of the essential mitochondrial respiratory complex II (succinate:ubiquinone oxidoreductase). Aerobically the bacterium synthesizes succinate:quinone reductase as part of its respiratory chain, whereas under microaerophilic conditions, the quinol:fumarate reductase can be utilized. All complex II enzymes harbor a covalently bound FAD co-factor that is essential for their ability to oxidize succinate. In eukaryotes and many bacteria, assembly of the covalent flavin linkage is facilitated by a small protein assembly factor, termed SdhE in E. coli. How SdhE assists with formation of the covalent flavin bond and how it binds the flavoprotein subunit of complex II remain unknown. Using photo-cross-linking, we report the interaction site between the flavoprotein of complex II and the SdhE assembly factor. These data indicate that SdhE binds to the flavoprotein between two independently folded domains and that this binding mode likely influences the interdomain orientation. In so doing, SdhE likely orients amino acid residues near the dicarboxylate and FAD binding site, which facilitates formation of the covalent flavin linkage. These studies identify how the conserved SdhE assembly factor and its homologs participate in complex II maturation. PMID:26644464

  7. Comparison of enzymatic and acid hydrolysis of bound flavor compounds in model system and grapes.

    PubMed

    Dziadas, Mariusz; Jeleń, Henryk H

    2016-01-01

    Four synthesized terpenyl-ß-D-glycopyranosides (geranyl, neryl, citronellyl, myrtenyl) were subjected to enzymatic (AR 2000, pH 5.5) and acid (citric buffer, pH 2.5) hydrolysis. Decrease of glycosides was measured by HPLC and the volatiles released--by comprehensive gas chromatography-mass spectrometry (GC × GC-ToF-MS). Enzymatic hydrolysis performed for 21 h yielded 100% degree of hydrolysis for all glycosides but citronellyl (97%). Degree of acid hydrolysis was highly dependent on type of aglycone and the conditions. The highest degree was achieved for geraniol, followed by citronellol and nerol. Myrtenylo-ß-D-glycopyranoside was the most resistant glycoside to hydrolysis. Acid hydrolysis degree was also related to temperature/time combination, the highest being for 100 °C and 2 h. In a result of enzymatic hydrolysis 85-91% of total peak areas was terpene aglycone, whereas for acid hydrolysis the area of released terpene aglycone did not exceed 1.3% of total peak area indicating almost complete decomposition/transformation of terpenyl aglycone. PMID:26212990

  8. Covalent organic frameworks.

    PubMed

    Feng, Xiao; Ding, Xuesong; Jiang, Donglin

    2012-09-21

    Covalent organic frameworks (COFs) are a class of crystalline porous polymers that allow the atomically precise integration of organic units to create predesigned skeletons and nanopores. They have recently emerged as a new molecular platform for designing promising organic materials for gas storage, catalysis, and optoelectronic applications. The reversibility of dynamic covalent reactions, diversity of building blocks, and geometry retention are three key factors involved in the reticular design and synthesis of COFs. This tutorial review describes the basic design concepts, the recent synthetic advancements and structural studies, and the frontiers of functional exploration. PMID:22821129

  9. Identification of amino acid residues that determine the substrate specificity of mammalian membrane-bound front-end fatty acid desaturases.

    PubMed

    Watanabe, Kenshi; Ohno, Makoto; Taguchi, Masahiro; Kawamoto, Seiji; Ono, Kazuhisa; Aki, Tsunehiro

    2016-01-01

    Membrane-bound desaturases are physiologically and industrially important enzymes that are involved in the production of diverse fatty acids such as polyunsaturated fatty acids and their derivatives. Here, we identified amino acid residues that determine the substrate specificity of rat Δ6 desaturase (D6d) acting on linoleoyl-CoA by comparing its amino acid sequence with that of Δ5 desaturase (D5d), which converts dihomo-γ-linolenoyl-CoA. The N-terminal cytochrome b5-like domain was excluded as a determinant by domain swapping analysis. Substitution of eight amino acid residues (Ser209, Asn211, Arg216, Ser235, Leu236, Trp244, Gln245, and Val344) of D6d with the corresponding residues of D5d by site-directed mutagenesis switched the substrate specificity from linoleoyl-CoA to dihomo-γ-linolenoyl-CoA. In addition, replacement of Leu323 of D6d with Phe323 on the basis of the amino acid sequence of zebra fish Δ5/6 bifunctional desaturase was found to render D6d bifunctional. Homology modeling of D6d using recent crystal structure data of human stearoyl-CoA (Δ9) desaturase revealed that Arg216, Trp244, Gln245, and Leu323 are located near the substrate-binding pocket. To our knowledge, this is the first report on the structural basis of the substrate specificity of a mammalian front-end fatty acid desaturase, which will aid in efficient production of value-added fatty acids. PMID:26590171

  10. Covalent immobilization of liposomes on plasma functionalized metallic surfaces.

    PubMed

    Mourtas, S; Kastellorizios, M; Klepetsanis, P; Farsari, E; Amanatides, E; Mataras, D; Pistillo, B R; Favia, P; Sardella, E; d'Agostino, R; Antimisiaris, S G

    2011-05-01

    A method was developed to functionalize biomedical metals with liposomes. The novelty of the method includes the plasma-functionalization of the metal surface with proper chemical groups to be used as anchor sites for the covalent immobilization of the liposomes. Stainless steel (SS-316) disks were processed in radiofrequency glow discharges fed with vapors of acrylic acid to coat them with thin adherent films characterized by surface carboxylic groups, where liposomes were covalently bound through the formation of amide bonds. For this, liposomes decorated with polyethylene glycol molecules bearing terminal amine-groups were prepared. After ensuring that the liposomes remain intact, under the conditions applying for immobilization; different attachment conditions were evaluated (incubation time, concentration of liposome dispersion) for optimization of the technique. Immobilization of calcein-entrapping liposomes was evaluated by monitoring the percent of calcein attached on the surfaces. Best results were obtained when liposome dispersions with 5mg/ml (liposomal lipid) concentration were incubated on each disk for 24h at 37°C. The method is proposed for developing drug-eluting biomedical materials or devices by using liposomes that have appropriate membrane compositions and are loaded with drugs or other bioactive agents. PMID:21273051

  11. Covalent organic frameworks: Crossing the channel

    NASA Astrophysics Data System (ADS)

    Xu, Hong; Jiang, Donglin

    2014-07-01

    The ordered one-dimensional nanochannels found in covalent organic frameworks (COFs) could render them able to conduct protons. However, the frameworks' instability in acid has thus far precluded any practical implementations. Now, a strategy to overcome this instability has enabled proton conduction using a COF for the first time.

  12. Tissue Plasminogen Activator Binding to Superparamagnetic Iron Oxide Nanoparticle-Covalent Versus Adsorptive Approach.

    PubMed

    Friedrich, Ralf P; Zaloga, Jan; Schreiber, Eveline; Tóth, Ildikó Y; Tombácz, Etelka; Lyer, Stefan; Alexiou, Christoph

    2016-12-01

    Functionalized superparamagnetic iron oxide nanoparticles are frequently used to develop vehicles for drug delivery, hyperthermia, and photodynamic therapy and as tools used for magnetic separation and purification of proteins or for biomolecular imaging. Depending on the application, there are various possible covalent and non-covalent approaches for the functionalization of particles, each of them shows different advantages and disadvantages for drug release and activity at the desired location.Particularly important for the production of adsorptive and covalent bound drugs to nanoparticles is the pureness of the involved formulation. Especially the covalent binding strategy demands defined chemistry of the drug, which is stabilized by excess free amino acids which could reduce reaction efficiency. In this study, we therefore used tangential flow filtration (TFF) method to purify the drugs before the reaction and used the frequently applied and clinically available recombinant tissue plasminogen activator (tPA; Actilyse(®)) as a proof of concept. We then coupled the tPA preparation to polyacrylic acid-co-maleic acid (PAM)-coated superparamagnetic iron oxide nanoparticles (SPIONs) using an amino-reactive activated ester reaction and compared these particles to PAM-coated SPIONs with electrostatically adsorbed tPA.Using dynamic light scattering (DLS) and pH-dependent electrokinetic mobility measurements, we showed that surface properties of the SPIONs were significantly greater affected after activation of the particles compared to the adsorption controls. Different in vitro assays were used to investigate the activity of tPA after coupling to the particles and purification of the ferrofluid. Covalent linkage significantly improves the reactivity and long-term stability of the conjugated SPION-tPA system compared to simple adsorption. In conclusion, we have shown an effective way to produce SPIONs with covalent and non-covalent ultra-filtrated drugs. We showed

  13. Uncertainty quantification and integration of machine learning techniques for predicting acid rock drainage chemistry: a probability bounds approach.

    PubMed

    Betrie, Getnet D; Sadiq, Rehan; Morin, Kevin A; Tesfamariam, Solomon

    2014-08-15

    Acid rock drainage (ARD) is a major pollution problem globally that has adversely impacted the environment. Identification and quantification of uncertainties are integral parts of ARD assessment and risk mitigation, however previous studies on predicting ARD drainage chemistry have not fully addressed issues of uncertainties. In this study, artificial neural networks (ANN) and support vector machine (SVM) are used for the prediction of ARD drainage chemistry and their predictive uncertainties are quantified using probability bounds analysis. Furthermore, the predictions of ANN and SVM are integrated using four aggregation methods to improve their individual predictions. The results of this study showed that ANN performed better than SVM in enveloping the observed concentrations. In addition, integrating the prediction of ANN and SVM using the aggregation methods improved the predictions of individual techniques. PMID:24852616

  14. Charge transport through dicarboxylic-acid-terminated alkanes bound to graphene-gold nanogap electrodes

    NASA Astrophysics Data System (ADS)

    Liu, Longlong; Zhang, Qian; Tao, Shuhui; Zhao, Cezhou; Almutib, Eman; Al-Galiby, Qusiy; Bailey, Steven W. D.; Grace, Iain; Lambert, Colin J.; Du, Jun; Yang, Li

    2016-07-01

    Graphene-based electrodes are attractive for single-molecule electronics due to their high stability and conductivity and reduced screening compared with metals. In this paper, we use the STM-based matrix isolation I(s) method to measure the performance of graphene in single-molecule junctions with one graphene electrode and one gold electrode. By measuring the length dependence of the electrical conductance of dicarboxylic-acid-terminated alkanes, we find that the transport is consistent with phase-coherent tunneling, but with an attenuation factor of βN = 0.69 per methyl unit, which is lower than the value measured for Au-molecule-Au junctions. Comparison with density-functional-theory calculations of electron transport through graphene-molecule-Au junctions and Au-molecule-Au junctions reveals that this difference is due to the difference in Fermi energies of the two types of junction, relative to the frontier orbitals of the molecules. For most molecules, their electrical conductance in graphene-molecule-Au junctions is higher than that in Au-molecule-Au junctions, which suggests that graphene offers superior electrode performance, when utilizing carboxylic acid anchor groups.Graphene-based electrodes are attractive for single-molecule electronics due to their high stability and conductivity and reduced screening compared with metals. In this paper, we use the STM-based matrix isolation I(s) method to measure the performance of graphene in single-molecule junctions with one graphene electrode and one gold electrode. By measuring the length dependence of the electrical conductance of dicarboxylic-acid-terminated alkanes, we find that the transport is consistent with phase-coherent tunneling, but with an attenuation factor of βN = 0.69 per methyl unit, which is lower than the value measured for Au-molecule-Au junctions. Comparison with density-functional-theory calculations of electron transport through graphene-molecule-Au junctions and Au

  15. Potent and selective activation of abscisic acid receptors in vivo by mutational stabilization of their agonist-bound conformation

    PubMed Central

    Mosquna, Assaf; Peterson, Francis C.; Park, Sang-Youl; Lozano-Juste, Jorge; Volkman, Brian F.; Cutler, Sean R.

    2011-01-01

    Pyrabactin resistance (PYR) 1 and its relatives belong to a family of soluble abscisic acid (ABA) receptors that inhibit type 2C protein phosphatases (PP2C) when in their agonist-stabilized conformation. Given their switch-like properties, we envisioned that mutations that stabilize their agonist-bound conformation could be used to activate signaling in vivo. To identify such mutations, we subjected PYR1 to site-saturation mutagenesis at 39 highly conserved residues that participate in ABA or PP2C contacts. All 741 possible single amino acid substitutions at these sites were tested to identify variants that increase basal PYR1-PP2C interactions, which uncovered activating mutations in 10 residues that preferentially cluster in PYR1's gate loop and C-terminal helix. The mutations cause measurable but incomplete receptor activation in vitro; however, specific triple and quadruple mutant combinations were constructed that promote an agonist-bound conformation, as measured by heteronuclear single quantum coherence NMR, and lead to full receptor activation. Moreover, these mutations retain functionality when introduced into divergent family members, and can therefore be used to dissect individual receptor function in vivo, which has been problematic because of redundancy and family size. Expression of activated PYL2 in Arabidopsis seeds activates ABA signaling by a number of measures: modulation of ABA-regulated gene expression, induction of hyperdormancy, and suppression of ABA deficiency phenotypes in the aba2-1 mutant. Our results set the stage for systematic gain-of-function studies of PYR1 and related ABA receptors and reveal that, despite the large number of receptors, activation of a single receptor is sufficient to activate signaling in planta. PMID:22139369

  16. Charge transport through dicarboxylic-acid-terminated alkanes bound to graphene-gold nanogap electrodes.

    PubMed

    Liu, Longlong; Zhang, Qian; Tao, Shuhui; Zhao, Cezhou; Almutib, Eman; Al-Galiby, Qusiy; Bailey, Steven W D; Grace, Iain; Lambert, Colin J; Du, Jun; Yang, Li

    2016-08-14

    Graphene-based electrodes are attractive for single-molecule electronics due to their high stability and conductivity and reduced screening compared with metals. In this paper, we use the STM-based matrix isolation I(s) method to measure the performance of graphene in single-molecule junctions with one graphene electrode and one gold electrode. By measuring the length dependence of the electrical conductance of dicarboxylic-acid-terminated alkanes, we find that the transport is consistent with phase-coherent tunneling, but with an attenuation factor of βN = 0.69 per methyl unit, which is lower than the value measured for Au-molecule-Au junctions. Comparison with density-functional-theory calculations of electron transport through graphene-molecule-Au junctions and Au-molecule-Au junctions reveals that this difference is due to the difference in Fermi energies of the two types of junction, relative to the frontier orbitals of the molecules. For most molecules, their electrical conductance in graphene-molecule-Au junctions is higher than that in Au-molecule-Au junctions, which suggests that graphene offers superior electrode performance, when utilizing carboxylic acid anchor groups. PMID:27412865

  17. Crystal Structure of Arachidonic Acid Bound to a Mutant of Prostaglandin Endoperoxide Synthase-1 that Forms Predominantly 11-HPETE

    SciTech Connect

    Harman, C.; Rieke, C.J.; Garavito, R.M.; Smith, W.L.

    2010-03-05

    Kinetic studies and analysis of the products formed by native and mutant forms of ovine prostaglandin endoperoxide H synthase-1 (oPGHS-1) have suggested that arachidonic acid (AA) can exist in the cyclooxygenase active site of the enzyme in three different, catalytically competent conformations that lead to prostaglandin G{sub 2} (PGG{sub 2}), 11Rhydroperoxyeicosatetraenoic acid (HPETE), and 15R,SHPETE, respectively. We have identified an oPGHS-1 mutant (V349A/W387F) that forms predominantly 11RHPETE. Thus, the preferred catalytically competent arrangement of AA in the cyclooxygenase site of this double mutant must be one that leads to 11-HPETE. The crystal structure of Co{sup 3+}-protoporphyrin IX V349A/W387F oPGHS-1 in a complex with AA was determined to 3.1 {angstrom}. Significant differences are observed in the positions of atoms C-3, C-4, C-5, C-6, C-10, C-11, and C-12 of bound AA between native and V349A/W387F oPGHS-1; in comparison, the positions of the side chains of cyclooxygenase active site residues are unchanged. The structure of the double mutant presented here provides structural insight as to how Val{sup 349} and Trp{sup 387} help position C-9 and C-11 of AA so that the incipient 11-peroxyl radical intermediate is able to add to C-9 to form the 9,11 endoperoxide group of PGG{sub 2}. In the V349A/W387F oPGHS-1 {center_dot} AA complex the locations of C-9 and C-11 of AA with respect to one another make it difficult to form the endoperoxide group from the 11-hydroperoxyl radical. Therefore, the reaction apparently aborts yielding 11R-HPETE instead of PGG{sub 2}. In addition, the observed differences in the positions of carbon atoms of AA bound to this mutant provides indirect support for the concept that the conformer of AA shown previously to be bound within the cyclooxygenase active site of native oPGHS-1 is the one that leads to PGG{sub 2}.

  18. Locking GTPases covalently in their functional states

    PubMed Central

    Wiegandt, David; Vieweg, Sophie; Hofmann, Frank; Koch, Daniel; Li, Fu; Wu, Yao-Wen; Itzen, Aymelt; Müller, Matthias P.; Goody, Roger S.

    2015-01-01

    GTPases act as key regulators of many cellular processes by switching between active (GTP-bound) and inactive (GDP-bound) states. In many cases, understanding their mode of action has been aided by artificially stabilizing one of these states either by designing mutant proteins or by complexation with non-hydrolysable GTP analogues. Because of inherent disadvantages in these approaches, we have developed acryl-bearing GTP and GDP derivatives that can be covalently linked with strategically placed cysteines within the GTPase of interest. Binding studies with GTPase-interacting proteins and X-ray crystallography analysis demonstrate that the molecular properties of the covalent GTPase–acryl–nucleotide adducts are a faithful reflection of those of the corresponding native states and are advantageously permanently locked in a defined nucleotide (that is active or inactive) state. In a first application, in vivo experiments using covalently locked Rab5 variants provide new insights into the mechanism of correct intracellular localization of Rab proteins. PMID:26178622

  19. Origin and vertical variation of the bound fatty acids in core sediments of Lake Dianchi in Southwest China.

    PubMed

    Wang, Lifang; Wu, Fengchang; Xiong, Yongqiang; Fang, Jidun

    2013-04-01

    Based on the molecular distribution of bound fatty acid (BFA) compound classes in core sediments of Lake Dianchi combined with the compound-specific δ(13)C values of the straight-chain BFAs, origin and vertical changes of organic matters in the sediments were investigated. The results indicated a significant change of BFA sources over the past 700 years. Contrast to the low concentrations of the terrestrial BFAs, the abundance of BFAs derived from the plankton/bacteria in the top sections (1944-recent) was more than 80%. The increasing proportions of the branched and unsaturated BFAs in total fatty acids were closely correlated with the heavy eutrophication and the frequent algal blooms in the decades. Furthermore, the positive shift of δ (13)C of C16 and C18 (~2‰) in the upper section might be an indicator of the excess phytoplankton productivity. However, it was found that the plankton/bacteria-derived BFAs were more easily degraded during the early diagenetic process. The special compound carbon isotopic compositions of the long straight-chain BFAs (C24 and C26) in the sediments showed a depletion of heavier δ (13)C values (ca. -30‰) in the midsections (1559-1787), reflecting a relatively growing contribution of C3 plants to C4 plants or that C4 plant growth was inhibited in cold and arid climates during the period. PMID:22903813

  20. Boron-11 pure quadrupole resonance investigation of peptide boronic acid inhibitors bound to alpha-lytic protease.

    PubMed

    Ivanov, Dmitri; Bachovchin, William W; Redfield, Alfred G

    2002-02-01

    Pure quadrupole resonance is a potentially useful spectroscopic approach to study the coordination of quadrupolar nuclei in biological systems. We used a field-cycling NMR method to observe boron pure quadrupole resonance of two peptide boronic acid inhibitors bound to alpha-lytic protease. The method is similar to our earlier field-cycling experiment [Ivanov, D., and Redfield, A. R. (1998) Z. Naturforsch. A 53, 269-272] but uses a simple Hartmann-Hahn transfer from proton to (11)B before field cycle and direct (11)B observe after it. Pure quadrupole resonance is sensitive to the boron coordination geometry. For example, trigonal boron in neutral phenylboronic acid, which was used as a model compound, resonates at 1450 kHz, while the resonance of the tetrahedral phenylboronic acid anion appears at approximately 600 kHz. In the complex of the MeOSuc-Ala-Ala-Pro-boroVal inhibitor with the enzyme the quadrupole resonance signal was observed at 600-650 kHz, which indicates tetrahedral boron coordination in the active site. The quadrupole frequency of the MeOSuc-Ala-Ala-Pro-boroPhe enzyme-inhibitor complex, in which a boron-histidine bond is known to be formed, was found to be the same within experimental error as in the MeOSuc-Ala-Ala-Pro-boroVal enzyme-inhibitor adduct, suggesting that the boron coordination geometry in the enzyme-MeOSuc-Ala-Ala-Pro-boroPhe adduct is also close to tetrahedral. PMID:11814352

  1. Loosely-Bound Diatomic Molecules.

    ERIC Educational Resources Information Center

    Balfour, W. J.

    1979-01-01

    Discusses concept of covalent bonding as related to homonuclear diatomic molecules. Article draws attention to the existence of bound rare gas and alkaline earth diatomic molecules. Summarizes their molecular parameters and offers spectroscopic data. Strength and variation with distance of interatomic attractive forces is given. (Author/SA)

  2. Bound Phenolics of Quinoa Seeds Released by Acid, Alkaline, and Enzymatic Treatments and Their Antioxidant and α-Glucosidase and Pancreatic Lipase Inhibitory Effects.

    PubMed

    Tang, Yao; Zhang, Bing; Li, Xihong; Chen, Peter X; Zhang, Hua; Liu, Ronghua; Tsao, Rong

    2016-03-01

    Unextractable phenolics from plant foods and their role in health benefits have become increasingly important. Meal residues of three quinoa seeds free of fat and extractable phenolics were subjected to acid, alkaline, and enzymatic hydrolyses. The total and individual phenolic compounds released were analyzed, and 19 phenolics, predominantly phenolic acids and several flavonoids, were identified. The concentration of bound phenolics was highest in black quinoa followed by red and white, regardless of the hydrolysis method. Higher phenolic contents also showed stronger antioxidant activities and inhibition of α-glucosidase and pancreatic lipase activities. Carbohydrases, that is, pectinase, xylanase and feruloyl esterase, which effectively liberated bound phenolics are known to be secreted by colonic bacteria, suggesting potential antioxidant and anti-inflammatory effects by these compounds in the large intestine during colonic fermentation. These results can also be applied to treat foods high in bound phenolics to enhance bioaccessibility. PMID:26853559

  3. Assessment of total (free and bound) phenolic compounds in spent coffee extracts.

    PubMed

    Monente, Carmen; Ludwig, Iziar A; Irigoyen, Angel; De Peña, María-Paz; Cid, Concepción

    2015-05-01

    Spent coffee is the main byproduct of the brewing process and a potential source of bioactive compounds, mainly phenolic acids easily extracted with water. Free and bound caffeoylquinic (3-CQA, 4-CQA, 5-CQA), dicaffeoylquinic (3,4-diCQA, 3,5-diCQA, 4,5-diCQA), caffeic, ferulic, p-coumaric, sinapic, and 4-hydroxybenzoic acids were measured by HPLC, after the application of three treatments (alkaline, acid, saline) to spent coffee extracts. Around 2-fold higher content of total phenolics has been estimated in comparison to free compounds. Phenolic compounds with one or more caffeic acid molecules were approximately 54% linked to macromolecules such as melanoidins, mainly by noncovalent interactions (up to 81% of bound phenolic compounds). The rest of the quantitated phenolic acids were mainly attached to other structures by covalent bonds (62-97% of total bound compounds). Alkaline hydrolysis and saline treatment were suitable to estimate total bound and ionically bound phenolic acids, respectively, whereas acid hydrolysis is an inadequate method to quantitate coffee phenolic acids. PMID:25891228

  4. Do acid volatile sulfides (AVS) influence the accumulation of sediment-bound metals to benthic invertebrates under natural field conditions?

    PubMed

    De Jonge, Maarten; Dreesen, Freja; De Paepe, Josefina; Blust, Ronny; Bervoets, Lieven

    2009-06-15

    The present study evaluates the influence of acid volatile sulfides (AVS) on accumulation of sediment-bound metals in benthic invertebrates under natural field conditions. Natural sediments, pore water, surface water, and two species of widespread benthic invertebrates (Chironomus gr. thummi and Tubifex tubifex) were collected from 17 historical polluted Flemish lowland rivers and measured for metal concentrations. Different sediment characteristics were determined (AVS, organic matter, clay content) and multiple regression was used to study their relationship with accumulated metals in the invertebrates. Physical and chemical analysis of the field samples indicated low metal concentrations in the water and pore water, but very high metal concentrations in the sediment and the invertebrates, especially for Pb (5.99 micromol/ g). In general, metal accumulation in chironomids and tubificid worms was most strongly correlated with total metal concentrations in the sediment and sediment metal concentrations normalized for organic matter and clay content. Following the results of the linear regression model, AVS did not turn out to be a significant variable in describing variation in metal accumulation. Our study clearly demonstrates that, in addition to the results gained from experiments under lab conditions, benthic invertebrates can accumulate metals from unspiked field sediments even when there's an excess of AVS. PMID:19603670

  5. Synthesis and mass-spectrometric characterization of human serum albumins modified by covalent binding of two non-steroidal anti-inflammatory drugs: tolmetin and zomepirac.

    PubMed Central

    Zia-Amirhosseini, P; Ding, A; Burlingame, A L; McDonagh, A F; Benet, L Z

    1995-01-01

    Human serum albumins modified by covalently bound tolmetin or zomepirac were synthesized as models for similar products formed in vivo from acyl glucuronides. Activated esters of both drugs were prepared with 1-ethyl-3-(3-dimethylaminopropyl)-carbodi-imide, and then allowed to react with human serum albumin. Tryptic digests of both protein products were analysed by HPLC to identify peptides containing covalently bound drugs, and binding sites on albumin were identified by high-performance tandem MS. Three binding sites were common to both products, i.e. lysine-195, -199 and -351. Three further modified residues were identified for the tolmetin-albumin product, i.e. aspartic acid 1, and lysine-524 and -536. PMID:7487878

  6. Fluorescence anisotropy metrology of electrostatically and covalently labelled silica nanoparticles

    NASA Astrophysics Data System (ADS)

    Yip, Philip; Karolin, Jan; Birch, David J. S.

    2012-08-01

    We compare determining the size of silica nanoparticles using the time-resolved fluorescence anisotropy decay of dye molecules when electrostatically and covalently bound to stable silica nanoparticles. Covalent labelling is shown to offer advantages by simplifying the dye rotational kinetics and the appropriateness of various kinetic models is discussed. Silica nanoparticles produced using Stöber synthesis of tetraethylorthosilicate (TEOS) are found to be controllable between ˜3.1 and 3.8 nm radius by adjusting the relative water:TEOS concentration. Covalent labelling with fluorescein 5(6)-isothiocyanate (FITC) bound to (3-aminopropyl) trimethoxysilane (FITC-APS) predicts a larger particle than electrostatically labelling with rhodamine 6G. The difference is attributed to the presence of an additional depolarization mechanism to Brownian rotation of the nanoparticle and dye wobbling with electrostatic labelling in the form of dye diffusion on the surface of the nanoparticle.

  7. Electronic Communication in Covalently vs. Non-Covalently Bonded Polyfluorene Systems: the Role of the Covalent Linker.

    NASA Astrophysics Data System (ADS)

    Uhler, Brandon; Reilly, Neil J.; Talipov, Marat R.; Ivanov, Maxim; Timerghazin, Qadir; Rathore, Rajendra; Reid, Scott

    2015-06-01

    The covalently linked polyfluorene molecules F1-F6 (see left) are prototypical molecular wires by virtue of their favorable electron/hole transport properties brought about by π-stacking. To understand the role of the covalent linker in facilitating electron transport in these systems, we have investigated several van der Waals (vdW) analogues by resonant mass spectroscopy. Electronic spectra and ion yield curves are reported for jet-cooled vdW clusters containing up to six fluorene units. The near-coincidence of the electronic band origins for the dimer and larger clusters suggests that a structure containing a central dimer chromophore is the predominant conformational motif. As for F1-F6, the threshold ionization potentials extracted from the ion yield measurements decrease linearly with inverse cluster size. Importantly, however, the rate of decrease is significantly smaller in the vdW clusters, indicating more efficient hole stabilization in the covalently bound systems. Results for similar vdW clusters that are locked into specific conformations by steric effects will also be reported.

  8. Characterization of 19 Genes Encoding Membrane-Bound Fatty Acid Desaturases and their Expression Profiles in Gossypium raimondii Under Low Temperature.

    PubMed

    Liu, Wei; Li, Wei; He, Qiuling; Daud, Muhammad Khan; Chen, Jinhong; Zhu, Shuijin

    2015-01-01

    To produce unsaturated fatty acids, membrane-bound fatty acid desaturases (FADs) can be exploited to introduce double bonds into the acyl chains of fatty acids. In this study, 19 membrane-bound FAD genes were identified in Gossypium raimondii through database searches and were classified into four different subfamilies based on phylogenetic analysis. All 19 membrane-bound FAD proteins shared three highly conserved histidine boxes, except for GrFAD2.1, which lost the third histidine box in the C-terminal region. In the G. raimondii genome, tandem duplication might have led to the increasing size of the FAD2 cluster in the Omega Desaturase subfamily, whereas segmental duplication appeared to be the dominant mechanism for the expansion of the Sphingolipid and Front-end Desaturase subfamilies. Gene expression analysis showed that seven membrane-bound FAD genes were significantly up-regulated and that five genes were greatly suppressed in G. raimondii leaves exposed to low temperature conditions. PMID:25894196

  9. Characterization of 19 Genes Encoding Membrane-Bound Fatty Acid Desaturases and their Expression Profiles in Gossypium raimondii Under Low Temperature

    PubMed Central

    He, Qiuling; Daud, Muhammad Khan; Chen, Jinhong; Zhu, Shuijin

    2015-01-01

    To produce unsaturated fatty acids, membrane-bound fatty acid desaturases (FADs) can be exploited to introduce double bonds into the acyl chains of fatty acids. In this study, 19 membrane-bound FAD genes were identified in Gossypium raimondii through database searches and were classified into four different subfamilies based on phylogenetic analysis. All 19 membrane-bound FAD proteins shared three highly conserved histidine boxes, except for GrFAD2.1, which lost the third histidine box in the C-terminal region. In the G. raimondii genome, tandem duplication might have led to the increasing size of the FAD2 cluster in the Omega Desaturase subfamily, whereas segmental duplication appeared to be the dominant mechanism for the expansion of the Sphingolipid and Front-end Desaturase subfamilies. Gene expression analysis showed that seven membrane-bound FAD genes were significantly up-regulated and that five genes were greatly suppressed in G. raimondii leaves exposed to low temperature conditions. PMID:25894196

  10. Transport of free and peptide-bound glycated amino acids: synthesis, transepithelial flux at Caco-2 cell monolayers, and interaction with apical membrane transport proteins.

    PubMed

    Hellwig, Michael; Geissler, Stefanie; Matthes, René; Peto, Anett; Silow, Christoph; Brandsch, Matthias; Henle, Thomas

    2011-05-16

    In glycation reactions, the side chains of protein-bound nucleophilic amino acids such as lysine and arginine are post-translationally modified to a variety of derivatives also known as Maillard reaction products (MRPs). Considerable amounts of MRPs are taken up in food. Here we have studied the interactions of free and dipeptide-bound MRPs with intestinal transport systems. Free and dipeptide-bound derivatives of N(6)-(1-fructosyl)lysine (FL), N(6)-(carboxymethyl)lysine (CML), N(6)-(1-carboxyethyl)lysine (CEL), formyline, argpyrimidine, and methylglyoxal-derived hydroimidazolone 1 (MG-H1) were synthesized. The inhibition of L-[(3)H]lysine and [(14) C]glycylsarcosine uptakes was measured in Caco-2 cells which express the H(+)/peptide transporter PEPT1 and lysine transport system(s). Glycated amino acids always displayed lower affinities than their unmodified analogues towards the L-[(3)H]lysine transporter(s). In contrast, all glycated dipeptides except Ala-FL were medium- to high-affinity inhibitors of [(14)C]Gly-Sar uptake. The transepithelial flux of the derivatives across Caco-2 cell monolayers was determined. Free amino acids and intact peptides derived from CML and CEL were translocated to very small extents. Application of peptide-bound MRPs, however, led to elevation (up to 80-fold) of the net flux and intracellular accumulation of glycated amino acids, which were hydrolyzed from the dipeptides inside the cells. We conclude 1) that free MRPs are not substrates for the intestinal lysine transporter(s), and 2) that dietary MRPs are absorbed into intestinal cells in the form of dipeptides, most likely by the peptide transporter PEPT1. After hydrolysis, hydrophobic glycated amino acids such as pyrraline, formyline, maltosine, and argpyrimidine undergo basolateral efflux, most likely by simple diffusion down their concentration gradients. PMID:21538757

  11. Fenofibrate, a PPARα agonist, protect proximal tubular cells from albumin-bound fatty acids induced apoptosis via the activation of NF-kB

    PubMed Central

    Zuo, Nan; Zheng, Xiaoyu; Liu, Hanzhe; Ma, Xiaoli

    2015-01-01

    Albumin-bound fatty acids is the main cause of renal damage, PPARα is responsible in the metabolism of fatty acids. Previous study found that PPARα played a protective role in fatty acids overload associated tubular injury. The aim of the present study is to investigate whether fenofibrate, a PPARα ligands, could contribute to the renoprotective action in fatty acids overload proximal tubule epithelial cells. We observed in HK-2 cells that fenofibrate significantly inhibited fatty acids bound albumin (FA-BSA) induced up-regulation of MCP-1 and IL-8. Treatment with fenofibrate attenuated renal oxidative stress induced by FA-BSA as evidenced by decreased MDA level, increased SOD activity and catalase, GPx-1 expression. FA-BSA induced apoptosis of HK-2 cells were also obviously prevented by fenofibrate. Furthermore, fenofibrate significantly increased the expression of PPARα mRNA and protein in FA-BSA treated cells. Finally, the activation of NF-kB induced by FA-BSA was markedly suppressed by fenofibrate. Taken together, our study describes a renoprotective role of fenofibrate in fatty acids associated tubular toxicity, and the transcriptional activation of PPARα and suppression of NF-kB were at least partially involved. PMID:26617775

  12. Fenofibrate, a PPARα agonist, protect proximal tubular cells from albumin-bound fatty acids induced apoptosis via the activation of NF-kB.

    PubMed

    Zuo, Nan; Zheng, Xiaoyu; Liu, Hanzhe; Ma, Xiaoli

    2015-01-01

    Albumin-bound fatty acids is the main cause of renal damage, PPARα is responsible in the metabolism of fatty acids. Previous study found that PPARα played a protective role in fatty acids overload associated tubular injury. The aim of the present study is to investigate whether fenofibrate, a PPARα ligands, could contribute to the renoprotective action in fatty acids overload proximal tubule epithelial cells. We observed in HK-2 cells that fenofibrate significantly inhibited fatty acids bound albumin (FA-BSA) induced up-regulation of MCP-1 and IL-8. Treatment with fenofibrate attenuated renal oxidative stress induced by FA-BSA as evidenced by decreased MDA level, increased SOD activity and catalase, GPx-1 expression. FA-BSA induced apoptosis of HK-2 cells were also obviously prevented by fenofibrate. Furthermore, fenofibrate significantly increased the expression of PPARα mRNA and protein in FA-BSA treated cells. Finally, the activation of NF-kB induced by FA-BSA was markedly suppressed by fenofibrate. Taken together, our study describes a renoprotective role of fenofibrate in fatty acids associated tubular toxicity, and the transcriptional activation of PPARα and suppression of NF-kB were at least partially involved. PMID:26617775

  13. Thiophene-based covalent organic frameworks

    PubMed Central

    Bertrand, Guillaume H. V.; Michaelis, Vladimir K.; Ong, Ta-Chung; Griffin, Robert G.; Dincă, Mircea

    2013-01-01

    We report the synthesis and characterization of covalent organic frameworks (COFs) incorporating thiophene-based building blocks. We show that these are amenable to reticular synthesis, and that bent ditopic monomers, such as 2,5-thiophenediboronic acid, are defect-prone building blocks that are susceptible to synthetic variations during COF synthesis. The synthesis and characterization of an unusual charge transfer complex between thieno[3,2-b]thiophene-2,5-diboronic acid and tetracyanoquinodimethane enabled by the unique COF architecture is also presented. Together, these results delineate important synthetic advances toward the implementation of COFs in electronic devices. PMID:23479656

  14. Thiophene-based covalent organic frameworks.

    PubMed

    Bertrand, Guillaume H V; Michaelis, Vladimir K; Ong, Ta-Chung; Griffin, Robert G; Dincă, Mircea

    2013-03-26

    We report the synthesis and characterization of covalent organic frameworks (COFs) incorporating thiophene-based building blocks. We show that these are amenable to reticular synthesis, and that bent ditopic monomers, such as 2,5-thiophenediboronic acid, are defect-prone building blocks that are susceptible to synthetic variations during COF synthesis. The synthesis and characterization of an unusual charge transfer complex between thieno[3,2-b]thiophene-2,5-diboronic acid and tetracyanoquinodimethane enabled by the unique COF architecture is also presented. Together, these results delineate important synthetic advances toward the implementation of COFs in electronic devices. PMID:23479656

  15. Reactions of a sulfonamide antimicrobial with model humic constituents: assessing pathways and stability of covalent bonding.

    PubMed

    Gulkowska, Anna; Krauss, Martin; Rentsch, Daniel; Hollender, Juliane

    2012-02-21

    The mechanism of covalent bond formation of the model sulfonamide sulfathiazole (STZ) and the stronger nucleophile para-ethoxyaniline was studied in reactions with model humic acid constituents (quinones and other carbonyl compounds) in the absence and presence of laccase. As revealed by high resolution mass spectrometry, the initial bonding of STZ occurred by 1,2- and 1,4-nucleophilic additions of the aromatic amino group to quinones resulting in imine and anilinoquinone formation, respectively. Experiments using the radical scavenger tert-butyl-alcohol provided the same products and similar formation rates as those without scavenger indicating that probably not radical coupling reactions were responsible for the initial covalent bond formation. No addition with nonquinone carbonyl compounds occurred within 76 days except for a slow 1,4-addition to the β-unsaturated carbonyl 1-penten-3-one. The stability of covalent bonds against desorption and pressurized liquid extraction (PLE) was assessed. The recovery rates showed no systematic differences in STZ extractability between the two product types. This suggests that the strength of bonding is not controlled by the initial type of bond, but by the extent of subsequent incorporation of the reaction product into the formed polymer. This incorporation was monitored for (15)N aniline by (1)H-(15)N HMBC NMR spectroscopy. The initial 1,2- and 1,4-addition bonds were replaced by stronger heterocyclic forms with increasing incubation time. These processes could also hold true for soils, and a slow nonextractable residue formation with time could be related to a slow increase of the amount of covalently bound sulfonamide and the strength of bonding. PMID:22260423

  16. Reversible Control of Nanoparticle Functionalization and Physicochemical Properties by Dynamic Covalent Exchange**

    PubMed Central

    della Sala, Flavio; Kay, Euan R

    2015-01-01

    Existing methods for the covalent functionalization of nanoparticles rely on kinetically controlled reactions, and largely lack the sophistication of the preeminent oligonucleotide-based noncovalent strategies. Here we report the application of dynamic covalent chemistry for the reversible modification of nanoparticle (NP) surface functionality, combining the benefits of non-biomolecular covalent chemistry with the favorable features of equilibrium processes. A homogeneous monolayer of nanoparticle-bound hydrazones can undergo quantitative dynamic covalent exchange. The pseudomolecular nature of the NP system allows for the in situ characterization of surface-bound species, and real-time tracking of the exchange reactions. Furthermore, dynamic covalent exchange offers a simple approach for reversibly switching—and subtly tuning—NP properties such as solvophilicity. PMID:25973468

  17. Reversible Control of Nanoparticle Functionalization and Physicochemical Properties by Dynamic Covalent Exchange†

    PubMed Central

    della Sala, Flavio

    2015-01-01

    Abstract Existing methods for the covalent functionalization of nanoparticles rely on kinetically controlled reactions, and largely lack the sophistication of the preeminent oligonucleotide‐based noncovalent strategies. Here we report the application of dynamic covalent chemistry for the reversible modification of nanoparticle (NP) surface functionality, combining the benefits of non‐biomolecular covalent chemistry with the favorable features of equilibrium processes. A homogeneous monolayer of nanoparticle‐bound hydrazones can undergo quantitative dynamic covalent exchange. The pseudomolecular nature of the NP system allows for the in situ characterization of surface‐bound species, and real‐time tracking of the exchange reactions. Furthermore, dynamic covalent exchange offers a simple approach for reversibly switching—and subtly tuning—NP properties such as solvophilicity.

  18. The influence of dissolved and surface-bound humic acid on the toxicity of TiO₂ nanoparticles to Chlorella sp.

    PubMed

    Lin, Daohui; Ji, Jing; Long, Zhifeng; Yang, Kun; Wu, Fengchang

    2012-09-15

    NOM is likely to coat TiO₂ nanoparticles (nano-TiO₂) discharged into the aquatic environment and influence the nanotoxicity to aquatic organisms, which however has not been well investigated. This study explored the influence of nanoparticle surface-bound humic acid (HA, as a model NOM) as well as dissolved HA on the toxicity of nano-TiO₂ to Chlorella sp., with a specific focus on adhesion of the nanoparticles to the algae. Results showed that nano-TiO₂ and the dissolved HA could inhibit the algal growth with an IC₅₀ of 4.9 and 8.4 mg L⁻¹, respectively, while both dissolved and nanoparticle surface-bound HA could significantly alleviate the algal toxicity of nano-TiO₂. IC₅₀ of nano-TiO₂ increased to 18 mg L⁻¹ in the presence of 5 mg L⁻¹ of the dissolved HA and to 48 mg L⁻¹ as the result of surface-saturation by HA. Co-precipitation experiment and transmission electron microscopy observation revealed that both dissolved and nanoparticle surface-bound HA prevented the adhesion of nano-TiO₂ to the algal cells due to the increased electrosteric repulsion. The generation of intracellular reactive oxygen species (ROS) was significantly limited by the dissolved and nanoparticle surface-bound HA. The prevention of adhesion and inhibition of ROS generation could account for the HA-mitigated nanotoxicity. PMID:22704133

  19. Immunogenicity and protective efficacy of Bacillus anthracis poly-gamma-D-glutamic acid capsule covalently coupled to a protein carrier using a novel triazine-based conjugation strategy.

    PubMed

    Joyce, Joseph; Cook, James; Chabot, Donald; Hepler, Robert; Shoop, Wesley; Xu, Qiuwei; Stambaugh, Thomas; Aste-Amezaga, Miguel; Wang, Su; Indrawati, Lani; Bruner, Mark; Friedlander, Arthur; Keller, Paul; Caulfield, Michael

    2006-02-24

    The capsular polypeptide of Bacillus anthracis is composed of a unique polyglutamic acid polymer in which D-glutamate monomers are joined by gamma-peptidyl bonds. The capsule is poorly immunogenic, and efforts at exploiting the polymer for vaccine development have focused on increasing its inherent immunogenicity through chemical coupling to immune-stimulating protein carriers. The usual strategy has employed carbodiimide-based condensing reagents for activation of free alpha-carboxyl groups, despite reports that this chemistry may lead to chain scission. We have purified the high molecular mass capsule to >95% homogeneity and have demonstrated that the polymer contains >99% poly-gamma-D-glutamic acid. The predominant structure of the polymer as assessed by circular dichroism and multiangle laser light scattering was unordered at near-neutral pH. We investigated the effects of various activation chemistries, and we demonstrated that carbodiimide treatment under aqueous conditions results in significant cleavage of the gamma-peptidyl bond, whereas scission is significantly reduced in nonaqueous polar solvents, although undesired side chain modification was still observed. An activation chemistry was developed using the triazine-based reagent 4-(4,6-dimethoxy (1,3,5)triazin-2-yl)-4-methylmorpholinium chloride, which allowed for controlled and reproducible derivatization of alpha-carbonyls. In a two-pot reaction scheme, activated capsule was derivatized with a sulfhydryl-reactive heterobifunctional moiety and was subsequently coupled to thiolated carrier protein. This conjugate elicited very high capsule-specific immune titers in mice. More importantly, mice immunized with conjugated capsule exhibited good protection against lethal challenge from a virulent B. anthracis strain in two models of infection. We also showed, for the first time, that treatment of capsule with carbodiimide significantly reduced recognition by capsule-specific antisera concurrent with the

  20. Covalent binding of aniline to humic substances. 2. 15N NMR studies of nucleophilic addition reactions

    USGS Publications Warehouse

    Thorn, K.A.; Pettigrew, P.J.; Goldenberg, W.S.; Weber, E.J.

    1996-01-01

    Aromatic amines are known to undergo covalent binding with humic substances in the environment. Although previous studies have examined reaction conditions and proposed mechanisms, there has been no direct spectroscopic evidence for the covalent binding of the amines to the functional groups in humic substances. In order to further elucidate the reaction mechanisms, the Suwannee River and IHSS soil fulvic and humic acids were reacted with 15N-labeled aniline at pH 6 and analyzed using 15N NMR spectrometry. Aniline underwent nucleophilic addition reactions with the quinone and other carbonyl groups in the samples and became incorporated in the form of anilinohydroquinone, anilinoquinone, anilide, imine, and heterocyclic nitrogen, the latter comprising 50% or more of the bound amine. The anilide and anilinohydroquinone nitrogens were determined to be susceptible to chemical exchange by ammonia. In the case of Suwannee River fulvic acid, reaction under anoxic conditions and pretreatment with sodium borohydride or hydroxylamine prior to reaction under oxic conditions resulted in a decrease in the proportion of anilinohydroquinone nitrogen incorporated. The relative decrease in the incorporation of anilinohydroquinone nitrogen with respect to anilinoquinone nitrogen under anoxic conditions suggested that inter- or intramolecular redox reactions accompanied the nucleophilic addition reactions.

  1. [Bounds of change in unsaturation index of fatty acid composition of phospholipids at adaptation of molluscs to biogenic and abiogenic factors of external medium].

    PubMed

    Chebotareva, M A; Zabelinskiĭ, S A; Shukoliukova, E P; Krivchenko, A I

    2011-01-01

    Comparative study of fatty acid composition of total phospholipids, as well as of phosphatidylcholine and phosphatidylethanolamine from hepatopancreas and leg muscle was performed on several representatives of gasteropods (Gastropoda) molluscs and bivalve (Bivalvia) mussel (Mytilus edulus). The objects of our study were marine litorins (Littorina saxsatilis) adapted to different temperature conditions of White Sea and Barents Sea, freshwater lymnaea (Lymnaea stagnalis) infested by Trematoda and mussels from White Sea and Black Sea. It was shown that depending on the existence conditions of studied tissue or lipid, the maximal change is observed in the percentage of saturated acids (4-83 %), the percentage of unsaturated acids was less expressed (1-14 %) and the changes in unsaturation index (UI) did not exceed 20 % on average. It was supposed that observed quantitative bounds of UI change under the action of different external factors is utmost for maintenance of membrane fluidity necessary for normal vital activity of cell, particularly in studied ectothermic molluscs. PMID:22145319

  2. Mouse Siglec-1 Mediates trans-Infection of Surface-bound Murine Leukemia Virus in a Sialic Acid N-Acyl Side Chain-dependent Manner.

    PubMed

    Erikson, Elina; Wratil, Paul R; Frank, Martin; Ambiel, Ina; Pahnke, Katharina; Pino, Maria; Azadi, Parastoo; Izquierdo-Useros, Nuria; Martinez-Picado, Javier; Meier, Chris; Schnaar, Ronald L; Crocker, Paul R; Reutter, Werner; Keppler, Oliver T

    2015-11-01

    Siglec-1 (sialoadhesin, CD169) is a surface receptor on human cells that mediates trans-enhancement of HIV-1 infection through recognition of sialic acid moieties in virus membrane gangliosides. Here, we demonstrate that mouse Siglec-1, expressed on the surface of primary macrophages in an interferon-α-responsive manner, captures murine leukemia virus (MLV) particles and mediates their transfer to proliferating lymphocytes. The MLV infection of primary B-cells was markedly more efficient than that of primary T-cells. The major structural protein of MLV particles, Gag, frequently co-localized with Siglec-1, and trans-infection, primarily of surface-bound MLV particles, efficiently occurred. To explore the role of sialic acid for MLV trans-infection at a submolecular level, we analyzed the potential of six sialic acid precursor analogs to modulate the sialylated ganglioside-dependent interaction of MLV particles with Siglec-1. Biosynthetically engineered sialic acids were detected in both the glycolipid and glycoprotein fractions of MLV producer cells. MLV released from cells carrying N-acyl-modified sialic acids displayed strikingly different capacities for Siglec-1-mediated capture and trans-infection; N-butanoyl, N-isobutanoyl, N-glycolyl, or N-pentanoyl side chain modifications resulted in up to 92 and 80% reduction of virus particle capture and trans-infection, respectively, whereas N-propanoyl or N-cyclopropylcarbamyl side chains had no effect. In agreement with these functional analyses, molecular modeling indicated reduced binding affinities for non-functional N-acyl modifications. Thus, Siglec-1 is a key receptor for macrophage/lymphocyte trans-infection of surface-bound virions, and the N-acyl side chain of sialic acid is a critical determinant for the Siglec-1/MLV interaction. PMID:26370074

  3. Covalent binding of reduced metabolites of [{sup 15}N{sub 3}]TNT to soil organic matter during a bioremediation process analyzed by {sup 15}N NMR spectroscopy

    SciTech Connect

    Achtnich, C.; Fernandes, E.; Bollag, J.M.; Knackmuss, H.J.; Lenke, H.

    1999-12-15

    Evidence is presented for the covalent binding of biologically reduced metabolites of 2,4,6-{sup 15}N{sub 3}-trinitrotoluene (TNT) to different soil fractions, using liquid {sup 15}N NMR spectroscopy. A silylation procedure was used to release soil organic matter from humin and whole soil for spectroscopic measurements. TNT-contaminated soil was spiked with 2,4,6-{sup 15}N{sub 3}-trinitrotoluene and {sup 14}C-ring labeled TNT, before treatment in a soil slurry reactor. During the anaerobic/aerobic incubation the amount of radioactivity detected in the fulvic and humic acid fractions did not change significantly whereas the radioactivity bound to humin increased to 71%. The {sup 15}N NMR spectra of the fulvic acid samples were dominated by a large peak that corresponded to aliphatic amines or ammonia. In the early stages of incubation, {sup 15}N NMR analysis of the humic acids indicated bound azoxy compounds. The signals arising from nitro and azoxy groups disappeared with further anaerobic treatment. At the end of incubation, the NMR shifts showed that nitrogen was covalently bound to humic acid as substituted amines and amides. The NMR spectra of the silylated humin suggest formation of azoxy compounds and imine linkages. Bound metabolites possessing nitro groups were also detected. Primary amines formed during the anaerobic incubation disappeared during the aerobic treatment. Simultaneously, the amount of amides and tertiary amines increased. Nitro and azoxy groups of bound molecules were still present in humin at the end of the incubation period. Formation of azoxy compounds from partially reduced TNT followed by binding and further reduction appears to be an important mechanism for the immobilization of metabolites of TNT to soil.

  4. Structural investigation of the covalent and electrostatic binding of yeast cytochrome c to the surface of various ultrathin lipid multilayers using x-ray diffraction.

    PubMed Central

    Pachence, J M; Blasie, J K

    1991-01-01

    X-Ray diffraction was used to characterize the profile structures of ultrathin lipid multilayers having a bound surface layer of cytochrome c. The lipid multilayers were formed on an alkylated glass surface, using the Langmuir-Blodgett method. The ultrathin lipid multilayers of this study were: five monolayers of arachidic acid, four monolayers of arachidic acid with a surface monolayer of dimyristoyl phosphatidylserine, and four monolayers of arachidic acid acid with a surface monolayer of thioethyl stearate. Both the phosphatidylserine and the thioethyl stearate surfaces were found previously to covalently bind yeast cytochrome c, while the arachidic acid surface electrostatically binds yeast cytochrome c. Meridional x-ray diffraction data were collected from these lipid multilayer films with and without a bound yeast cytochrome c surface layer. A box refinement technique, previously shown to be effective in deriving the profile structures of ultrathin multilayer lipid films with and without electrostatically bound cytochrome c, was used to determine the multilayer electron density profiles. The surface monolayer of bound cytochrome c was readily apparent upon comparison of the multilayer electron density profiles for the various pairs of ultrathin multilayer films plus/minus cytochrome c for all cases. In addition, cytochrome c binding to the multilayer surface significantly perturbs the underlying lipid monolayers. PMID:1648415

  5. Strategies for discovering and derisking covalent, irreversible enzyme inhibitors

    PubMed Central

    Johnson, Douglas S; Weerapana, Eranthie; Cravatt, Benjamin F

    2010-01-01

    This article presents several covalent inhibitors, including examples of successful drugs, as well as highly selective, irreversible inhibitors of emerging therapeutic targets, such as fatty acid amide hydolase. Covalent inhibitors have many desirable features, including increased biochemical efficiency of target disruption, less sensitivity toward pharmacokinetic parameters and increased duration of action that outlasts the pharmacokinetics of the compound. Safety concerns that must be mitigated include lack of specificity and the potential immunogenicity of protein–inhibitor adduct(s). Particular attention will be given to recent technologies, such as activity-based protein profiling, which allow one to define the proteome-wide selectivity patterns for covalent inhibitors in vitro and in vivo. For instance, any covalent inhibitor can, in principle, be modified with a ‘clickable’ tag to generate an activity probe that is almost indistinguishable from the original agent. These probes can be applied to any living system across a broad dose range to fully inventory their on and off targets. The substantial number of drugs on the market today that act by a covalent mechanism belies historical prejudices against the development of irreversibly acting therapeutic small molecules. Emerging proteomic technologies offer a means to systematically discriminate safe (selective) versus deleterious (nonselective) covalent inhibitors and thus should inspire their future design and development. PMID:20640225

  6. A Heteromeric Membrane-Bound Prenyltransferase Complex from Hop Catalyzes Three Sequential Aromatic Prenylations in the Bitter Acid Pathway1[OPEN

    PubMed Central

    Li, Haoxun; Ban, Zhaonan; Qin, Hao; Ma, Liya; King, Andrew J.

    2015-01-01

    Bitter acids (α and β types) account for more than 30% of the fresh weight of hop (Humulus lupulus) glandular trichomes and are well known for their contribution to the bitter taste of beer. These multiprenylated chemicals also show diverse biological activities, some of which have potential benefits to human health. The bitter acid biosynthetic pathway has been investigated extensively, and the genes for the early steps of bitter acid synthesis have been cloned and functionally characterized. However, little is known about the enzyme(s) that catalyze three sequential prenylation steps in the β-bitter acid pathway. Here, we employed a yeast (Saccharomyces cerevisiae) system for the functional identification of aromatic prenyltransferase (PT) genes. Two PT genes (HlPT1L and HlPT2) obtained from a hop trichome-specific complementary DNA library were functionally characterized using this yeast system. Coexpression of codon-optimized PT1L and PT2 in yeast, together with upstream genes, led to the production of bitter acids, but no bitter acids were detected when either of the PT genes was expressed by itself. Stepwise mutation of the aspartate-rich motifs in PT1L and PT2 further revealed the prenylation sequence of these two enzymes in β-bitter acid biosynthesis: PT1L catalyzed only the first prenylation step, and PT2 catalyzed the two subsequent prenylation steps. A metabolon formed through interactions between PT1L and PT2 was demonstrated using a yeast two-hybrid system, reciprocal coimmunoprecipitation, and in vitro biochemical assays. These results provide direct evidence of the involvement of a functional metabolon of membrane-bound prenyltransferases in bitter acid biosynthesis in hop. PMID:25564559

  7. Determination of covalent binding to intact DNA, RNA, and oligonucleotides by intercalating anticancer drugs using high-performance liquid chromatography. Studies with doxorubicin and NADPH cytochrome P-450 reductase

    SciTech Connect

    Cummings, J.; Bartoszek, A.; Smyth, J.F. )

    1991-04-01

    An HPLC method is described which can determine covalent binding to intact nucleic acid by intercalating anticancer drugs and at the same time remove noncovalently bound intercalated drug. The method uses a column containing a nonporous 2-microns DEAE anion-exchange resin capable of chromatographing nucleic acids greater than 50,000 bases in size in under 1 h. After priming with 1 mg of DNA, the column behaves as an intercalator affinity column, strongly retaining the drug while allowing the nucleic acid to pass through normally. Retained drug is released with an injection of 0.1 M potassium hydroxide. Incubations were performed with the intercalator doxorubicin, which is also believed to bind covalently to DNA. When (14C)doxorubicin was mixed with DNA, at a concentration where all the drug would bind by intercalation, the column retained 82% of the total radioactivity, only 18% migrated with the nucleic acid. If the DNA was mildly denatured by treatment with 2 M sodium chloride at 50 degrees C for 45 min before chromatography, then 99.8% of total radioactivity was retained, only background counts migrated with the nucleic acid, as was the case with single-stranded DNA and RNA without any treatment. Purified NADPH cytochrome P-450 reductase was used to activate doxorubicin. DNA inhibited the metabolism of the drug by the enzyme, no covalent binding occurred with RNA, low levels occurred with single-stranded DNA, and the highest levels were recorded with oligonucleotides. The assay was sufficiently sensitive to measure covalent binding to DNA extracted from MCF-7 human breast cancer cells treated with 50 microM (14C)doxorubicin. Thus, covalent binding to DNA, RNA, and oligonucleotides by intercalators can be measured quickly (20 min) without the need to either digest the nucleic acid or subject it to long sample preparation techniques.

  8. Concurrent Covalent and Supramolecular Polymerization.

    PubMed

    Hou, Xisen; Ke, Chenfeng; Zhou, Yu; Xie, Zhuang; Alngadh, Ahmed; Keane, Denis T; Nassar, Majed S; Botros, Youssry Y; Mirkin, Chad A; Stoddart, J Fraser

    2016-08-22

    Covalent and supramolecular polymerizations, both of which offer their own unique advantages, have emerged as popular strategies for making artificial materials. Herein, we describe a concurrent covalent and supramolecular polymerization strategy-namely, one which utilizes 1) a bis-azide-functionalized diazaperopyrenium dication that undergoes polymeriation covalently with a bis-alkyne-functionalized biphenyl derivative in one dimension as a result of a rapid and efficient β-cyclodextrin(CD)-accelerated, cucurbit[6]uril(CB)-templated azide-alkyne cycloaddition, while 2) the aromatic core of the dication is able to dimerize in a criss-cross fashion by dint of π-π interactions, enabling simultaneous supramolecular assembly, resulting in an extended polymer network in an orthogonal dimension. PMID:27338246

  9. Crystal Structure of Fatty Acid Amide Hydrolase Bound to the Carbamate Inhibitor URB597: Discovery of a Deacylating Water Molecule and Insight into Enzyme Inactivation

    SciTech Connect

    Mileni, Mauro; Kamtekar, Satwik; Wood, David C.; Benson, Timothy E.; Cravatt, Benjamin F.; Stevens, Raymond C.

    2010-08-12

    The endocannabinoid system regulates a wide range of physiological processes including pain, inflammation, and cognitive/emotional states. URB597 is one of the best characterized covalent inhibitors of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH). Here, we report the structure of the FAAH-URB597 complex at 2.3 {angstrom} resolution. The structure provides insights into mechanistic details of enzyme inactivation and experimental evidence of a previously uncharacterized active site water molecule that likely is involved in substrate deacylation. This water molecule is part of an extensive hydrogen-bonding network and is coordinated indirectly to residues lining the cytosolic port of the enzyme. In order to corroborate our hypothesis concerning the role of this water molecule in FAAH's catalytic mechanism, we determined the structure of FAAH conjugated to a urea-based inhibitor, PF-3845, to a higher resolution (2.4 {angstrom}) than previously reported. The higher-resolution structure confirms the presence of the water molecule in a virtually identical location in the active site. Examination of the structures of serine hydrolases that are non-homologous to FAAH, such as elastase, trypsin, or chymotrypsin, shows a similarly positioned hydrolytic water molecule and suggests a functional convergence between the amidase signature enzymes and serine proteases.

  10. Reductive transformation of bound trinitrophenyl residues and free TNT during a bioremediation process analyzed by immunoassay

    SciTech Connect

    Achtnich, C.; Pfortner, P.; Weller, M.G.; Niessner, R.; Lenke, H.; Knackmuss, H.J.

    1999-10-01

    To follow the fate of bound metabolites of TNT in soil, a synthetic trinitrophenyl residue covalently linked to humic acids was used as model compound. A selective monoclonal antibody was able to detect chemical changes of the nitro groups of the bound residues. The general possibility of reductive transformations of nitro groups of bound molecules and the reduction rates should be determined. In comparison to the reduction of free TNT and its metabolites, the reductive transformation of the bound trinitrophenyl residue was delayed, and the transformation rate was considerably slower. Trinitrophenyl residues also could be detected by the immunoassay in humic acids extracted from TNT contaminated soil. The reductive transformation of these trinitrophenyl residues started after the reduction of free TNT. At the end of the treatment, small amounts of these residues were still detectable indicating that some of these structures were not completely reduced during the process. From present results one can conclude that the further reduction of nitro groups of bound metabolites requires a prolonged anaerobic treatment. Not only the monitoring of free nitroaromatic compounds is recommended during the bioremediation process but also the measurement of bound residues to determine the optimal conditions and duration of the treatment.

  11. Covalent binding of aniline to humic substances. 1. Kinetic studies

    USGS Publications Warehouse

    Weber, E.J.; Spidle, D.L.; Thorn, K.A.

    1996-01-01

    The reaction kinetics for the covalent binding of aniline with reconstituted IHSS humic and fulvic acids, unfractionated DOM isolated from Suwannee River water, and whole samples of Suwannee River water have been investigated. The reaction kinetics in each of these systems can be adequately described by a simple second-order rate expression. The effect of varying the initial concentration of aniline on reaction kinetics suggested that approximately 10% of the covalent binding sites associated with Suwannee River fulvic acid are highly reactive sites that are quickly saturated. Based on the kinetic parameters determined for the binding of aniline with the Suwannee River fulvic and humic acid isolates, it was estimated that 50% of the aniline concentration decrease in a Suwannee River water sample could be attributed to reaction with the fulvic and humic acid components of the whole water sample. Studies with Suwannee River fulvic acid demonstrated that the rate of binding decreased with decreasing pH, which parallels the decrease in the effective concentration of the neutral form, or reactive nucleophilic species of aniline. The covalent binding of aniline with Suwannee River fulvic acid was inhibited by prior treatment of the fulvic acid with hydrogen sulfide, sodium borohydride, or hydroxylamine. These observations are consistent with a reaction pathway involving nucleophilic addition of aniline to carbonyl moieties present in the fulvic acid.

  12. Gas chromatographic mass spectrometric detection of dihydroxy fatty acids preserved in the 'bound' phase of organic residues of archaeological pottery vessels.

    PubMed

    Hansel, Fabricio A; Bull, Ian D; Evershed, Richard P

    2011-07-15

    A methodology is presented for the determination of dihydroxy fatty acids preserved in the 'bound' phase of organic residues preserved in archaeological potsherds. The method comprises saponification, esterification, silica gel column chromatographic fractionation, and analysis by gas chromatography/mass spectrometry. The electron ionisation mass spectra of the trimethylsilyl ether methyl ester derivatives are characterised by fragment ions arising from cleavage of the bond between the two vicinal trimethylsiloxy groups. Other significant fragment ions are [M-15](+.), [M-31](+.), m/z 147 and ions characteristic of vicinal disubstituted (trimethylsiloxy) TMSO- groups (Δ(7,8), Δ(9,10), Δ(11,12) and Δ(13,14): m/z 304, 332, 360 and 388, respectively). The dihydroxy fatty acids identified in archaeological extracts exhibited carbon numbers ranging from C(16) to C(22) and concentrations varying from 0.05 to 14.05 µg g(-1) . The wide range of dihydroxy fatty acids observed indicates that this approach may be applied confidently in screening archaeological potsherds for the degradation products of monounsaturated fatty acids derived from commodities processed in archaeological pottery vessels. PMID:21638365

  13. Chemistry of Covalent Organic Frameworks.

    PubMed

    Waller, Peter J; Gándara, Felipe; Yaghi, Omar M

    2015-12-15

    Linking organic molecules by covalent bonds into extended solids typically generates amorphous, disordered materials. The ability to develop strategies for obtaining crystals of such solids is of interest because it opens the way for precise control of the geometry and functionality of the extended structure, and the stereochemical orientation of its constituents. Covalent organic frameworks (COFs) are a new class of porous covalent organic structures whose backbone is composed entirely of light elements (B, C, N, O, Si) that represent a successful demonstration of how crystalline materials of covalent solids can be achieved. COFs are made by combination of organic building units covalently linked into extended structures to make crystalline materials. The attainment of crystals is done by several techniques in which a balance is struck between the thermodynamic reversibility of the linking reactions and their kinetics. This success has led to the expansion of COF materials to include organic units linked by these strong covalent bonds: B-O, C-N, B-N, and B-O-Si. Since the organic constituents of COFs, when linked, do not undergo significant change in their overall geometry, it has been possible to predict the structures of the resulting COFs, and this advantage has facilitated their characterization using powder X-ray diffraction (PXRD) techniques. It has also allowed for the synthesis of COF structures by design and for their formation with the desired composition, pore size, and aperture. In practice, the modeled PXRD pattern for a given expected COF is compared with the experimental one, and depending on the quality of the match, this is used as a starting point for solving and then refining the crystal structure of the target COF. These characteristics make COFs an attractive class of new porous materials. Accordingly, they have been used as gas storage materials for energy applications, solid supports for catalysis, and optoelectronic devices. A large and

  14. Effect of feeding lipids recovered from fish processing waste by lactic acid fermentation and enzymatic hydrolysis on antioxidant and membrane bound enzymes in rats.

    PubMed

    Rai, Amit Kumar; Bhaskar, N; Baskaran, V

    2015-06-01

    Fish oil recovered from fresh water fish visceral waste (FVW-FO) through lactic acid fermentation (FO-LAF) and enzymatic hydrolysis (FO-EH) were fed to rats to study their influence on lipid peroxidation and activities of antioxidant and membrane bound enzyme in liver, heart and brain. Feeding of FO-LAF and FO-EH resulted in increase (P < 0.05) in lipid peroxides level in serum, liver, brain and heart tissues compared to ground nut oil (control). Activity of catalase (40-235 %) and superoxide dismutase (17-143 %) also increased (P < 0.05) with incremental level of EPA + DHA in diet. The increase was similar to cod liver oil fed rats at same concentration of EPA + DHA. FO-LAF and FO-EH increased (P < 0.05) the Na(+)K(+) ATPase activity in liver and brain microsomes, Ca(+)Mg(+) ATPase in heart microsome and acetylcholine esterase in brain microsomes when fed with 5 % EPA + DHA. There was also significant change in fatty acid composition and cholesterol/phospholipid ratio in microsomes of rat fed with FVW-FO. Feeding FVW-FO recovered by biotechnological approaches enhanced the activity of antioxidant enzymes in tissues, modulates the activities of membrane bound enzymes and improved the fatty acid composition in microsomes of tissues similar to CLO. Utilization of these processing wastes for the production of valuable biofunctional products can reduce the mounting economic values of fish oil and minimize the environmental pollution problems. PMID:26028754

  15. Improved Quantification of Free and Ester-Bound Gallic Acid in Foods and Beverages by UHPLC-MS/MS.

    PubMed

    Newsome, Andrew G; Li, Yongchao; van Breemen, Richard B

    2016-02-17

    Hydrolyzable tannins are measured routinely during the characterization of food and beverage samples. Most methods for the determination of hydrolyzable tannins use hydrolysis or methanolysis to convert complex tannins to small molecules (gallic acid, methyl gallate, and ellagic acid) for quantification by HPLC-UV. Often unrecognized, analytical limitations and variability inherent in these approaches for the measurement of hydrolyzable tannins include the variable mass fraction (0-0.90) that is released as analyte, contributions of sources other than tannins to hydrolyzable gallate (can exceed >10 wt %/wt), the measurement of both free and total analyte, and lack of controls to account for degradation. An accurate, specific, sensitive, and higher-throughput approach for the determination of hydrolyzable gallate based on ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) that overcomes these limitations was developed. PMID:26804199

  16. Fatty acids bound to human serum albumin and its structural variants modulate apolipoprotein B secretion in HepG2 cells.

    PubMed

    Ha, Ji-Sook; Theriault, Andre; Bhagavan, Nadhipuram V; Ha, Chung-Eun

    2006-07-01

    Epidemiologic studies have shown an inverse relationship between human serum albumin (HSA) levels and coronary heart disease (CHD). However, no mechanisms have been identified to explain this relationship. We hypothesized that this relationship is due to differences in binding affinity of fatty acids to HSA and subsequent atherogenic lipoprotein synthesis and secretion from hepatocytes. To test our hypothesis we undertook the current study. Using HepG2 cells, we demonstrated that oleic acid (OA) bound to HSA in a molar ratio of 4:1 and after incubation for 24 h stimulated apolipoprotein B (apoB) secretion. We also tested whether mutant forms of HSA could alter the binding affinity for fatty acids and change the availability of substrate for lipoprotein secretion. Based on the results obtained in this study using 11 HSA mutant proteins complexed with OA, we were able to classify into three major mutant groups based on their effects on apoB secretion. One group in particular (R410Q/Y411W, R410A/Y411A, and W214L/Y411W) showed a significantly diminished effect on apoB secretion when compared to the wild type HSA/OA complex. Furthermore, the amount of free OA internalized in HepG2 cells in the presence of HSA mutant proteins was in good agreement with the effects seen on apoB secretion by the various HSA mutants. This suggests that some mutant forms of HSA might potentially bind fatty acids with a much higher binding affinity and thus deprive fatty acids available for lipoprotein assembly in hepatocytes. In conclusion, our data illustrate that certain HSA polymorphic forms may be protective against the development of CHD and warrants further investigation. PMID:16843720

  17. Atomic Covalent Functionalization of Graphene

    PubMed Central

    Johns, James E.; Hersam, Mark C.

    2012-01-01

    Conspectus Although graphene’s physical structure is a single atom thick, two-dimensional, hexagonal crystal of sp2 bonded carbon, this simple description belies the myriad interesting and complex physical properties attributed to this fascinating material. Because of its unusual electronic structure and superlative properties, graphene serves as a leading candidate for many next generation technologies including high frequency electronics, broadband photodetectors, biological and gas sensors, and transparent conductive coatings. Despite this promise, researchers could apply graphene more routinely in real-world technologies if they could chemically adjust graphene’s electronic properties. For example, the covalent modification of graphene to create a band gap comparable to silicon (~1 eV) would enable its use in digital electronics, and larger band gaps would provide new opportunities for graphene-based photonics. Towards this end, researchers have focused considerable effort on the chemical functionalization of graphene. Due to its high thermodynamic stability and chemical inertness, new methods and techniques are required to create covalent bonds without promoting undesirable side reactions or irreversible damage to the underlying carbon lattice. In this Account, we review and discuss recent theoretical and experimental work studying covalent modifications to graphene using gas phase atomic radicals. Atomic radicals have sufficient energy to overcome the kinetic and thermodynamic barriers associated with covalent reactions on the basal plane of graphene but lack the energy required to break the C-C sigma bonds that would destroy the carbon lattice. Furthermore, because they are atomic species, radicals substantially reduce the likelihood of unwanted side reactions that confound other covalent chemistries. Overall, these methods based on atomic radicals show promise for the homogeneous functionalization of graphene and the production of new classes of two

  18. Atomic covalent functionalization of graphene.

    PubMed

    Johns, James E; Hersam, Mark C

    2013-01-15

    Although graphene's physical structure is a single atom thick, two-dimensional, hexagonal crystal of sp(2) bonded carbon, this simple description belies the myriad interesting and complex physical properties attributed to this fascinating material. Because of its unusual electronic structure and superlative properties, graphene serves as a leading candidate for many next generation technologies including high frequency electronics, broadband photodetectors, biological and gas sensors, and transparent conductive coatings. Despite this promise, researchers could apply graphene more routinely in real-world technologies if they could chemically adjust graphene's electronic properties. For example, the covalent modification of graphene to create a band gap comparable to silicon (∼1 eV) would enable its use in digital electronics, and larger band gaps would provide new opportunities for graphene-based photonics. Toward this end, researchers have focused considerable effort on the chemical functionalization of graphene. Due to its high thermodynamic stability and chemical inertness, new methods and techniques are required to create covalent bonds without promoting undesirable side reactions or irreversible damage to the underlying carbon lattice. In this Account, we review and discuss recent theoretical and experimental work studying covalent modifications to graphene using gas phase atomic radicals. Atomic radicals have sufficient energy to overcome the kinetic and thermodynamic barriers associated with covalent reactions on the basal plane of graphene but lack the energy required to break the C-C sigma bonds that would destroy the carbon lattice. Furthermore, because they are atomic species, radicals substantially reduce the likelihood of unwanted side reactions that confound other covalent chemistries. Overall, these methods based on atomic radicals show promise for the homogeneous functionalization of graphene and the production of new classes of two

  19. Gallic acid-based alkyl esters synthesis in a water-free system by celite-bound lipase of Bacillus licheniformis SCD11501.

    PubMed

    Sharma, Shivika; Kanwar, Shamsher S; Dogra, Priyanka; Chauhan, Ghanshyam S

    2015-01-01

    Gallic acid (3, 4, 5- trihydroxybenzoic acid) is an important antioxidant, anti-inflammatory, and radical scavenging agent. In the present study, a purified thermo-tolerant extra-cellular lipase of Bacillus licheniformis SCD11501 was successfully immobilized by adsorption on Celite 545 gel matrix followed by treatment with a cross-linking agent, glutaraldehyde. The celite-bound lipase treated with glutaraldehyde showed 94.8% binding/retention of enzyme activity (36 U/g; specific activity 16.8 U/g matrix; relative increase in enzyme activity 64.7%) while untreated matrix resulted in 88.1% binding/retention (28.0 U/g matrix; specific activity 8.5 U/g matrix) of lipase. The celite-bound lipase was successfully used to synthesis methyl gallate (58.2%), ethyl gallate (66.9%), n-propyl gallate (72.1%), and n-butyl gallate (63.8%) at 55(o) C in 10 h under shaking (150 g) in a water-free system by sequentially optimizing various reaction parameters. The low conversion of more polar alcohols such as methanol and ethanol into their respective gallate esters might be due to the ability of these alcohols to severely remove water from the protein hydration shell, leading to enzyme inactivation. Molecular sieves added to the reaction mixture resulted in enhanced yield of the alkyl ester(s). The characterization of synthesised esters was done through fourier transform infrared (FTIR) spectroscopy and (1) H NMR spectrum analysis. PMID:25737230

  20. Stochastic sensing through covalent interactions

    SciTech Connect

    Bayley, Hagan; Shin, Seong-Ho; Luchian, Tudor; Cheley, Stephen

    2013-03-26

    A system and method for stochastic sensing in which the analyte covalently bonds to the sensor element or an adaptor element. If such bonding is irreversible, the bond may be broken by a chemical reagent. The sensor element may be a protein, such as the engineered P.sub.SH type or .alpha.HL protein pore. The analyte may be any reactive analyte, including chemical weapons, environmental toxins and pharmaceuticals. The analyte covalently bonds to the sensor element to produce a detectable signal. Possible signals include change in electrical current, change in force, and change in fluorescence. Detection of the signal allows identification of the analyte and determination of its concentration in a sample solution. Multiple analytes present in the same solution may be detected.

  1. Highly Emissive Covalent Organic Frameworks.

    PubMed

    Dalapati, Sasanka; Jin, Enquan; Addicoat, Matthew; Heine, Thomas; Jiang, Donglin

    2016-05-11

    Highly luminescent covalent organic frameworks (COFs) are rarely achieved because of the aggregation-caused quenching (ACQ) of π-π stacked layers. Here, we report a general strategy to design highly emissive COFs by introducing an aggregation-induced emission (AIE) mechanism. The integration of AIE-active units into the polygon vertices yields crystalline porous COFs with periodic π-stacked columnar AIE arrays. These columnar AIE π-arrays dominate the luminescence of the COFs, achieve exceptional quantum yield via a synergistic structural locking effect of intralayer covalent bonding and interlayer noncovalent π-π interactions and serve as a highly sensitive sensor to report ammonia down to sub ppm level. Our strategy breaks through the ACQ-based mechanistic limitations of COFs and opens a way to explore highly emissive COF materials. PMID:27108740

  2. Outward Bound.

    ERIC Educational Resources Information Center

    Outward Bound, Inc., Andover, MA.

    The Outward Bound concept was developed in Germany and Great Britain with the saving of human life as the ultimate goal. Courses are designed to help students discover their true physical and mental limits through development of skills including emergency medical aid, firefighting, search and rescue, mountaineering, and sailing. Five Outward Bound…

  3. Thermochemolysis of the Murchison meteorite: identification of oxygen bound and occluded units in the organic macromolecule

    NASA Astrophysics Data System (ADS)

    Watson, Jonathan S.; Sephton, Mark A.; Gilmour, Iain

    2010-10-01

    An organic macromolecular residue, prepared from the Murchison meteorite by treatment with hydrofluoric and hydrochloric acids, was subjected to online thermochemolysis with tetramethylammonium hydroxide (TMAH). The most abundant compound released by thermochemolysis was benzoic acid. Other abundant compounds include methyl and dimethyl benzoic acids as well as methoxy benzoic acids. Short chain dicarboxylic acids (C4-8) were also released from the organic macromolecule. Within the C1 and C2 benzoic acids all possible structural isomers are present reflecting the abiotic origin of these units. The most abundant isomers include 3,4-dimethylbenzoic acid (DMBA), 3,5-DMBA, 2,6-DMBA and phenylacetic acid. Thermochemolysis also liberates hydrocarbons that are not observed during thermal desorption; these compounds include naphthalene, methylnaphthalenes, biphenyl, methylbiphenyls, acenaphthylene, acenaphthene, phenanthrene, anthracene, fluoranthene and pyrene. The lack of oxygen containing functional groups in these hydrocarbons indicates that they represent non-covalently bound, occluded molecules within the organic framework. This data provides a valuable insight into oxygen bound and physically occluded moieties in the Murchison organic macromolecule and implies a relative order of synthesis or agglomeration for the detected organic constituents.

  4. The biliverdin chromophore binds covalently to a conserved cysteine residue in the N-terminus of Agrobacterium phytochrome Agp1.

    PubMed

    Lamparter, Tilman; Carrascal, Montserrat; Michael, Norbert; Martinez, Enriqueta; Rottwinkel, Gregor; Abian, Joaquin

    2004-03-30

    Phytochromes are widely distributed biliprotein photoreceptors. Typically, the chromophore becomes covalently linked to the protein during an autocatalytic lyase reaction. Plant and cyanobacterial phytochromes incorporate bilins with a ring A ethylidene side chain, whereas other bacterial phytochromes utilize biliverdin as chromophore, which has a vinyl ring A side chain. For Agrobacterium phytochrome Agp1, site-directed mutagenesis provided evidence that biliverdin is bound to cysteine 20. This cysteine is highly conserved within bacterial homologues, but its role as attachment site has as yet not been proven. We therefore performed mass spectrometry studies on proteolytic holopeptide fragments. For that purpose, an Agp1 expression vector was re-engineered to produce a protein with an N-terminal affinity tag. Following proteolysis, the chromophore co-purified with a ca. 5 kDa fragment during affinity chromatography, showing that the attachment site is located close to the N-terminus. Mass spectrometry analyses performed with the purified chromopeptide confirmed the role of the cysteine 20 as biliverdin attachment site. We also analyzed the role of the highly conserved histidine 250 by site-directed mutagenesis. The homologous amino acid plays an important but yet undefined role in plant phytochromes and has been proposed as chromophore attachment site of Deinococcus phytochrome. We found that in Agp1, this amino acid is dispensable for covalent attachment, but required for tight chromophore-protein interaction. PMID:15035636

  5. Factors Affecting Peptide Interactions with Surface-Bound Microgels.

    PubMed

    Nyström, Lina; Nordström, Randi; Bramhill, Jane; Saunders, Brian R; Álvarez-Asencio, Rubén; Rutland, Mark W; Malmsten, Martin

    2016-02-01

    Effects of electrostatics and peptide size on peptide interactions with surface-bound microgels were investigated with ellipsometry, confocal microscopy, and atomic force microscopy (AFM). Results show that binding of cationic poly-L-lysine (pLys) to anionic, covalently immobilized, poly(ethyl acrylate-co-methacrylic acid) microgels increased with increasing peptide net charge and microgel charge density. Furthermore, peptide release was facilitated by decreasing either microgel or peptide charge density. Analogously, increasing ionic strength facilitated peptide release for short peptides. As a result of peptide binding, the surface-bound microgels displayed pronounced deswelling and increased mechanical rigidity, the latter quantified by quantitative nanomechanical mapping. While short pLys was found to penetrate the entire microgel network and to result in almost complete charge neutralization, larger peptides were partially excluded from the microgel network, forming an outer peptide layer on the microgels. As a result of this difference, microgel flattening was more influenced by the lower Mw peptide than the higher. Peptide-induced deswelling was found to be lower for higher Mw pLys, the latter effect not observed for the corresponding microgels in the dispersed state. While the effects of electrostatics on peptide loading and release were similar to those observed for dispersed microgels, there were thus considerable effects of the underlying surface on peptide-induced microgel deswelling, which need to be considered in the design of surface-bound microgels as carriers of peptide loads, for example, in drug delivery or in functionalized biomaterials. PMID:26750986

  6. Protein covalent immobilization via its scarce thiol versus abundant amine groups: Effect on orientation, cell binding domain exposure and conformational lability.

    PubMed

    Ba, O M; Hindie, M; Marmey, P; Gallet, O; Anselme, K; Ponche, A; Duncan, A C

    2015-10-01

    Quantity, orientation, conformation and covalent linkage of naturally cell adhesive proteins adsorbed or covalently linked to a surface, are known to influence the preservation of their subsequent long term cell adhesion properties and bioactivity. In the present work, we explore two different strategies for the covalent linking of plasma fibronectin (pFN) - used as a cell adhesive model protein, onto a polystyrene (PS) surface. One is aimed at tethering the protein to the surface in a semi-oriented fashion (via one of the 4 free thiol reactive groups on the protein) with a heterofunctional coupling agent (SSMPB method). The other aims to immobilize the protein in a more random fashion by reaction between the abundant pendant primary amine bearing amino acids of the pFN and activated carboxylic surface functions obtained after glutaric anhydride surface treatment (GA method). The overall goal will be to verify the hypothesis of a correlation between covalent immobilization of a model cell adhesive protein to a PS surface in a semi-oriented configuration (versus randomly oriented) with promotion of enhanced exposure of the protein's cell binding domain. This in turn would lead to enhanced cell adhesion. Ideally the goal is to elaborate substrates exhibiting a long term stable protein monolayer with preserved cell adhesive properties and bioactivity for biomaterial and/or cell adhesion commercial plate applications. However, the initial restrictive objective of this paper is to first quantitatively and qualitatively investigate the reversibly (merely adsorbed) versus covalently irreversibly bound protein to the surface after the immobilization procedure. Although immobilized surface amounts were similar (close to the monolayer range) for all immobilization approaches, covalent grafting showed improved retention and stronger "tethering" of the pFN protein to the surface (roughly 40%) after SDS rinsing compared to that for mere adsorption (0%) suggesting an added value

  7. Chemically stable multilayered covalent organic nanosheets from covalent organic frameworks via mechanical delamination.

    PubMed

    Chandra, Suman; Kandambeth, Sharath; Biswal, Bishnu P; Lukose, Binit; Kunjir, Shrikant M; Chaudhary, Minakshi; Babarao, Ravichandar; Heine, Thomas; Banerjee, Rahul

    2013-11-27

    A series of five thermally and chemically stable functionalized covalent organic frameworks (COFs), namely, TpPa-NO2, TpPa-F4, TpBD-(NO2)2, TpBD-Me2, and TpBD-(OMe)2 were synthesized by employing the solvothermal aldehyde-amine Schiff base condensation reaction. In order to complete the series, previously reported TpPa-1, TpPa-2, and TpBD have also been synthesized, and altogether, eight COFs were fully characterized through powder X-ray diffraction (PXRD), Fourier transform IR (FT-IR) spectroscopy, (13)C solid-state NMR spectroscopy, and thermogravimetric analysis. These COFs are crystalline, permanently porous, and stable in boiling water, acid (9 N HCl), and base (3 N NaOH). The synthesized COFs (all eight) were successfully delaminated using a simple, safe, and environmentally friendly mechanical grinding route to transform into covalent organic nanosheets (CONs) and were well characterized via transmission electron microscopy and atomic force microscopy. Further PXRD and FT-IR analyses confirm that these CONs retain their structural integrity throughout the delamination process and also remain stable in aqueous, acidic, and basic media like the parent COFs. These exfoliated CONs have graphene-like layered morphology (delaminated layers), unlike the COFs from which they were synthesized. PMID:24168521

  8. Capillary electrophoresis methods for the determination of covalent polyphenol-protein complexes.

    PubMed

    Trombley, John D; Loegel, Thomas N; Danielson, Neil D; Hagerman, Ann E

    2011-09-01

    The bioactivities and bioavailability of plant polyphenols including proanthocyanidins and other catechin derivatives may be affected by covalent reaction between polyphenol and proteins. Both processing conditions and gastrointestinal conditions may promote formation of covalent complexes for polyphenol-rich foods and beverages such as wine. Little is known about covalent reactions between proteins and tannin, because suitable methods for quantitating covalent complexes have not been developed. We established capillary electrophoresis methods that can be used to distinguish free protein from covalently bound protein-polyphenol complexes and to monitor polyphenol oxidation products. The methods are developed using the model protein bovine serum albumin and the representative polyphenol (-)epigallocatechin gallate. By pairing capillaries with different diameters with appropriate alkaline borate buffers, we are able to optimize resolution of either the protein-polyphenol complexes or the polyphenol oxidation products. This analytical method, coupled with purification of the covalent complexes by diethylaminoethyl cellulose chromatography, should facilitate characterization of covalent complexes in polyphenol-rich foods and beverages such as wine. PMID:21400190

  9. Functional systems with orthogonal dynamic covalent bonds.

    PubMed

    Wilson, Adam; Gasparini, Giulio; Matile, Stefan

    2014-03-21

    This review summarizes the use of orthogonal dynamic covalent bonds to build functional systems. Dynamic covalent bonds are unique because of their dual nature. They can be as labile as non-covalent interactions or as permanent as covalent bonds, depending on conditions. Examples from nature, reaching from the role of disulfides in protein folding to thioester exchange in polyketide biosynthesis, indicate how dynamic covalent bonds are best used in functional systems. Several synthetic functional systems that employ a single type of dynamic covalent bonds have been reported. Considering that most functional systems make simultaneous use of several types of non-covalent interactions together, one would expect the literature to contain many examples in which different types of dynamic covalent bonds are similarly used in tandem. However, the incorporation of orthogonal dynamic covalent bonds into functional systems is a surprisingly rare and recent development. This review summarizes the available material comprehensively, covering a remarkably diverse collection of functions. However, probably more revealing than the specific functions addressed is that the questions asked are consistently quite unusual, very demanding and highly original, focusing on molecular systems that can self-sort, self-heal, adapt, exchange, replicate, transcribe, or even walk and "think" (logic gates). This focus on adventurous chemistry off the beaten track supports the promise that with orthogonal dynamic covalent bonds we can ask questions that otherwise cannot be asked. The broad range of functions and concepts covered should appeal to the supramolecular organic chemist but also to the broader community. PMID:24287608

  10. Mechanism of Orlistat Hydrolysis by the Thioesterase of Human Fatty Acid Synthase

    PubMed Central

    2015-01-01

    Fatty acid synthase (FASN), the sole protein capable of de novo synthesis of free fatty acids, is overexpressed in a wide variety of human cancers and is associated with poor prognosis and aggressiveness of these cancers. Orlistat, an FDA-approved drug for obesity treatment that inhibits pancreatic lipases in the GI tract, also inhibits the thioesterase (TE) of human FASN. The cocrystal structure of TE with orlistat shows a pseudo TE dimer containing two different forms of orlistat in the active site, an intermediate that is covalently bound to a serine residue (Ser2308) and a hydrolyzed and inactivated product. In this study, we attempted to understand the mechanism of TE-catalyzed orlistat hydrolysis by examining the role of the hexyl tail of the covalently bound orlistat in water activation for hydrolysis using molecular dynamics simulations. We found that the hexyl tail of the covalently bound orlistat undergoes a conformational transition, which is accompanied by destabilization of a hydrogen bond between a hydroxyl moiety of orlistat and the catalytic His2481 of TE that in turn leads to an increased hydrogen bonding between water molecules and His2481 and increased chance for water activation to hydrolyze the covalent bond between orlistat and Ser2308. Thus, the conformation of the hexyl tail of orlistat plays an important role in orlistat hydrolysis. Strategies that stabilize the hexyl tail may lead to the design of more potent irreversible inhibitors that target FASN and block TE activity with greater endurance. PMID:25309810

  11. Stimulation of Tetrabromobisphenol A Binding to Soil Humic Substances by Birnessite and the Chemical Structure of the Bound Residues.

    PubMed

    Tong, Fei; Gu, Xueyuan; Gu, Cheng; Xie, Jinyu; Xie, Xianchuan; Jiang, Bingqi; Wang, Yongfeng; Ertunc, Tanya; Schäffer, Andreas; Ji, Rong

    2016-06-21

    Studies have shown the main fate of the flame retardant tetrabromobisphenol A (TBBPA) in soils is the formation of bound residues, and mechanisms on it are less-understood. This study investigated the effect of birnessite (δ-MnO2), a naturally occurring oxidant in soils, on the formation of bound residues. (14)C-labeled TBBPA was used to investigate the pH dependency of TBBPA bound-residue formation to two soil humic acids (HAs), Elliott soil HA and Steinkreuz soil HA, in the presence of δ-MnO2. The binding of TBBPA and its transformation products to both HAs was markedly increased (3- to 17-fold) at all pH values in the presence of δ-MnO2. More bound residues were formed with the more aromatic Elliott soil HA than with Steinkreuz soil HA. Gel-permeation chromatography revealed a uniform distribution of the bound residues within Steinkreuz soil HA and a nonuniform distribution within Elliott soil HA. (13)C NMR spectroscopy of (13)C-TBBPA residues bound to (13)C-depleted HA suggested that in the presence of δ-MnO2, binding occurred via ester and ether and other types of covalent bonds besides HA sequestration. The insights gained in this study contribute to an understanding of the formation of TBBPA bound residues facilitated by δ-MnO2. PMID:27223831

  12. Covalent bulk functionalization of graphene.

    PubMed

    Englert, Jan M; Dotzer, Christoph; Yang, Guang; Schmid, Martin; Papp, Christian; Gottfried, J Michael; Steinrück, Hans-Peter; Spiecker, Erdmann; Hauke, Frank; Hirsch, Andreas

    2011-04-01

    Graphene, a truly two-dimensional and fully π-conjugated honeycomb carbon network, is currently evolving into the most promising successor to silicon in micro- and nanoelectronic applications. However, its wider application is impeded by the difficulties in opening a bandgap in its gapless band-structure, as well as the lack of processability in the resultant intrinscially insoluble material. Covalent chemical modification of the π-electron system is capable of addressing both of these issues through the introduction of variable chemical decoration. Although there has been significant research activity in the field of functionalized graphene, most work to date has focused on the use of graphene oxide. In this Article, we report on the first wet chemical bulk functionalization route beginning with pristine graphite that does not require initial oxidative damage of the graphene basal planes. Through effective reductive activation, covalent functionalization of the charged graphene is achieved by organic diazonium salts. Functionalization was observed spectroscopically, and successfully prevents reaggregation while providing solubility in common organic media. PMID:21430685

  13. Simultaneous covalent and noncovalent hybrid polymerizations

    NASA Astrophysics Data System (ADS)

    Yu, Zhilin; Tantakitti, Faifan; Yu, Tao; Palmer, Liam C.; Schatz, George C.; Stupp, Samuel I.

    2016-01-01

    Covalent and supramolecular polymers are two distinct forms of soft matter, composed of long chains of covalently and noncovalently linked structural units, respectively. We report a hybrid system formed by simultaneous covalent and supramolecular polymerizations of monomers. The process yields cylindrical fibers of uniform diameter that contain covalent and supramolecular compartments, a morphology not observed when the two polymers are formed independently. The covalent polymer has a rigid aromatic imine backbone with helicoidal conformation, and its alkylated peptide side chains are structurally identical to the monomer molecules of supramolecular polymers. In the hybrid system, covalent chains grow to higher average molar mass relative to chains formed via the same polymerization in the absence of a supramolecular compartment. The supramolecular compartments can be reversibly removed and re-formed to reconstitute the hybrid structure, suggesting soft materials with novel delivery or repair functions.

  14. Simultaneous covalent and noncovalent hybrid polymerizations.

    PubMed

    Yu, Zhilin; Tantakitti, Faifan; Yu, Tao; Palmer, Liam C; Schatz, George C; Stupp, Samuel I

    2016-01-29

    Covalent and supramolecular polymers are two distinct forms of soft matter, composed of long chains of covalently and noncovalently linked structural units, respectively. We report a hybrid system formed by simultaneous covalent and supramolecular polymerizations of monomers. The process yields cylindrical fibers of uniform diameter that contain covalent and supramolecular compartments, a morphology not observed when the two polymers are formed independently. The covalent polymer has a rigid aromatic imine backbone with helicoidal conformation, and its alkylated peptide side chains are structurally identical to the monomer molecules of supramolecular polymers. In the hybrid system, covalent chains grow to higher average molar mass relative to chains formed via the same polymerization in the absence of a supramolecular compartment. The supramolecular compartments can be reversibly removed and re-formed to reconstitute the hybrid structure, suggesting soft materials with novel delivery or repair functions. PMID:26823427

  15. Constructing monocrystalline covalent organic networks by polymerization

    NASA Astrophysics Data System (ADS)

    Beaudoin, Daniel; Maris, Thierry; Wuest, James D.

    2013-10-01

    An emerging strategy for making ordered materials is modular construction, which connects preformed molecular subunits to neighbours through interactions of properly selected reactive sites. This strategy has yielded remarkable materials, including metal-organic frameworks joined by coordinative bonds, supramolecular networks linked by strong non-covalent interactions, and covalent organic frameworks in which atoms of carbon and other light elements are bonded covalently. However, the strategy has not yet produced covalently bonded organic materials in the form of large single crystals. Here we show that such materials can result from reversible self-addition polymerizations of suitably designed monomers. In particular, monomers with four tetrahedrally oriented nitroso groups polymerize to form diamondoid azodioxy networks that can be fully characterized by single-crystal X-ray diffraction. This work forges a strong new link between polymer science and supramolecular chemistry by showing how predictably ordered covalent or non-covalent structures can both be built using a single modular strategy.

  16. Cell wall bound anionic peroxidases from asparagus byproducts.

    PubMed

    Jaramillo-Carmona, Sara; López, Sergio; Vazquez-Castilla, Sara; Jimenez-Araujo, Ana; Rodriguez-Arcos, Rocio; Guillen-Bejarano, Rafael

    2014-10-01

    Asparagus byproducts are a good source of cationic soluble peroxidases (CAP) useful for the bioremediation of phenol-contaminated wastewaters. In this study, cell wall bound peroxidases (POD) from the same byproducts have been purified and characterized. The covalent forms of POD represent >90% of the total cell wall bound POD. Isoelectric focusing showed that whereas the covalent fraction is constituted primarily by anionic isoenzymes, the ionic fraction is a mixture of anionic, neutral, and cationic isoenzymes. Covalently bound peroxidases were purified by means of ion exchange chromatography and affinity chromatography. In vitro detoxification studies showed that although CAP are more effective for the removal of 4-CP and 2,4-DCP, anionic asparagus peroxidase (AAP) is a better option for the removal of hydroxytyrosol (HT), the main phenol present in olive mill wastewaters. PMID:25195693

  17. Interaction of functionally bound vitamins in the distribution and metabolism of (/sup 14/C)nicotinic acid in tissues and blood cells

    SciTech Connect

    Rozanov, A.Ya.; Yakubik, E.Yu.

    1986-03-10

    Leukocytes absorb two orders of magnitude more of labeled nicotinic acid ((/sup 14/C)NA) than erythrocytes (recalculated per cell). The dynamics of the binding of the labeled vitamin by leukocytes is biphasic, with the formation chiefly of (/sup 14/C)-nicotinic coenzymes in the shortest periods after its injection into rats. At the same time, injected thiamine, riboflavin, lipoate, and pantothenate increase the accumulation of labeled metabolites of nicotinate in the blood and leukocytes of rats by a factor of 2.1 and 4.1, respectively. The metabolism of subcutaneously injected (/sup 14/C)NA occurs chiefly in the digestive system, with a pronounced biphasic dynamics of the changes in the content of labeled metabolites in the liver and small intestine, with secretion of substantial amounts of them with the digestive juices. At the same time, functionally bound vitamins introduced increase the incorporation of the total label into liver tissue (to 45% of the introduced dose, versus 33% in the control) and the rise in the content of (/sup 14/C)-pyridine nucleotides. Analogous effects were also observed in the accumulation of labeled metabolites of (/sup 14/C)NA in the membranes of the small intestine.

  18. Repeating covalent structure of streptococcal M protein.

    PubMed Central

    Beachey, E H; Seyer, J M; Kang, A H

    1978-01-01

    We have attempted to identify the covalent structure of the M protein molecule of group A streptococci that is responsible for inducing type-specific, protective immunity. M protein was extracted from type 24 streptococci, purified, and cleaved with cyanogen bromide. Seven cyanogen bromide peptides were purified and further characterized. Together, the peptides account for the entire amino acid content of the M protein molecule. Each of the purified peptides possessed the type-specific determinant that inhibits opsonic antibodies for group A streptococci. The primary structures of the amino-terminal regions of each of the purified peptides was studied by automated Edman degradation. The partial sequences of two of the peptides were found to be identical to each other and to that of the uncleaved M protein molecule through at least the first 27 residues. The amino-terminal sequences of the remaining five peptides were identical to each other through the twentieth residue but completely different from the amino-terminal region of the other two peptides. However, the type-specific immunoreactivity and the incomplete analysis of the primary structure of the seven peptides suggest that the antiphagocytic determinant resides in a repeating amino acid sequence in the M protein molecule. PMID:80011

  19. Covalent organic/inorganic hybrid proton-conductive membrane with semi-interpenetrating polymer network: Preparation and characterizations

    NASA Astrophysics Data System (ADS)

    Fu, Rong-Qiang; Woo, Jung-Je; Seo, Seok-Jun; Lee, Jae-Suk; Moon, Seung-Hyeon

    2008-05-01

    A series of new covalent organic/inorganic hybrid proton-conductive membranes, each with a semi-interpenetrating polymer network (semi-IPN), for direct methanol fuel cell (DMFC) applications is prepared through the following sequence: (i) copolymerization of impregnated styrene (St), p-vinylbenzyl chloride (VBC) and divinylbenzene (DVB) within a supporting polyvinyl chloride (PVC) film; (ii) reaction of the chloromethyl group with 3-(methylamine)propyl-trimethoxysilane (MAPTMS); (ii) a sol-gel process under acidic conditions; (iv) a sulfonation reaction. The developed membranes are characterized in terms of Fourier transform infrared/attenuated total reflectance (FTIR/ATR), scanning electron microscopy/energy-dispersive X-ray analysis (SEM/EDXA), elemental analysis (EA) and thermogravimetric analysis (TGA), which confirm the formation of the target membranes. The developed copolymer chains are interpenetrating with the PVC matrix to form the semi-IPN structure, and the inorganic silica is covalently bound to the copolymers. These features provide the membranes with high mechanical strength. The effect of silica content is investigated. As the silica content increases, proton conductivity and water content decrease, whereas oxidative stability is improved. In particular, methanol permeability and methanol uptake are reduced largely by the silica. The ratio of proton conductivity to methanol permeability for the hybrid membranes is higher than that of Nafion 117. All these properties make the hybrid membranes a potential candidate for DMFC applications.

  20. Covalent complex of microperoxidase with a 21-residue synthetic peptide as a maquette for low-molecular-mass redox proteins.

    PubMed Central

    Ippoliti, R; Picciau, A; Santucci, R; Antonini, G; Brunori, M; Ranghino, G

    1997-01-01

    Here we report the structural and functional characterization of a covalent complex (MKP) obtained by cross-linking microperoxidase (Mp), the haem-undecapeptide obtained by the peptic digestion of cytochrome c, with a 21-residue synthetic peptide (P21) analogous to the S-peptide of the RNase A. The covalent complex has been prepared by introducing a disulphide bond between Cys-1 of P21 and Lys-13 of Mp, previously modified with a thiol-containing reagent. On formation of the complex (which is a monomer), the helical content of P21 increases significantly. The results obtained indicate that His-13 of P21 co-ordinates to the sixth co-ordination position of the haem iron, thus leading to the formation of a complex characterized by an equilibrium between an 'open' and a 'closed' structure, as confirmed by molecular dynamics simulations. Under acidic pH conditions, where His-13 of P21 is loosely bound to the haem iron ('open' conformation), MKP displays appreciable, quasi-reversible electrochemical activity; in contrast, at neutral pH ('closed' conformation) electrochemical behaviour is negligible, indicating that P21 interferes with the electron-transfer properties typical of Mp. On the whole, MKP is a suitable starting material for building a miniature haem system, with interesting potential for application to biosensor technology. PMID:9396728

  1. Athermal fracture of covalent bonds

    SciTech Connect

    Gilman, J.J.

    1999-08-01

    Most fracture is athermal. Either because it occurs at low temperatures or because it occurs too fast for thermal activation to be effective. Thus it must be directly activated by applied stresses. This can occur via quantum tunneling when the chemical bonding of a solid resides in localized (covalent) bonds. Then applied stresses can cause the bonding electrons to become delocalized (anti-bonded) through quantum tunneling. That is, the bonds become broken. The process is related to the Zener tunneling process that is thought to be responsible for dielectric breakdown in semiconductors. Under a driving force, bonding electrons tunnel at constant energy from their bonding states into anti-bonding states through the forbidden gap in the bonding energy spectrum.

  2. Covalent Binding on the Femtometer Scale: Nuclear Molecules

    NASA Astrophysics Data System (ADS)

    von Oertzen, Wolfram; Milin, Matko

    Nuclear molecules are objects having two or more individual clusters as centres with extra nucleons (usually neutrons) binding them. The clusters have to be strongly bound themselves, while they get bound into molecules due to the specific properties of the nucleus-nucleus potentials and exchange of nucleons. The molecular, quantum mechanical covalent binding effect via the exchange of neutrons is the dominant source of binding in many light nuclei, overcoming the Coulomb repulsion in such structures. This results in states having valence neutrons in the π and σ orbitals, known from molecules in atomic physics; in this paper we discuss the structural properties of such states in light nuclei. The interaction between clusters resulting in nuclear molecules should show a repulsive interaction at smaller distances. This is generally the case for strongly bound clusters, like α-particles (but also for the 12C and 16O nuclei)—due to the Pauli principle, nucleons penetrating the second cluster show then a strong repulsion effect. The particular properties of nuclear clusters needed for the formation of bound molecules is their intrinsic stiffness (a large gap to the first excited states), which inhibits their excitation and allows their survival in a two-centre configuration. A large number of strongly deformed nuclear states in light nuclei with neutron excess have been experimentally identified in the last decades, and some of them have been associated with covalent structures, mainly via their grouping into rotational bands. These results have been confirmed in many theoretical studies with nuclear cluster models, but also in model independent calculations, e.g. in the Antisymmetrized Molecular Dynamics approach, where no a priori cluster structure is assumed.

  3. Covalent immobilisation of VEGF on plasma-coated electrospun scaffolds for tissue engineering applications.

    PubMed

    Guex, A G; Hegemann, D; Giraud, M N; Tevaearai, H T; Popa, A M; Rossi, R M; Fortunato, G

    2014-11-01

    Recent findings in the field of biomaterials and tissue engineering provide evidence that surface immobilised growth factors display enhanced stability and induce prolonged function. Cell response can be regulated by material properties and at the site of interest. To this end, we developed scaffolds with covalently bound vascular endothelial growth factor (VEGF) and evaluated their mitogenic effect on endothelial cells in vitro. Nano- (254±133 nm) or micro-fibrous (4.0±0.4 μm) poly(ɛ-caprolactone) (PCL) non-wovens were produced by electrospinning and coated in a radio frequency (RF) plasma process to induce an oxygen functional hydrocarbon layer. Implemented carboxylic acid groups were converted into amine-reactive esters and covalently coupled to VEGF by forming stable amide bonds (standard EDC/NHS chemistry). Substrates were analysed by X-ray photoelectron spectroscopy (XPS), enzyme-linked immuno-assays (ELISA) and immunohistochemistry (anti-VEGF antibody and VEGF-R2 binding). Depending on the reaction conditions, immobilised VEGF was present at 127±47 ng to 941±199 ng per substrate (6mm diameter; concentrations of 4.5 ng mm(-2) or 33.3 ng mm(-2), respectively). Immunohistochemistry provided evidence for biological integrity of immobilised VEGF. Endothelial cell number of primary endothelial cells or immortalised endothelial cells were significantly enhanced on VEGF-functionalised scaffolds compared to native PCL scaffolds. This indicates a sustained activity of immobilised VEGF over a culture period of nine days. We present a versatile method for the fabrication of growth factor-loaded scaffolds at specific concentrations. PMID:25454657

  4. Electron tunneling through covalent and noncovalent pathways in proteins

    NASA Technical Reports Server (NTRS)

    Beratan, David N.; Onuchic, Jose Nelson; Hopfield, J. J.

    1987-01-01

    A model is presented for electron tunneling in proteins which allows the donor-acceptor interaction to be mediated by the covalent bonds between amino acids and noncovalent contacts between amino acid chains. The important tunneling pathways are predicted to include mostly bonded groups with less favorable nonbonded interactions being important when the through bond pathway is prohibitively long. In some cases, vibrational motion of nonbonded groups along the tunneling pathway strongly influences the temperature dependence of the rate. Quantitative estimates for the sizes of these noncovalent interactions are made and their role in protein mediated electron transport is discussed.

  5. Exceptional ammonia uptake by a covalent organic framework

    NASA Astrophysics Data System (ADS)

    Doonan, Christian J.; Tranchemontagne, David J.; Glover, T. Grant; Hunt, Joseph R.; Yaghi, Omar M.

    2010-03-01

    Covalent organic frameworks (COFs) are porous crystalline materials composed of light elements linked by strong covalent bonds. A number of these materials contain a high density of Lewis acid boron sites that can strongly interact with Lewis basic guests, which makes them ideal for the storage of corrosive chemicals such as ammonia. We found that a member of the covalent organic framework family, COF-10, shows the highest uptake capacity (15 mol kg-1, 298 K, 1 bar) of any porous material, including microporous 13X zeolite (9 mol kg-1), Amberlyst 15 (11 mol kg-1) and mesoporous silica, MCM-41 (7.9 mol kg-1). Notably, ammonia can be removed from the pores of COF-10 by heating samples at 200 °C under vacuum. In addition, repeated adsorption of ammonia into COF-10 causes a shift in the interlayer packing, which reduces its apparent surface area to nitrogen. However, owing to the strong Lewis acid-base interactions, the total uptake capacity of ammonia and the structural integrity of the COF are maintained after several cycles of adsorption/desorption.

  6. Cellular delivery of quantum dot-bound hybridization probe for detection of intracellular pre-microRNA using chitosan/poly(γ-glutamic acid) complex as a carrier.

    PubMed

    Geng, Yao; Lin, Dajie; Shao, Lijia; Yan, Feng; Ju, Huangxian

    2013-01-01

    A quantum dot (QD)-bound hybridization probe was designed for detection of intracellular pre-miRNA using chitosan (CS)/poly(γ-glutamic acid) (γ-PGA) complex as a gene vector. The probe was prepared by assembling thiolated RNA to gold nanoparticle (Au NP) via Au-S bond and then binding 3'-end amine of the RNA to the carboxy group capped on quantum dot surface. The QD-RNA-Au NP probe was assembled on the vector by mixing with aqueous γ-PGA solution and then CS solution to construct a gene delivery system for highly effective cellular uptake and delivery. After the probe was released from CS/γ-PGA complex to the cytoplasm by electrostatic repulsion at intracellular pH, it hybridized with pre-miRNA precursor as target. The formed product was then cleaved by RNase III Dicer, leading to the separation of QDs from Au NPs and fluorescence emission of QDs, which could be detected by confocal microscopic imaging to monitor the amount of the intracellular pre-miRNA precursor. The in vitro assays revealed that the QD-RNA-Au NP was a robust, sensitive and selective probe for quantitative detection of target pre-miRNA. Using MDA-MB231 and MCF-7 breast cancer cells as models, the relative amount of pre-miRNA let-7a could be successfully compared. Since the amount of miRNA is related to the progress and prognosis of cancer, this strategy could be expected to hold promising application potential in medical research and clinical diagnostics. PMID:23762388

  7. Theoretical insights into covalency driven f element separations.

    PubMed

    Roy, Lindsay E; Bridges, Nicholas J; Martin, Leigh R

    2013-02-21

    Through Density Function Theory (DFT) calculations, we set out to understand the structures and stabilities of the aqueous phase complexes [M(III)(DTPA)-H(2)O](2-) (M = Nd, Am) as well as the changes in Gibbs free energy for complexation in the gas phase and aqueous solution. All bonding analyses suggest that the preference of the DTPA(5-) ligand for Am over Nd is mainly due to electrostatic and covalent interactions from the oxygen atoms with the nitrogen chelates providing an additional, yet small, covalent interaction. These results question the exclusive use of hard and soft acids and bases (HSAB) concepts for the design of extracting reagents and suggest that hard-soft interactions may play more of a role in the separations process than previously thought. PMID:23223573

  8. Production of a covalent flavin linkage in lipoamide dehydrogenase. Reaction with 8-Cl-FAD.

    PubMed

    Moore, E G; Cardemil, E; Massey, V

    1978-09-25

    A method is described for preparation of apolipoamide dehydrogenase which gives quantitative removal of FAD. Active holoenzyme can be reconstituted by incubation with FAD. Reconstitution of apoenzyme with 8-Cl-FAD results in the fixation of most of the flavin to the protein in a covalently bound form. The portion noncovalently bound was shown to be unmodified 8-Cl-FAD. The covalently bound flavin has an absorption spectrum quite different from that of 8-Cl-FAD. It has a single band in the visible with a maximum at 459 nm (extinction coefficient of 22 mM-1 cm-1) and a shoulder at 480 nm. Model reactions between 8-Cl-Flavin (riboflavin or FAD) and organic thiols (thiophenol, beta-mercaptoethanol, or N-acetylcysteine) give products with spectra which are similar to that of FAD covalently bound to lipoamide dehydrogenase. The products of the model reactions have a single visible band with a maximum at 480 nm (extinction coefficient of 23.6 mM-1 cm-1 to 28.4 mM-1 cm-1) and a shoulder at 460 nm. The products of the model reaction and the covalently bound FAD of lipoamide dehydrogenase appear to be the result of a nucleophilic attack on the carbon at position 8 of the flavin ring by a thiolate anion, displacing the chloride. Thus, the product of the model reaction is 8-(RS)-flavin, and the product of the reaction between 8-Cl-FAD and protein probably has a cysteinyl residue covalently attacked at position 8 of FAD. Reconstitution of apoliopoamide dehydrogenase with 8-Cl-FAD gives two enzyme products which are fractionated by ammonium sulfate. Enzyme fractionating between 20% and 45% ammonium sulfate is monomeric and contains covanently bound FAD. Enzyme fractionating between 55% and 75% ammonium sulfate is dimeric and contains both covalently bound FAD and noncovalently bound 8-Cl-FAD. Both protein fractions contain one FAD per protein subunit and both are active with physiological substrates with Km values for NAD and dihydrolipoamide similar to those of native lipoamide

  9. Covalently crosslinked diels-alder polymer networks.

    SciTech Connect

    Bowman, Christopher; Adzima, Brian J.; Anderson, Benjamin John

    2011-09-01

    This project examines the utility of cycloaddition reactions for the synthesis of polymer networks. Cycloaddition reactions are desirable because they produce no unwanted side reactions or small molecules, allowing for the formation of high molecular weight species and glassy crosslinked networks. Both the Diels-Alder reaction and the copper-catalyzed azide-alkyne cycloaddition (CuAAC) were studied. Accomplishments include externally triggered healing of a thermoreversible covalent network via self-limited hysteresis heating, the creation of Diels-Alder based photoresists, and the successful photochemical catalysis of CuAAC as an alternative to the use of ascorbic acid for the generation of Cu(I) in click reactions. An analysis of the results reveals that these new methods offer the promise of efficiently creating robust, high molecular weight species and delicate three dimensional structures that incorporate chemical functionality in the patterned material. This work was performed under a Strategic Partnerships LDRD during FY10 and FY11 as part of a Sandia National Laboratories/University of Colorado-Boulder Excellence in Science and Engineering Fellowship awarded to Brian J. Adzima, a graduate student at UC-Boulder. Benjamin J. Anderson (Org. 1833) was the Sandia National Laboratories point-of-contact for this fellowship.

  10. Proteins and Amino Acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins are the most abundant substances in living organisms and cells. All proteins are constructed from the same twenty amino acids that are linked together by covalent bonds. Shorter chains of two or more amino acids can be linked by covalent bonds to form polypeptides. There are twenty amino...

  11. Design and pharmacological characterization of VUF14480, a covalent partial agonist that interacts with cysteine 983.36 of the human histamine H4 receptor

    PubMed Central

    Nijmeijer, S; Engelhardt, H; Schultes, S; van de Stolpe, A C; Lusink, V; de Graaf, C; Wijtmans, M; Haaksma, E E J; de Esch, I J P; Stachurski, K; Vischer, H F; Leurs, R

    2013-01-01

    Background and Purpose The recently proposed binding mode of 2-aminopyrimidines to the human (h) histamine H4 receptor suggests that the 2-amino group of these ligands interacts with glutamic acid residue E1825.46 in the transmembrane (TM) helix 5 of this receptor. Interestingly, substituents at the 2-position of this pyrimidine are also in close proximity to the cysteine residue C983.36 in TM3. We hypothesized that an ethenyl group at this position will form a covalent bond with C983.36 by functioning as a Michael acceptor. A covalent pyrimidine analogue will not only prove this proposed binding mode, but will also provide a valuable tool for H4 receptor research. Experimental Approach We designed and synthesized VUF14480, and pharmacologically characterized this compound in hH4 receptor radioligand binding, G protein activation and β-arrestin2 recruitment experiments. The ability of VUF14480 to act as a covalent binder was assessed both chemically and pharmacologically. Key Results VUF14480 was shown to be a partial agonist of hH4 receptor-mediated G protein signalling and β-arrestin2 recruitment. VUF14480 bound covalently to the hH4 receptor with submicromolar affinity. Serine substitution of C983.36 prevented this covalent interaction. Conclusion and Implications VUF14480 is thought to bind covalently to the hH4 receptor-C983.36 residue and partially induce hH4 receptor-mediated G protein activation and β-arrestin2 recruitment. Moreover, these observations confirm our previously proposed binding mode of 2-aminopyrimidines. VUF14480 will be a useful tool to stabilize the receptor into an active confirmation and further investigate the structure of the active hH4 receptor. Linked Articles This article is part of a themed issue on Histamine Pharmacology Update. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2013.170.issue-1 PMID:23347159

  12. Synergistic Assembly of Covalent and Supramolecular Polymers.

    PubMed

    Bai, Linyi; Zhao, Yanli

    2016-06-01

    Integrating irreplaceable features of both covalent chemistry and noncovalent interactions into a single entity to maximize the applicability is highly desired. Here, a discovery of this type of hybrid, developed by Stupp and co-workers, is developed, where a synergistic combination of covalent and noncovalent compartments enables them to assemble by each other perfectively. The covalent compartments can grow into polymer chains assisted by a supramolecular compartment. The supramolecular compartments can be reversibly removed and re-formed to reconstitute the hybrid structure. The obtained soft materials can serve as functional platforms for molecular delivery or self-repairing materials. PMID:27076255

  13. Formation of the syn isomer of [Fe(IV)(Oanti)(TMC)(NCMe)](2+) in the reaction of Lewis acids with the side-on bound peroxo ligand in [Fe(III)(η(2)-O2)(TMC)](.).

    PubMed

    Prakash, Jai; Que, Lawrence

    2016-06-21

    We demonstrate that the reactions of [Fe(III)(η(2)-O2)(TMC)](+) (TMC = tetramethylcyclam) with Lewis acids (H(+) and NO(+)) afford the recently described syn isomer of [Fe(IV)(O)(TMC)(NCMe)](2+) (and not the anti isomer as had been tacitly assumed). This outcome is a logical consequence of the fact that the side-on peroxo ligand is bound to the syn face of the Fe(TMC) unit in the precursor. PMID:27273175

  14. Breast cancer photothermal therapy based on gold nanorods targeted by covalently-coupled bombesin peptide

    NASA Astrophysics Data System (ADS)

    Heidari, Zahra; Salouti, Mojtaba; Sariri, Reyhaneh

    2015-05-01

    Photothermal therapy, a minimally invasive treatment method for killing cancers cells, has generated a great deal of interest. In an effort to improve treatment efficacy and reduce side effects, better targeting of photoabsorbers to tumors has become a new concept in the battle against cancer. In this study, a bombesin (BBN) analog that can bind to all gastrin-releasing peptide (GRP) receptor subtypes was bound covalently with gold nanorods (GNRs) using Nanothinks acid as a link. The BBN analog was also coated with poly(ethylene glycol) to increase its stability and biocompatibility. The interactions were confirmed by ultraviolet-visible and Fourier transform infrared spectroscopy. A methylthiazol tetrazolium assay showed no cytotoxicity of the PEGylated GNR-BBN conjugate. The cell binding and internalization studies showed high specificity and uptake of the GNR-BBN-PEG conjugate toward breast cancer cells of the T47D cell line. The in vitro study revealed destruction of the T47D cells exposed to the new photothermal agent combined with continuous-wave near-infrared laser irradiation. The biodistribution study showed significant accumulation of the conjugate in the tumor tissue of mice with breast cancer. The in vivo photothermal therapy showed the complete disappearance of xenographted breast tumors in the mouse model.

  15. Structure of EstA esterase from psychrotrophic Pseudoalteromonas sp. 643A covalently inhibited by monoethylphosphonate

    SciTech Connect

    Brzuszkiewicz, Anna; Nowak, Elzbieta; Dauter, Zbigniew; Dauter, Miroslawa; Cieslinski, Hubert; Dlugolecka, Anna; Kur, Józef

    2010-10-28

    The crystal structure of the esterase EstA from the cold-adapted bacterium Pseudoalteromonas sp. 643A was determined in a covalently inhibited form at a resolution of 1.35 {angstrom}. The enzyme has a typical SGNH hydrolase structure consisting of a single domain containing a five-stranded {beta}-sheet, with three helices at the convex side and two helices at the concave side of the sheet, and is ornamented with a couple of very short helices at the domain edges. The active site is located in a groove and contains the classic catalytic triad of Ser, His and Asp. In the structure of the crystal soaked in diethyl p-nitrophenyl phosphate (DNP), the catalytic serine is covalently connected to a phosphonate moiety that clearly has only one ethyl group. This is the only example in the Protein Data Bank of a DNP-inhibited enzyme with covalently bound monoethylphosphate.

  16. How Cellulose Stretches: Synergism between Covalent and Hydrogen Bonding

    PubMed Central

    2014-01-01

    Cellulose is the most familiar and most abundant strong biopolymer, but the reasons for its outstanding mechanical performance are not well understood. Each glucose unit in a cellulose chain is joined to the next by a covalent C–O–C linkage flanked by two hydrogen bonds. This geometry suggests some form of cooperativity between covalent and hydrogen bonding. Using infrared spectroscopy and X-ray diffraction, we show that mechanical tension straightens out the zigzag conformation of the cellulose chain, with each glucose unit pivoting around a fulcrum at either end. Straightening the chain leads to a small increase in its length and is resisted by one of the flanking hydrogen bonds. This constitutes a simple form of molecular leverage with the covalent structure providing the fulcrum and gives the hydrogen bond an unexpectedly amplified effect on the tensile stiffness of the chain. The principle of molecular leverage can be directly applied to certain other carbohydrate polymers, including the animal polysaccharide chitin. Related but more complex effects are possible in some proteins and nucleic acids. The stiffening of cellulose by this mechanism is, however, in complete contrast to the way in which hydrogen bonding provides toughness combined with extensibility in protein materials like spider silk. PMID:24568640

  17. The effect of Plantago major Linnaeus on serum total sialic acid, lipid-bound sialic acid, some trace elements and minerals after administration of 7,12-dimethylbenz(a)anthracene in rats.

    PubMed

    Oto, Gokhan; Ekin, Suat; Ozdemir, Hulya; Levent, Abdulkadir; Berber, Ismet

    2012-05-01

    The present study was designed to evaluate the effect of Plantago major Linnaeus (PM) extract on serum total sialic acid (TSA), lipid-bound sialic acid (LSA), some trace elements (copper (Cu), zinc (Zn) and iron) and mineral levels (magnesium, calcium and sodium) in Wistar albino rat administrated 7,12-dimethylbenz(a)anthracene (DMBA). Rats were divided into three equal groups (n = 6). Group I comprised the control group, group II was treated with DMBA (100 mg/kg, single dose) and group III was treated with DMBA (100 mg/kg single dose) and aqueous extract of PM 100 mg/kg/day for 60 days. After 60 days, statistical analyses showed that TSA and LSA levels in DMBA and DMBA + PM groups were significantly higher compared to the control group (TSA: p < 0.01, p < 0.05; LSA: p < 0.05, p < 0.05, respectively). Serum Zn levels were decreased in subjects treated with DMBA (p < 0.01) and DMBA + PM (p < 0.05) compared to the control group values. Serum Cu levels were increased in DMBA group and PM-treated group compared to the control group values. The results of this investigation showed that the levels of TSA and LSA changed significantly, which are sensitive markers for detecting the toxic effects of DMBA. On the other hand, observed decline in Zn levels in rats from DMBA + PM group might be due to decreased generation of free radicals and oxidative stress. Results from this study suggest that PM may be partially effective in preventing carcinogenesis initiated by environmental carcinogen DMBA. PMID:21996710

  18. Acetaminophen structure-toxicity studies: In vivo covalent binding of a nonhepatotoxic analog, 3-hydroxyacetanilide

    SciTech Connect

    Roberts, S.A.; Price, V.F.; Jollow, D.J. )

    1990-09-01

    High doses of 3-hydroxyacetanilide (3HAA), a structural isomer of acetaminophen, do not produce hepatocellular necrosis in normal male hamsters or in those sensitized to acetaminophen-induced liver damage by pretreatment with a combination of 3-methylcholanthrene, borneol, and diethyl maleate. Although 3HAA was not hepatotoxic, the administration of acetyl-labeled (3H or 14C)3HAA (400 mg/kg, ip) produced levels of covalently bound radiolabel that were similar to those observed after an equimolar, hepatotoxic dose of (G-3H)acetaminophen. The covalent nature of 3HAA binding was demonstrated by retention of the binding after repetitive organic solvent extraction following protease digestion. Hepatic and renal covalent binding after 3HAA was approximately linear with both dose and time. In addition, 3HAA produced only a modest depletion of hepatic glutathione, suggesting the lack of a glutathione threshold. 3-Methylcholanthrene pretreatment increased and pretreatment with cobalt chloride and piperonyl butoxide decreased the hepatic covalent binding of 3HAA, indicating the involvement of cytochrome P450 in the formation of the 3HAA reactive metabolite. The administration of multiple doses or a single dose of (ring-3H)3HAA to hamsters pretreated with a combination of 3-methylcholanthrene, borneol, and diethyl maleate produced hepatic levels of 3HAA covalent binding that were in excess of those observed after a single, hepatotoxic acetaminophen dose. These data suggest that the nature and/or the intracellular processing of the reactive metabolites of acetaminophen and 3HAA are different. These data also demonstrate that absolute levels of covalently bound xenobiotic metabolites cannot be utilized as absolute predictors of cytotoxic potential.

  19. Functionalized membrane supports for covalent protein microsequence analysis

    SciTech Connect

    Coull, J.M.; Pappin, D.J.; Mark, J.; Aebersold, R.; Koester, H. )

    1991-04-01

    Methods were developed for high yield covalent attachment of peptides and proteins to isothiocyanate and arylamine-derivatized poly(vinylidene difluoride) membranes for solid-phase sequence analysis. Solutions of protein or peptide were dried onto 8-mm membrane disks such that the functional groups on the surface and the polypeptide were brought into close proximity. In the case of the isothiocyanate membrane, reaction between polypeptide amino groups and the surface isothiocyanate moieties was promoted by application of aqueous N-methylmorpholine. Attachment of proteins and peptides to the arylamine surface was achieved by application of water-soluble carbodiimide in a pH 5.0 buffer. Edman degradation of covalently bound polypeptides was accomplished with initial and repetitive sequence yields ranging from 33 to 75% and 88.5 to 98.5%, respectively. The yields were independent of the sample load (20 pmol to greater than 1 nmol) for either surface. Significant loss of material was not observed when attachment residues were encountered during sequence runs. Application of bovine beta-lactoglobulin A chain, staphylococcus protein A, or the peptide melittin to the isothiocyanate membrane allowed for extended N-terminal sequence identification (35 residues from 20 pmol of beta-lactoglobulin). A number of synthetic and naturally occurring peptides were sequenced to the C-terminal residue following attachment to the arylamine surface. In one example, 10 micrograms of bovine alpha-casein was digested with staphylococcal protease V8 and the peptides were separated by reverse-phase chromatography. Peptide fractions were then directly applied to arylamine membrane disks for covalent sequence analysis. From as little as 2 pmol of initial signal it was possible to determine substantial sequence information (greater than 10 residues).

  20. Functionalized membrane supports for covalent protein microsequence analysis.

    PubMed

    Coull, J M; Pappin, D J; Mark, J; Aebersold, R; Köster, H

    1991-04-01

    Methods were developed for high yield covalent attachment of peptides and proteins to isothiocyanate and arylamine-derivatized poly(vinylidene difluoride) membranes for solid-phase sequence analysis. Solutions of protein or peptide were dried onto 8-mm membrane disks such that the functional groups on the surface and the polypeptide were brought into close proximity. In the case of the isothiocyanate membrane, reaction between polypeptide amino groups and the surface isothiocyanate moieties was promoted by application of aqueous N-methylmorpholine. Attachment of proteins and peptides to the arylamine surface was achieved by application of water-soluble carbodiimide in a pH 5.0 buffer. Edman degradation of covalently bound polypeptides was accomplished with initial and repetitive sequence yields ranging from 33 to 75% and 88.5 to 98.5%, respectively. The yields were independent of the sample load (20 pmol to greater than 1 nmol) for either surface. Significant loss of material was not observed when attachment residues were encountered during sequence runs. Application of bovine beta-lactoglobulin A chain, staphylococcus protein A, or the peptide melittin to the isothiocyanate membrane allowed for extended N-terminal sequence identification (35 residues from 20 pmol of beta-lactoglobulin). A number of synthetic and naturally occurring peptides were sequenced to the C-terminal residue following attachment to the arylamine surface. In one example, 10 micrograms of bovine alpha-casein was digested with staphylococcal protease V8 and the peptides were separated by reverse-phase chromatography. Peptide fractions were then directly applied to arylamine membrane disks for covalent sequence analysis. From as little as 2 pmol of initial signal it was possible to determine substantial sequence information (greater than 10 residues). PMID:1867375

  1. Binding of cationic peptides (KX)4K to DPPG bilayers. Increasing the hydrophobicity of the uncharged amino acid X drives formation of membrane bound β-sheets: A DSC and FT-IR study.

    PubMed

    Hädicke, André; Blume, Alfred

    2016-06-01

    The binding of cationic peptides of the sequence (KX)4K to lipid vesicles of negatively charged dipalmitoyl-phosphatidylglycerol (DPPG) was investigated by differential scanning calorimetry (DSC) and temperature dependent Fourier-transformed infrared (FT-IR) spectroscopy. The hydrophobicity of the uncharged amino acid X was changed from G (glycine) over A (alanine), Abu (α-aminobutyric acid), V (valine) to L (leucine). The binding of the peptides caused an increase of the phase transition temperature (Tm) of DPPG by up to 20°C. The shift depended on the charge ratio and on the hydrophobicity of the amino acid X. Unexpectedly, the upward shift of Tm increased with increasing hydrophobicity of X. FT-IR spectroscopy showed a shift of the CH2 stretching vibrations of DPPG to lower frequency, particularly for bilayers in the liquid-crystalline phase, indicating an ordering of the hydrocarbon chains when the peptides were bound. Changes in the lipid C=O vibrational band indicated a dehydration of the lipid headgroup region after peptide binding. (KG)4K was bound in an unordered structure at all temperatures. All other peptides formed intermolecular antiparallel β-sheets, when bound to gel phase DPPG. However, for (KA)4K and (KAbu)4K, the β-sheets converted into an unordered structure above Tm. In contrast, the β-sheet structures of (KV)4K and (KL)4K remained stable even at 80°C when bound to the liquid-crystalline phase of DPPG. Strong aggregation of DPPG vesicles occurred after peptide binding. For the aggregates, we suggest a structure, where aggregated single β-sheets are sandwiched between opposing DPPG bilayers with a dehydrated interfacial region. PMID:26903220

  2. Synthesis of Polymers Containing Covalently Bonded NLO Chromophores

    NASA Technical Reports Server (NTRS)

    Denga, Xiao-Hua; Sanghadasa, Mohan; Walton, Connie; Penn, Benjamin B.; Amai, Robert L. S.; Clark, Ronald D.

    1998-01-01

    Polymers containing covalently bonded nonlinear optical (NLO) chromophores are expected to possess special properties such as greater stability, better mechanical processing, and easier film formation than their non-polymeric equivalent. For the present work, polymethylmethacrylate (PMMA) was selected as the basic polymer unit on which to incorporate different NLO chromophores. The NLO components were variations of DIVA {[2-methoxyphenyl methylidene]-propanedinitrile} which we prepared from vanillin derivatives and malononitrile. These were esterified with methacrylic acid and polymerized either directly or with methyl methacrylate to form homopolymers or copolymers respectively. Characterization of the polymers and NLO property studies are underway.

  3. Covalent Bond between Ligand and Receptor Required for Efficient Activation in Rhodopsin*

    PubMed Central

    Matsuyama, Take; Yamashita, Takahiro; Imai, Hiroo; Shichida, Yoshinori

    2010-01-01

    Rhodopsin is an extensively studied member of the G protein-coupled receptors (GPCRs). Although rhodopsin shares many features with the other GPCRs, it exhibits unique features as a photoreceptor molecule. A hallmark in the molecular structure of rhodopsin is the covalently bound chromophore that regulates the activity of the receptor acting as an agonist or inverse agonist. Here we show the pivotal role of the covalent bond between the retinal chromophore and the lysine residue at position 296 in the activation pathway of bovine rhodopsin, by use of a rhodopsin mutant K296G reconstituted with retinylidene Schiff bases. Our results show that photoreceptive functions of rhodopsin, such as regiospecific photoisomerization of the ligand, and its quantum yield were not affected by the absence of the covalent bond, whereas the activation mechanism triggered by photoisomerization of the retinal was severely affected. Furthermore, our results show that an active state similar to the Meta-II intermediate of wild-type rhodopsin did not form in the bleaching process of this mutant, although it exhibited relatively weak G protein activity after light irradiation because of an increased basal activity of the receptor. We propose that the covalent bond is required for transmitting structural changes from the photoisomerized agonist to the receptor and that the covalent bond forcibly keeps the low affinity agonist in the receptor, resulting in a more efficient G protein activation. PMID:20042594

  4. Prolonged and tunable residence time using reversible covalent kinase inhibitors

    PubMed Central

    Bradshaw, J. Michael; McFarland, Jesse M.; Paavilainen, Ville O.; Bisconte, Angelina; Tam, Danny; Phan, Vernon T.; Romanov, Sergei; Finkle, David; Shu, Jin; Patel, Vaishali; Ton, Tony; Li, Xiaoyan; Loughhead, David G.; Nunn, Philip A.; Karr, Dane E.; Gerritsen, Mary E.; Funk, Jens Oliver; Owens, Timothy D.; Verner, Erik; Brameld, Ken A.; Hill, Ronald J.; Goldstein, David M.; Taunton, Jack

    2015-01-01

    Drugs with prolonged, on-target residence time often show superior efficacy, yet general strategies for optimizing drug-target residence time are lacking. Here, we demonstrate progress toward this elusive goal by targeting a noncatalytic cysteine in Bruton's tyrosine kinase (BTK) with reversible covalent inhibitors. Utilizing an inverted orientation of the cysteine-reactive cyanoacrylamide electrophile, we identified potent and selective BTK inhibitors that demonstrate biochemical residence times spanning from minutes to 7 days. An inverted cyanoacrylamide with prolonged residence time in vivo remained bound to BTK more than 18 hours after clearance from the circulation. The inverted cyanoacrylamide strategy was further utilized to discover fibroblast growth factor receptor (FGFR) kinase inhibitors with residence times of several days, demonstrating generalizability of the approach. Targeting noncatalytic cysteines with inverted cyanoacrylamides may serve as a broadly applicable platform that facilitates “residence time by design”, the ability to modulate and improve the duration of target engagement in vivo. PMID:26006010

  5. Non-covalent interactions of nitrous oxide with aromatic compounds: Spectroscopic and computational evidence for the formation of 1:1 complexes

    NASA Astrophysics Data System (ADS)

    Cao, Qian; Gor, Gennady Y.; Krogh-Jespersen, Karsten; Khriachtchev, Leonid

    2014-04-01

    We present the first study of intermolecular interactions between nitrous oxide (N2O) and three representative aromatic compounds (ACs): phenol, cresol, and toluene. The infrared spectroscopic experiments were performed in a Ne matrix and were supported by high-level quantum chemical calculations. Comparisons of the calculated and experimental vibrational spectra provide direct identification and characterization of the 1:1 N2O-AC complexes. Our results show that N2O is capable of forming non-covalently bonded complexes with ACs. Complex formation is dominated by dispersion forces, and the interaction energies are relatively low (about -3 kcal mol-1); however, the complexes are clearly detected by frequency shifts of the characteristic bands. These results suggest that N2O can be bound to the amino-acid residues tyrosine or phenylalanine in the form of π complexes.

  6. Non-covalent interactions of nitrous oxide with aromatic compounds: Spectroscopic and computational evidence for the formation of 1:1 complexes

    SciTech Connect

    Cao, Qian; Gor, Gennady Y.; Krogh-Jespersen, Karsten; Khriachtchev, Leonid

    2014-04-14

    We present the first study of intermolecular interactions between nitrous oxide (N{sub 2}O) and three representative aromatic compounds (ACs): phenol, cresol, and toluene. The infrared spectroscopic experiments were performed in a Ne matrix and were supported by high-level quantum chemical calculations. Comparisons of the calculated and experimental vibrational spectra provide direct identification and characterization of the 1:1 N{sub 2}O-AC complexes. Our results show that N{sub 2}O is capable of forming non-covalently bonded complexes with ACs. Complex formation is dominated by dispersion forces, and the interaction energies are relatively low (about −3 kcal mol{sup −1}); however, the complexes are clearly detected by frequency shifts of the characteristic bands. These results suggest that N{sub 2}O can be bound to the amino-acid residues tyrosine or phenylalanine in the form of π complexes.

  7. Platinum-modified covalent triazine frameworks hybridized with carbon nanoparticles as methanol-tolerant oxygen reduction electrocatalysts.

    PubMed

    Kamiya, Kazuhide; Kamai, Ryo; Hashimoto, Kazuhito; Nakanishi, Shuji

    2014-01-01

    Covalent triazine frameworks, which are crosslinked porous polymers with two-dimensional molecular structures, are promising materials for heterogeneous catalysts. However, the application of the frameworks as electrocatalysts has not been achieved to date because of their poor electrical conductivity. Here we report that platinum-modified covalent triazine frameworks hybridized with conductive carbon nanoparticles are successfully synthesized by introducing carbon nanoparticles during the polymerization process of covalent triazine frameworks. The resulting materials exhibit clear electrocatalytic activity for oxygen reduction reactions in acidic solutions. More interestingly, the platinum-modified covalent triazine frameworks show almost no activity for methanol oxidation, in contrast to commercial carbon-supported platinum. Thus, platinum-modified covalent triazine frameworks hybridized with carbon nanoparticles exhibit selective activity for oxygen reduction reactions even in the presence of high concentrations of methanol, which indicates potential utility as a cathode catalyst in direct methanol fuel cells. PMID:25242214

  8. Platinum-modified covalent triazine frameworks hybridized with carbon nanoparticles as methanol-tolerant oxygen reduction electrocatalysts

    PubMed Central

    Kamiya, Kazuhide; Kamai, Ryo; Hashimoto, Kazuhito; Nakanishi, Shuji

    2014-01-01

    Covalent triazine frameworks, which are crosslinked porous polymers with two-dimensional molecular structures, are promising materials for heterogeneous catalysts. However, the application of the frameworks as electrocatalysts has not been achieved to date because of their poor electrical conductivity. Here we report that platinum-modified covalent triazine frameworks hybridized with conductive carbon nanoparticles are successfully synthesized by introducing carbon nanoparticles during the polymerization process of covalent triazine frameworks. The resulting materials exhibit clear electrocatalytic activity for oxygen reduction reactions in acidic solutions. More interestingly, the platinum-modified covalent triazine frameworks show almost no activity for methanol oxidation, in contrast to commercial carbon-supported platinum. Thus, platinum-modified covalent triazine frameworks hybridized with carbon nanoparticles exhibit selective activity for oxygen reduction reactions even in the presence of high concentrations of methanol, which indicates potential utility as a cathode catalyst in direct methanol fuel cells. PMID:25242214

  9. Platinum-modified covalent triazine frameworks hybridized with carbon nanoparticles as methanol-tolerant oxygen reduction electrocatalysts

    NASA Astrophysics Data System (ADS)

    Kamiya, Kazuhide; Kamai, Ryo; Hashimoto, Kazuhito; Nakanishi, Shuji

    2014-09-01

    Covalent triazine frameworks, which are crosslinked porous polymers with two-dimensional molecular structures, are promising materials for heterogeneous catalysts. However, the application of the frameworks as electrocatalysts has not been achieved to date because of their poor electrical conductivity. Here we report that platinum-modified covalent triazine frameworks hybridized with conductive carbon nanoparticles are successfully synthesized by introducing carbon nanoparticles during the polymerization process of covalent triazine frameworks. The resulting materials exhibit clear electrocatalytic activity for oxygen reduction reactions in acidic solutions. More interestingly, the platinum-modified covalent triazine frameworks show almost no activity for methanol oxidation, in contrast to commercial carbon-supported platinum. Thus, platinum-modified covalent triazine frameworks hybridized with carbon nanoparticles exhibit selective activity for oxygen reduction reactions even in the presence of high concentrations of methanol, which indicates potential utility as a cathode catalyst in direct methanol fuel cells.

  10. Benchmarking in vitro covalent binding burden as a tool to assess potential toxicity caused by nonspecific covalent binding of covalent drugs.

    PubMed

    Dahal, Upendra P; Obach, R Scott; Gilbert, Adam M

    2013-11-18

    Despite several advantages of covalent inhibitors (such as increased biochemical efficiency, longer duration of action on the target, and lower efficacious doses) over their reversible binding counterparts, there is a reluctance to use covalent inhibitors as a drug design strategy in pharmaceutical research. This reluctance is due to their anticipated reactions with nontargeted macromolecules. We hypothesized that there may be a threshold limit for nonspecific covalent binding, below which a covalent binding drug may be less likely to cause toxicity due to irreversible binding to off-target macromolecules. Estimation of in vivo covalent binding burden from in vitro data has previously been used as an approach to distinguish those agents more likely to cause toxicity (e.g., hepatotoxicity) via metabolic activation to reactive metabolites. We have extended this approach to nine covalent binding drugs to determine in vitro covalent binding burden. In vitro covalent binding burden was determined by incubating radiolabeled drugs with pooled human hepatocytes. These data were scaled to an estimate of in vivo covalent binding burden by combining the in vitro data with daily dose. Scaled in vivo daily covalent binding burden of marketed covalent drugs was found to be under 10 mg/day, which is in agreement with previously reported threshold value for metabolically activated reversible drugs. Covalent binding was also compared to the intrinsic reactivities of the covalent inhibitors assessed using nucleophiles glutathione and N-α-acetyl lysine. The intrinsic reactivity did not correlate with observed in vitro covalent binding, which demonstrated that the intrinsic reactivity of the electrophilic groups of covalent drugs does not exclusively account for the extent of covalent binding. The ramifications of these findings for consideration of using a covalent strategy in drug design are discussed. PMID:24164572

  11. CovalentDock Cloud: a web server for automated covalent docking.

    PubMed

    Ouyang, Xuchang; Zhou, Shuo; Ge, Zemei; Li, Runtao; Kwoh, Chee Keong

    2013-07-01

    Covalent binding is an important mechanism for many drugs to gain its function. We developed a computational algorithm to model this chemical event and extended it to a web server, the CovalentDock Cloud, to make it accessible directly online without any local installation and configuration. It provides a simple yet user-friendly web interface to perform covalent docking experiments and analysis online. The web server accepts the structures of both the ligand and the receptor uploaded by the user or retrieved from online databases with valid access id. It identifies the potential covalent binding patterns, carries out the covalent docking experiments and provides visualization of the result for user analysis. This web server is free and open to all users at http://docking.sce.ntu.edu.sg/. PMID:23677616

  12. Multiple-component covalent organic frameworks

    NASA Astrophysics Data System (ADS)

    Huang, Ning; Zhai, Lipeng; Coupry, Damien E.; Addicoat, Matthew A.; Okushita, Keiko; Nishimura, Katsuyuki; Heine, Thomas; Jiang, Donglin

    2016-07-01

    Covalent organic frameworks are a class of crystalline porous polymers that integrate molecular building blocks into periodic structures and are usually synthesized using two-component [1+1] condensation systems comprised of one knot and one linker. Here we report a general strategy based on multiple-component [1+2] and [1+3] condensation systems that enable the use of one knot and two or three linker units for the synthesis of hexagonal and tetragonal multiple-component covalent organic frameworks. Unlike two-component systems, multiple-component covalent organic frameworks feature asymmetric tiling of organic units into anisotropic skeletons and unusually shaped pores. This strategy not only expands the structural complexity of skeletons and pores but also greatly enhances their structural diversity. This synthetic platform is also widely applicable to multiple-component electron donor-acceptor systems, which lead to electronic properties that are not simply linear summations of those of the conventional [1+1] counterparts.

  13. Use of (2-/sup 14/C)mevalonate and saponin-bound (/sup 14/C)-3-hydroxy-3-methylglutaric acid for the biosynthesis of terpenoids in leaves of Dioscorea deltoidea

    SciTech Connect

    Gurielidze, K.G.; Paseshnichenko, V.A.; Vasil'eva, I.S.

    1986-03-20

    After the introduction of (2-/sup 14/C)acetate into leaves of Dioscorea deltoidea, a radioactive furonanalog of deltafolin - protodeltofolin, containing two-thirds of the label in the 3-hydroxy-3-methylglutaryl portion - was isolated from them. Radioactive ..beta..-careotene and sterols were isolated from cut young leaves of Dioscorea 24 h after the introduction of (/sup 14/C) protodeltofolin into them, using chromatography on a column of silica gel and precipitation of sterols in the form of digitonins for this purpose. The incorporation of radioactivity from (/sup 14/C)-3-hydroxy-3-methyl-glutaric acid, bound in the form of a saponin, and ..beta..-carotene came to 0.18-0.80%, while incorporation into sterols came to 0.07-2.86% of the radioactivity of the alcohol extract. Thereby it was shown that 3-hydroxyl-3-methylglutaric acid, bound in the form of the saponin, can be used to form terpenoids in Dioscorea leaves. It was suggested that the binding of hydroxymethylglutaric acid to saponin represents one of the mechanisms of regulation of the rate of terpenoid biosynthesis in Dioscorea leaves.

  14. Theoretical Insights into Covalency Driven f Element Separations

    SciTech Connect

    Lindsay E. Roy; Nicholas J. Bridges; Leigh R. Martin

    2013-02-01

    The lanthanide series, Am, and Cm are predominantly found in the trivalent oxidation state in aqueous solutions making their separation very difficult to achieve. To date, one of the mostly promising separation processes for transplutonium elements from the lanthanides is the TALSPEAK process. Though the mechanism of the TALSPEAK process is not fully understood, it has been demonstrated to provide excellent separation factors between the lanthanides and the trivalent lanthanides. Through Density Function Theory (DFT) calculations of di 2-ethylenetriamine-N,N,N',N”,N”-pentaacetic acid (DTPA), we set out to understand the structures and stabilities of the aqueous phase complexes [MIII(DTPA)-H2O]2- (M = Nd, Am) as well as the changes in Gibbs free energy for complexation in the gas phase and aqueous solution. Mulliken population analysis, Bader’s Atoms-in-Molecules (AIM) approach, and Natural Bond Orbital (NBO) analysis were then used to analyze the bonding in both molecules. The results discussed below suggest that the preference of the DTPA5- ligand for Am over Nd is mainly due to electrostatic and covalent interactions from the oxygen atoms with the nitrogen chelates provide an additional, yet small, covalent interaction. These results question the exclusive use of hard and soft acids and bases (HSAB) concepts for the design of extracting reagents and suggest that hard-soft interactions play more of a role in the separations process than previously thought.

  15. Enhanced stability of catalase covalently immobilized on functionalized titania submicrospheres.

    PubMed

    Wu, Hong; Liang, Yanpeng; Shi, Jiafu; Wang, Xiaoli; Yang, Dong; Jiang, Zhongyi

    2013-04-01

    In this study, a novel approach combing the chelation and covalent binding was explored for facile and efficient enzyme immobilization. The unique capability of titania to chelate with catecholic derivatives at ambient conditions was utilized for titania surface functionalization. The functionalized titania was then used for enzyme immobilization. Titania submicrospheres (500-600 nm) were synthesized by a modified sol-gel method and functionalized with carboxylic acid groups through a facile chelation method by using 3-(3,4-dihydroxyphenyl) propionic acid as the chelating agent. Then, catalase (CAT) was covalently immobilized on these functionalized titania submicrospheres through 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) coupling reaction. The immobilized CAT retained 65% of its free form activity with a loading capacity of 100-150 mg/g titania. The pH stability, thermostability, recycling stability and storage stability of the immobilized CAT were evaluated. A remarkable enhancement in enzyme stability was achieved. The immobilized CAT retained 90% and 76% of its initial activity after 10 and 16 successive cycles of decomposition of hydrogen peroxide, respectively. Both the Km and the Vmax values of the immobilized CAT (27.4 mM, 13.36 mM/min) were close to those of the free CAT (25.7 mM, 13.46 mM/min). PMID:23827593

  16. Formation of 4-keto-D-aldopentoses and 4-pentulosonates (4-keto-D-pentonates) with unidentified membrane-bound enzymes from acetic acid bacteria.

    PubMed

    Adachi, Osao; Hours, Roque A; Shinagawa, Emiko; Akakabe, Yoshihiko; Yakushi, Toshiharu; Matsushita, Kazunobu

    2011-01-01

    In our previous study, a new microbial reaction yielding 4-keto-D-arabonate from 2,5-diketo-D-gluconate was identified with Gluconacetobacter liquefaciens RCTMR 10. It appeared that decarboxylation and dehydrogenation took place together in the reaction. To analyze the nature of the reaction, investigations were done with the membrane fraction of the organism, and 4-keto-D-arabinose was confirmed as the direct precursor of 4-keto-D-arabonate. Two novel membrane-bound enzymes, 2,5-diketo-D-gluconate decarboxylase and 4-keto-D-aldopentose 1-dehydrogenase, were involved in the reaction. Alternatively, D-arabonate was oxidized to 4-keto-D-arabonate by another membrane-bound enzyme, D-arabonate 4-dehydrogenase. More directly, D-arabinose oxidation was examined with growing cells and with the membrane fraction of G. suboxydans IFO 12528. 4-Keto-D-arabinose, the same intermediate as that from 2,5-diketo-D-gluconate, was detected, and it was oxidized to 4-keto-D-arabonate. Likewise, D-ribose was oxidized to 4-keto-D-ribose and then it was oxidized to 4-keto-D-ribonate. In addition to 4-keto-D-aldopentose 1-dehydrogenase, the presence of a novel membrane-bound enzyme, D-aldopentose 4-dehydrogenase, was confirmed in the membrane fraction. The formation of 4-keto-D-aldopentoses and 4-keto-D-pentonates (4-pentulosonates) was finally confirmed as reaction products of four different novel membrane-bound enzymes. PMID:21897028

  17. Interfacial welding of dynamic covalent network polymers

    NASA Astrophysics Data System (ADS)

    Yu, Kai; Shi, Qian; Li, Hao; Jabour, John; Yang, Hua; Dunn, Martin L.; Wang, Tiejun; Qi, H. Jerry

    2016-09-01

    Dynamic covalent network (or covalent adaptable network) polymers can rearrange their macromolecular chain network by bond exchange reactions (BERs) where an active unit replaces a unit in an existing bond to form a new bond. Such macromolecular events, when they occur in large amounts, can attribute to unusual properties that are not seen in conventional covalent network polymers, such as shape reforming and surface welding; the latter further enables the important attributes of material malleability and powder-based reprocessing. In this paper, a multiscale modeling framework is developed to study the surface welding of thermally induced dynamic covalent network polymers. At the macromolecular network level, a lattice model is developed to describe the chain density evolution across the interface and its connection to bulk stress relaxation due to BERs. The chain density evolution rule is then fed into a continuum level interfacial model that takes into account surface roughness and applied pressure to predict the effective elastic modulus and interfacial fracture energy of welded polymers. The model yields particularly accessible results where the moduli and interfacial strength of the welded samples as a function of temperature and pressure can be predicted with four parameters, three of which can be measured directly. The model identifies the dependency of surface welding efficiency on the applied thermal and mechanical fields: the pressure will affect the real contact area under the consideration of surface roughness of dynamic covalent network polymers; the chain density increment on the real contact area of interface is only dependent on the welding time and temperature. The modeling approach shows good agreement with experiments and can be extended to other types of dynamic covalent network polymers using different stimuli for BERs, such as light and moisture etc.

  18. Covalent bonding of polycations to small polymeric particles

    NASA Technical Reports Server (NTRS)

    Rembaum, A.

    1975-01-01

    Process produces small spherical polymeric particles which have polycations bound to them. In emulsion form, particles present large positively charged surface which is available to absorb polyanions. This properly can be used in removing heparin from blood or bile acids from the digestive tract. Other anions, such as DNA and RNA, can also be removed from aqueous solutions.

  19. Protein-RNA networks revealed through covalent RNA marks.

    PubMed

    Lapointe, Christopher P; Wilinski, Daniel; Saunders, Harriet A J; Wickens, Marvin

    2015-12-01

    Protein-RNA networks are ubiquitous and central in biological control. We present an approach termed RNA Tagging that enables the user to identify protein-RNA interactions in vivo by analyzing purified cellular RNA, without protein purification or cross-linking. An RNA-binding protein of interest is fused to an enzyme that adds uridines to the end of RNA. RNA targets bound by the chimeric protein in vivo are covalently marked with uridines and subsequently identified from extracted RNA via high-throughput sequencing. We used this approach to identify hundreds of RNAs bound by a Saccharomyces cerevisiae PUF protein, Puf3p. The results showed that although RNA-binding proteins productively bind specific RNAs to control their function, they also 'sample' RNAs without exerting a regulatory effect. We used the method to uncover hundreds of new and likely regulated targets for a protein without canonical RNA-binding domains, Bfr1p. RNA Tagging is well suited to detect and analyze protein-RNA networks in vivo. PMID:26524240

  20. Protein-RNA networks revealed through covalent RNA marks

    PubMed Central

    Lapointe, Christopher P.; Wilinski, Daniel; Saunders, Harriet A. J.; Wickens, Marvin

    2015-01-01

    Protein-RNA networks are ubiquitous and central in biological control. We present an approach, termed “RNA Tagging,” that identifies protein-RNA interactions in vivo by analyzing purified cellular RNA, without protein purification or crosslinking. An RNA-binding protein of interest is fused to an enzyme that adds uridines to the end of RNA. RNA targets bound by the chimeric protein in vivo are covalently marked with uridines and subsequently identified from extracted RNA using high-throughput sequencing. We used this approach to identify hundreds of RNAs bound by a Saccharomyces cerevisiae PUF protein, Puf3p. The method revealed that while RNA-binding proteins productively bind specific RNAs to control their function, they also “sample” RNAs without exerting a regulatory effect. We exploited the method to uncover hundreds of new and likely regulated targets for a protein without canonical RNA-binding domains, Bfr1p. The RNA Tagging approach is well-suited to detect and analyze protein-RNA networks in vivo. PMID:26524240

  1. β-Lactoglobulin as nanotransporter--Part II: Characterization of the covalent protein modification by allicin and diallyl disulfide.

    PubMed

    Wilde, Sandra Catharina; Treitz, Christian; Keppler, Julia Katharina; Koudelka, Tomas; Palani, Kalpana; Tholey, Andreas; Rawel, Harshadrai M; Schwarz, Karin

    2016-04-15

    The whey protein β-lactoglobulin has been proposed as a transporter for covalent bound bioactive compounds in order to enhance their stability and reduce their sensory perception. The garlic derived compounds allicin and diallyl disulfide were bound covalently to the native and heat denatured protein. The binding site and the influence of the modification on the digestibility were determined by mass spectrometric analysis of the modified β-lactoglobulin. Further, the conformation of the modified protein was assessed by circular dichroism and dynamic light scattering. The free thiol group of Cys(121) turned out to be the major binding site. After proteolysis with trypsin at pH 7 but not with pepsin at pH 2, a limited transfer to other cysteinyl residues was observed. The covalently bound ligands did not mask any proteolytic cleavage sites of pepsin, trypsin or chymotrypsin. The modified β-lactoglobulin showed a native like conformation, besides a moderate loosening of protein folding. The covalent binding of organosulfur compounds to β-lactoglobulin provides a bioactive ingredient without impairing the digestibility and functional properties of the protein. PMID:26617049

  2. Effect of solution chemistry on the extent of binding of phenanthrene by a soil humic acid: A comparison of dissolved and clay bound humic

    SciTech Connect

    Jones, K.D.; Tiller, C.L.

    1999-02-15

    The effect of pH, ionic strength, and cation in solution on the binding of phenanthrene by a soil humic acid in the aqueous phase was determined using fluorescence quenching. The phenanthrene binding coefficient with the dissolved soil humic, K{sub oc}, decreased with increasing ionic strength and solution cation valence. At low values of ionic strength, K{sub oc} values for this soil humic acid increased with increasing pH. For this humic sample, the experimental results were consistent with a conformational model of the humic substance in aqueous solution where, depending on solution conditions, some parts of the humic structure may be more open to allow increased PAH access to attachment sites. After sorption onto clays, supernatant solutions of the unadsorbed humic fraction yielded lower K{sub oc} values than the original bulk humic acid, suggesting that the humic substance was fractionating during its sorption onto the clays. Additionally, the extent of phenanthrene binding with the adsorbed humic fraction was lower than the results determined for the bulk humic acid prior to adsorption. The conformation of the humic substance when sorbed onto the inorganic surface appears to be affecting the level of phenanthrene binding by the humic acid.

  3. Crystal Structure and Promiscuous Partitioning of a Covalent Intermediate Common in the Pentein Superfamily

    PubMed Central

    Linsky, Thomas W.; Monzingo, Arthur F.; Stone, Everett M.; Robertus, Jon D.; Fast, Walter

    2008-01-01

    Summary Many enzymes in the pentein superfamily use a transient covalent intermediate in their catalytic mechanisms. Here, we use a mutant (H162G) dimethylarginine dimethylaminohydrolase from Pseudomonas aeruginosa and an alternative substrate, S-methyl-L-thiocitrulline, to trap, crystallize and determine the 2.8 Å resolution structure of a stable covalent adduct which mimics this reaction intermediate. Observed interactions between the trapped adduct and active site residues along with comparison to a previously known product-bound structure provide insight into the normal catalytic mechanism. The plane of the trapped thiouronium intermediate is angled away from that seen in the product and substrate complexes, allowing for an altered angle of attack between the nucleophiles of the first and second half reactions. The stable covalent adduct is also capable of further reaction. Addition of exogenous imidazole can rescue the original hydrolytic activity. Notably, addition of other exogenous amines can instead yield substituted arginine products. These alternative products arise from partitioning of the trapped intermediate into the evolutionarily related amidinotransferase reaction pathway. The enzyme scaffold provides both selectivity and catalysis for the amidinotransferase reaction, underscoring commonalities between different reaction pathways found in this mechanistically diverse enzyme superfamily. The promiscuous partitioning of this covalent intermediate may also help to illuminate the evolutionary history of these enzymes. PMID:18482699

  4. Short peptide tag for covalent protein labeling based on coiled coils.

    PubMed

    Wang, Jianpeng; Yu, Yongsheng; Xia, Jiang

    2014-01-15

    To label proteins covalently, one faces a trade-off between labeling a protein specifically and using a small tag. Often one must compromise one parameter for the other or use additional components, such as an enzyme, to satisfy both requirements. Here, we report a new reaction that covalently labels proteins by using engineered coiled-coil peptides. Harnessing the concept of "proximity-induced reactivity", the 21-amino-acid three-heptad peptides CCE/CCK were modified with a nucleophilic cysteine and an α-chloroacetyl group at selected positions. When pairs of coiled coils associated, an irreversible covalent bond spontaneously formed between the peptides. The specificity of the cross-linking reaction was characterized, the probes were improved by making them bivalent, and the system was used to label a protein in vitro and receptors on the surface of mammalian cells. PMID:24341800

  5. Bound states and the Bekenstein bound

    SciTech Connect

    Bousso, Raphael

    2003-10-16

    We explore the validity of the generalized Bekenstein bound, S<= pi M a. We define the entropy S as the logarithm of the number of states which have energy eigenvalue below M and are localized to a flat space region of width alpha. If boundary conditions that localize field modes are imposed by fiat, then the bound encounters well-known difficulties with negative Casimir energy and large species number, as well as novel problems arising only in the generalized form. In realistic systems, however, finite-size effects contribute additional energy. We study two different models for estimating such contributions. Our analysis suggests that the bound is both valid and nontrivial if interactions are properly included, so that the entropy S counts the bound states of interacting fields.

  6. Prevention of sulfide mineral leaching through covalent coating

    SciTech Connect

    K.M. Zaman; C. Chusuei; L.Y. Blue; D.A. Atwood

    2007-09-15

    The use of benzene-1,3-diamidoethanethiol as a covalent surface coating for the prevention of metal leaching was demonstrated with several sulfide minerals including cinnabar (HgS), pyrite (FeS{sub 2}), chalcopyrite (CuFeS{sub 2}), covellite (CuS), galena (PbS), realgar (As{sub 4}S{sub 4}) and sphalerite (ZnS). The minerals were coated with sufficient H2BDT to bind the surface metals in a 1:1 ratio. Leaching at pH 1, 3 and 7 was then conducted on both treated and untreated minerals. ICP and CVAFS (for mercury) analyses revealed that the coated minerals showed a dramatic reduction in metal leaching as compared to uncoated control samples. X-ray photoelectron spectroscopy indicated the formation of covalent bonds between the sulphur of the ligand and the metals from the minerals. Results indicate that it would be possible to prevent acid mine drainage through the binding of the metals in coal. 51 refs., 4 figs., 8 tabs.

  7. Covalent immobilization of glucose oxidase onto new modified acrylonitrile copolymer/silica gel hybrid supports.

    PubMed

    Godjevargova, Tzonka; Nenkova, Ruska; Dimova, Nedyalka

    2005-08-12

    New polymer/silica gel hybrid supports were prepared by coating high surface area of silica gel with modified acrylonitrile copolymer. The concentrations of the modifying agent (NaOH) and the modified polymer were varied. GOD was covalently immobilized on these hybrid supports and the relative activity and the amount of bound protein were determined. The highest relative activity and sufficient amount of bound protein of the immobilized GOD were achieved in 10% NaOH and 2% solution of modified acrylonitrile copolymer. The influence of glutaraldehyde concentration and the storage time on enzyme efficiency were examined. Glutaraldehyde concentration of 0.5% is optimal for the immobilized GOD. It was shown that the covalently bound enzyme (using 0.5% glutaraldehyde) had higher relative activity than the activity of the adsorbed enzyme. Covalently immobilized GOD with 0.5% glutaraldehyde was more stable for four months in comparison with the one immobilized on pure silica gel, hybrid support with 10% glutaraldehyde and the free enzyme. The effect of the pore size on the enzyme efficiency was studied on four types of silica gel with different pore size. Silica with large pores (CPC-Silica carrier, 375 A) presented higher relative activity than those with smaller pore size (Silica gel with 4, 40 and 100 A). The amount of bound protein was also reduced with decreasing the pore size. The effect of particle size was studied and it was found out that the smaller the particle size was, the greater the activity and the amount of immobilized enzyme were. The obtained results proved that these new polymer/silica gel hybrid supports were suitable for GOD immobilization. PMID:16080168

  8. Photophysical and theoretical insights on non-covalently linked fullerene-zinc phthalocyanine complexes

    NASA Astrophysics Data System (ADS)

    Ray, A.; Chattopadhyay, S.; Bhattacharya, S.

    2011-09-01

    The photo-physical aspects of non-covalently linked assemblies of a series of fullerenes, namely, C 60, C 70, tert-butyl-(1,2-methanofullerene)-61-carboxylate ( 1) and [6,6]-phenyl C 70 butyric acid methyl ester ( 2) with a designed zinc phthalocyanine (ZnPc), viz., zinc-1,4,8,11,15,18,22,25-octabutoxy-29 H,31 H-phthalocyanine ( 3) in toluene medium are studied employing absorption spectrophotometric, steady state and time resolved fluorescence spectroscopic measurements. Of central interest in these investigations is the preferential binding of various fullerenes with ZnPc in toluene. The ground state interaction between fullerenes and 3 is first evidenced from UV-Vis measurements. Steady state fluorescence experiment reveals efficient quenching of the excited singlet state of 3 in presence of both underivatized and derivatized fullerenes. K values for the complexes of C 60, C 70, 1 and 2 with 3 are determined to be 6500, 22,230, 47,800 and 54,770 dm 3 mol -1, respectively. The magnitude of K suggests that 3 preferentially binds C 70 and derivatized C 70 in comparison to C 60 and 1. Time resolved emission measurements establish that C 70- 3 and 2- 3 complexes are stabilized much more in comparison to C 60- 3 and 1- 3 systems in terms of charge separation process. Semi empirical calculations employing third parametric method substantiate the strong binding of C 70 and its derivative with 3 in terms of heat of formation values of the respective complexes, and at the same time, determine the orientation of bound guest (here fullerenes) with the molecular plane of 3.

  9. Photophysical and theoretical insights on non-covalently linked fullerene-zinc phthalocyanine complexes.

    PubMed

    Ray, A; Chattopadhyay, S; Bhattacharya, S

    2011-09-01

    The photo-physical aspects of non-covalently linked assemblies of a series of fullerenes, namely, C60, C70, tert-butyl-(1,2-methanofullerene)-61-carboxylate (1) and [6,6]-phenyl C70 butyric acid methyl ester (2) with a designed zinc phthalocyanine (ZnPc), viz., zinc-1,4,8,11,15,18,22,25-octabutoxy-29H,31H-phthalocyanine (3) in toluene medium are studied employing absorption spectrophotometric, steady state and time resolved fluorescence spectroscopic measurements. Of central interest in these investigations is the preferential binding of various fullerenes with ZnPc in toluene. The ground state interaction between fullerenes and 3 is first evidenced from UV-Vis measurements. Steady state fluorescence experiment reveals efficient quenching of the excited singlet state of 3 in presence of both underivatized and derivatized fullerenes. K values for the complexes of C60, C70, 1 and 2 with 3 are determined to be 6500, 22,230, 47,800 and 54,770 dm3 mol(-1), respectively. The magnitude of K suggests that 3 preferentially binds C70 and derivatized C70 in comparison to C60 and 1. Time resolved emission measurements establish that C(70)-3 and 2-3 complexes are stabilized much more in comparison to C(60)-3 and 1-3 systems in terms of charge separation process. Semi empirical calculations employing third parametric method substantiate the strong binding of C70 and its derivative with 3 in terms of heat of formation values of the respective complexes, and at the same time, determine the orientation of bound guest (here fullerenes) with the molecular plane of 3. PMID:21640637

  10. Nanoparticle mediated non-covalent drug delivery☆

    PubMed Central

    Doane, Tennyson; Burda, Clemens

    2013-01-01

    The use of nanoparticles (NPs) for enhanced drug delivery has been heavily explored during the last decade. Within the field, it is has become increasingly apparent that the physical properties of the particles themselves dictate their efficacy, and the relevant non-covalent chemistry at the NP interface also influences how drugs are immobilized and delivered. In this review, we reflect on the physical chemistry of NP mediated drug delivery (and more specifically, non-covalent drug delivery) at the three main experimental stages of drug loading, NP–drug conjugate transport, and the resulting cellular drug delivery. Through a critical evaluation of advances in drug delivery within the last decade, an outlook for biomedical applications of nanoscale transport vectors will be presented. PMID:22664231

  11. A matrix lower bound

    SciTech Connect

    Grcar, Joseph F.

    2002-02-04

    A matrix lower bound is defined that generalizes ideas apparently due to S. Banach and J. von Neumann. The matrix lower bound has a natural interpretation in functional analysis, and it satisfies many of the properties that von Neumann stated for it in a restricted case. Applications for the matrix lower bound are demonstrated in several areas. In linear algebra, the matrix lower bound of a full rank matrix equals the distance to the set of rank-deficient matrices. In numerical analysis, the ratio of the matrix norm to the matrix lower bound is a condition number for all consistent systems of linear equations. In optimization theory, the matrix lower bound suggests an identity for a class of min-max problems. In real analysis, a recursive construction that depends on the matrix lower bound shows that the level sets of continuously differential functions lie asymptotically near those of their tangents.

  12. Covalent Sidewall Functionalization of Carbon Nanotubes

    NASA Technical Reports Server (NTRS)

    Chiang, I.W.; Saini, R. K.; Mickelson, E. T.; Billups, W. E.; Hauge, R. H.; Margrave, J. L.

    2001-01-01

    Progress of fluorination of single-wall carbon nanotubes is being reported. Covalent attachment of alkyl groups including methyl, n-butyl and n-hexyl groups to the sidewalls of single wall carbon nanotubes (SWNTs) has been achieved. Quantitative measurement of the alkylation was done by thermal gravimetric analysis. FTIR, Raman and UV-Vis-NIR were used to characterize these alkylated SWNTs. Application of these nanotubes are being investigated-fibers, composites, batteries, lubricants, etc.

  13. Crystal structure of a new benzoic acid inhibitor of influenza neuraminidase bound with a new tilt induced by overpacking subsite C6

    PubMed Central

    2012-01-01

    Background Influenza neuraminidase (NA) is an important target for antiviral inhibitors since its active site is highly conserved such that inhibitors can be cross-reactive against multiple types and subtypes of influenza. Here, we discuss the crystal structure of neuraminidase subtype N9 complexed with a new benzoic acid based inhibitor (2) that was designed to add contacts by overpacking one side of the active site pocket. Inhibitor 2 uses benzoic acid to mimic the pyranose ring, a bis-(hydroxymethyl)-substituted 2-pyrrolidinone ring in place of the N-acetyl group of the sialic acid, and a branched aliphatic structure to fill the sialic acid C6 subsite. Results Inhibitor 2 {4-[2,2-bis(hydroxymethyl)-5-oxo-pyrrolidin-1-yl]-3-[(dipropylamino)methyl)]benzoic acid} was soaked into crystals of neuraminidase of A/tern/Australia/G70c/75 (N9), and the structure refined with 1.55 Å X-ray data. The benzene ring of the inhibitor tilted 8.9° compared to the previous compound (1), and the number of contacts, including hydrogen bonds, increased. However, the IC50 for compound 2 remained in the low micromolar range, likely because one propyl group was disordered. In this high-resolution structure of NA isolated from virus grown in chicken eggs, we found electron density for additional sugar units on the N-linked glycans compared to previous neuraminidase structures. In particular, seven mannoses and two N-acetylglucosamines are visible in the glycan attached to Asn200. This long, branched high-mannose glycan makes significant contacts with the neighboring subunit. Conclusions We designed inhibitor 2 with an extended substituent at C4-corresponding to C6 of sialic acid-to increase the contact surface in the C6-subsite and to force the benzene ring to tilt to maximize these interactions while retaining the interactions of the carboxylate and the pyrolidinone substituents. The crystal structure at 1.55 Å showed that we partially succeeded in that the ring in 2 is tilted

  14. Multiple-component covalent organic frameworks

    PubMed Central

    Huang, Ning; Zhai, Lipeng; Coupry, Damien E.; Addicoat, Matthew A.; Okushita, Keiko; Nishimura, Katsuyuki; Heine, Thomas; Jiang, Donglin

    2016-01-01

    Covalent organic frameworks are a class of crystalline porous polymers that integrate molecular building blocks into periodic structures and are usually synthesized using two-component [1+1] condensation systems comprised of one knot and one linker. Here we report a general strategy based on multiple-component [1+2] and [1+3] condensation systems that enable the use of one knot and two or three linker units for the synthesis of hexagonal and tetragonal multiple-component covalent organic frameworks. Unlike two-component systems, multiple-component covalent organic frameworks feature asymmetric tiling of organic units into anisotropic skeletons and unusually shaped pores. This strategy not only expands the structural complexity of skeletons and pores but also greatly enhances their structural diversity. This synthetic platform is also widely applicable to multiple-component electron donor–acceptor systems, which lead to electronic properties that are not simply linear summations of those of the conventional [1+1] counterparts. PMID:27460607

  15. Multiple-component covalent organic frameworks.

    PubMed

    Huang, Ning; Zhai, Lipeng; Coupry, Damien E; Addicoat, Matthew A; Okushita, Keiko; Nishimura, Katsuyuki; Heine, Thomas; Jiang, Donglin

    2016-01-01

    Covalent organic frameworks are a class of crystalline porous polymers that integrate molecular building blocks into periodic structures and are usually synthesized using two-component [1+1] condensation systems comprised of one knot and one linker. Here we report a general strategy based on multiple-component [1+2] and [1+3] condensation systems that enable the use of one knot and two or three linker units for the synthesis of hexagonal and tetragonal multiple-component covalent organic frameworks. Unlike two-component systems, multiple-component covalent organic frameworks feature asymmetric tiling of organic units into anisotropic skeletons and unusually shaped pores. This strategy not only expands the structural complexity of skeletons and pores but also greatly enhances their structural diversity. This synthetic platform is also widely applicable to multiple-component electron donor-acceptor systems, which lead to electronic properties that are not simply linear summations of those of the conventional [1+1] counterparts. PMID:27460607

  16. Construct Polyoxometalate Frameworks through Covalent Bonds.

    PubMed

    Chen, Hong; Zhao, Huishuang; Yu, Zheng-Bao; Wang, Lei; Sun, Licheng; Sun, Junliang

    2015-09-01

    An emerging strategy for exploring the application of polyoxometalates (POMs) is to assemble POM clusters into open-framework materials, especially inorganic-organic hybrid three-dimensional (3D) open-framework materials, via the introduction of different organic linkers between the POM clusters. This strategy has yielded a few 3D crystalline POMs of which a typical class is the group of polyoxometalate metal-organic frameworks (POMMOFs). However, for reported POMMOFs, only coordination bonds are involved between the linkers and POM clusters, and it has not yet produced any covalently bonded polyoxometalate frameworks. Here, the concept of "covalently bonded POMs (CPOMs)" is developed. By using vanadoborates as an example, we showed that the 3D CPOMs can be obtained by a condensation reaction through the oxolation mechanism of polymer chemistry. In particular, suitable single crystals were harvested and characterized by single-crystal X-ray diffraction. This work forges a link among polymer science, POM chemistry, and open-framework materials by demonstrating that it is possible to use covalent bonds according to polymer chemistry principles to construct crystalline 3D open-framework POM materials. PMID:26286321

  17. Dual-Mode HDAC Prodrug for Covalent Modification and Subsequent Inhibitor Release

    PubMed Central

    2016-01-01

    Histone deacetylase inhibitors (HDACi) target abnormal epigenetic states associated with a variety of pathologies, including cancer. Here, the development of a prodrug of the canonical broad-spectrum HDACi suberoylanilide hydroxamic acid (SAHA) is described. Although hydroxamic acids are utilized universally in the development of metalloenzyme inhibitors, they are considered to be poor pharmacophores with reduced activity in vivo. We developed a prodrug of SAHA by appending a promoiety, sensitive to thiols, to the hydroxamic acid warhead (termed SAHA-TAP). After incubation of SAHA-TAP with an HDAC, the thiol of a conserved HDAC cysteine residue becomes covalently tagged with the promoiety, initiating a cascade reaction that leads to the release of SAHA. Mass spectrometry and enzyme kinetics experiments validate that the cysteine residue is covalently appended with the TAP promoiety. SAHA-TAP demonstrates cytotoxicity activity against various cancer cell lines. This strategy represents an original prodrug design with a dual mode of action for HDAC inhibition. PMID:25974739

  18. Post-Synthetic Decoupling of On-Surface-Synthesized Covalent Nanostructures from Ag(111).

    PubMed

    Rastgoo-Lahrood, Atena; Björk, Jonas; Lischka, Matthias; Eichhorn, Johanna; Kloft, Stephan; Fritton, Massimo; Strunskus, Thomas; Samanta, Debabrata; Schmittel, Michael; Heckl, Wolfgang M; Lackinger, Markus

    2016-06-27

    The on-surface synthesis of covalent organic nanosheets driven by reactive metal surfaces leads to strongly adsorbed organic nanostructures, which conceals their intrinsic properties. Hence, reducing the electronic coupling between the organic networks and commonly used metal surfaces is an important step towards characterization of the true material. We demonstrate that post-synthetic exposure to iodine vapor leads to the intercalation of an iodine monolayer between covalent polyphenylene networks and Ag(111) surfaces. The experimentally observed changes from surface-bound to detached nanosheets are reproduced by DFT simulations. These findings suggest that the intercalation of iodine provides a material that shows geometric and electronic properties substantially closer to those of the freestanding network. PMID:27125328

  19. Covalent immobilization of Pseudomonas cepacia lipase on semiconducting materials

    NASA Astrophysics Data System (ADS)

    Fernandez, Renny Edwin; Bhattacharya, Enakshi; Chadha, Anju

    2008-05-01

    Lipase from Pseudomonas cepacia was covalently immobilized on crystalline silicon, porous silicon and silicon nitride surfaces. The various stages of immobilization were characterized using FTIR (Fourier transform infrared) spectroscopy. The surface topography of the enzyme immobilized surfaces was investigated using scanning electron microscopy (SEM). The quantity of the immobilized active enzyme was estimated by the para-nitrophenyl palmitate (pNPP) assay. The immobilized lipase was used for triglyceride hydrolysis and the acid produced was detected by a pH sensitive silicon nitride surface as a shift in the C- V (capacitance-voltage) characteristics of an electrolyte-insulator-semiconductor capacitor (EISCAP) thus validating the immobilization method for use as a biosensor.

  20. Mechanochemical synthesis of chemically stable isoreticular covalent organic frameworks.

    PubMed

    Biswal, Bishnu P; Chandra, Suman; Kandambeth, Sharath; Lukose, Binit; Heine, Thomas; Banerjee, Rahul

    2013-04-10

    Three thermally and chemically stable isoreticular covalent organic frameworks (COFs) were synthesized via room-temperature solvent-free mechanochemical grinding. These COFs were successfully compared with their solvothermally synthesized counterparts in all aspects. These solvent-free mechanochemically synthesized COFs have moderate crystallinity with remarkable stability in boiling water, acid (9 N HCl), and base [TpBD (MC) in 3 N NaOH and TpPa-2 (MC) in 9 N NaOH]. Exfoliation of COF layers was simultaneously observed with COF formation during mechanochemical synthesis. The structures thus obtained seemed to have a graphene-like layered morphology (exfoliated layers), unlike the parent COFs synthesized solvothermally. PMID:23521070

  1. 5-hydroxymethyl-2-furfural and derivatives formed during acid hydrolysis of conjugated and bound phenolics in plant foods and the effects on phenolic content and antioxidant capacity.

    PubMed

    Chen, Peter X; Tang, Yao; Zhang, Bing; Liu, Ronghua; Marcone, Massimo F; Li, Xihong; Tsao, Rong

    2014-05-21

    A common protocol for the extraction of phenolic aglycons or bound phenolics in plants generally involves hydrothermal hydrolysis in an aqueous methanol or ethanol solution containing 2-4 N HCl. However, as shown in the present study, this process also forms 5-(hydroxymethyl)furan-2-carbaldehyde (HMF) and its derivative products 5-(methoxymethyl)furan-2-carbaldehyde (MMF) and 5-(ethoxymethyl)furan-2-carbaldehyde (EMF), as identified by HPLC-DAD-ESI-MS/MS and NMR. These compounds are commonly misidentified as phenolics due to similar UV absorption at 280 nm. In this study, production of HMF, MMF, and EMF was shown to be dependent on the solvent condition and duration and temperature of hydrolysis. Fruits and vegetables produced HMF more readily than grains. HMF and its derivatives were subjected to various spectrophotometric antioxidant assays [2-diphenyl-1-picryhydrazyl radical scavenging activity (DPPH), ferric-reducing antioxidant power (FRAP), and oxygen radical absorbing capacity (ORAC)] and displayed antioxidant activity mainly in the ORAC assay. Results of this study help avoid overestimation of phenolic content and antioxidant activities of plant foods. PMID:24796380

  2. Modulation of UvrD helicase activity by covalent DNA-protein cross-links.

    PubMed

    Kumari, Anuradha; Minko, Irina G; Smith, Rebecca L; Lloyd, R Stephen; McCullough, Amanda K

    2010-07-01

    UvrD (DNA helicase II) has been implicated in DNA replication, DNA recombination, nucleotide excision repair, and methyl-directed mismatch repair. The enzymatic function of UvrD is to translocate along a DNA strand in a 3' to 5' direction and unwind duplex DNA utilizing a DNA-dependent ATPase activity. In addition, UvrD interacts with many other proteins involved in the above processes and is hypothesized to facilitate protein turnover, thus promoting further DNA processing. Although UvrD interactions with proteins bound to DNA have significant biological implications, the effects of covalent DNA-protein cross-links on UvrD helicase activity have not been characterized. Herein, we demonstrate that UvrD-catalyzed strand separation was inhibited on a DNA strand to which a 16-kDa protein was covalently bound. Our sequestration studies suggest that the inhibition of UvrD activity is most likely due to a translocation block and not helicase sequestration on the cross-link-containing DNA substrate. In contrast, no inhibition of UvrD-catalyzed strand separation was apparent when the protein was linked to the complementary strand. The latter result is surprising given the earlier observations that the DNA in this covalent complex is severely bent ( approximately 70 degrees ), with both DNA strands making multiple contacts with the cross-linked protein. In addition, UvrD was shown to be required for replication of plasmid DNAs containing covalent DNA-protein complexes. Combined, these data suggest a critical role for UvrD in the processing of DNA-protein cross-links. PMID:20444702

  3. Irreversible covalent modification of type I dehydroquinase with a stable Schiff base.

    PubMed

    Tizón, Lorena; Maneiro, María; Peón, Antonio; Otero, José M; Lence, Emilio; Poza, Sergio; van Raaij, Mark J; Thompson, Paul; Hawkins, Alastair R; González-Bello, Concepción

    2015-01-21

    The irreversible inhibition of type I dehydroquinase (DHQ1), the third enzyme of the shikimic acid pathway, is investigated by structural, biochemical and computational studies. Two epoxides, which are mimetics of the natural substrate, were designed as irreversible inhibitors of the DHQ1 enzyme and to study the binding requirements of the linkage to the enzyme. The epoxide with the S configuration caused the covalent modification of the protein whereas no reaction was obtained with its epimer. The first crystal structure of DHQ1 from Salmonella typhi covalently modified by the S epoxide, which is reported at 1.4 Å, revealed that the modified ligand is surprisingly covalently attached to the essential Lys170 by the formation of a stable Schiff base. The experimental and molecular dynamics simulation studies reported here highlight the huge importance of the conformation of the C3 carbon of the ligand for covalent linkage to this type of aldolase I enzyme, revealed the key role played by the essential His143 as a Lewis acid in this process and show the need for a neatly closed active site for catalysis. PMID:25370445

  4. Comparative immunogenicity of conjugates composed of the Staphylococcus aureus type 8 capsular polysaccharide bound to carrier proteins by adipic acid dihydrazide or N-succinimidyl-3-(2-pyridyldithio)propionate.

    PubMed Central

    Fattom, A; Shiloach, J; Bryla, D; Fitzgerald, D; Pastan, I; Karakawa, W W; Robbins, J B; Schneerson, R

    1992-01-01

    Staphylococcus aureus type 8 capsular polysaccharide (CP) was conjugated either to diphtheria toxoid or to Pseudomonas aeruginosa recombinant exoprotein A by using adipic acid dihydrazide (ADH) or N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) as the joining reagent. The polysaccharide/protein ratios of these two pairs of conjugates were similar. The two synthetic schemes bound the linker to the carboxyls of the type 8 CP by carbodiimide-mediated condensation. ADH was bound to the carboxyls of the protein, whereas SPDP reacted with the amino groups of the protein. Intermolecular linking of the carrier protein, caused by the carbodiimide during the conjugation reaction with the type 8 CP derivative, probably accounts for the larger size of the conjugates formed with ADH compared with those formed with SPDP. Both conjugates synthesized with ADH elicited higher levels of CP antibodies, especially after the first immunization, than did those prepared with SPDP. Similar levels of exoprotein A antibodies were elicited by both conjugates. Higher levels of diphtheria toxoid antibodies were elicited by the conjugate prepared with SPDP than by the one prepared with ADH. The basis for the differences in the immunogenicities of these two pairs of S. aureus type 8 CP conjugates is discussed. PMID:1730492

  5. Comparative immunogenicity of conjugates composed of the Staphylococcus aureus type 8 capsular polysaccharide bound to carrier proteins by adipic acid dihydrazide or N-succinimidyl-3-(2-pyridyldithio)propionate.

    PubMed

    Fattom, A; Shiloach, J; Bryla, D; Fitzgerald, D; Pastan, I; Karakawa, W W; Robbins, J B; Schneerson, R

    1992-02-01

    Staphylococcus aureus type 8 capsular polysaccharide (CP) was conjugated either to diphtheria toxoid or to Pseudomonas aeruginosa recombinant exoprotein A by using adipic acid dihydrazide (ADH) or N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) as the joining reagent. The polysaccharide/protein ratios of these two pairs of conjugates were similar. The two synthetic schemes bound the linker to the carboxyls of the type 8 CP by carbodiimide-mediated condensation. ADH was bound to the carboxyls of the protein, whereas SPDP reacted with the amino groups of the protein. Intermolecular linking of the carrier protein, caused by the carbodiimide during the conjugation reaction with the type 8 CP derivative, probably accounts for the larger size of the conjugates formed with ADH compared with those formed with SPDP. Both conjugates synthesized with ADH elicited higher levels of CP antibodies, especially after the first immunization, than did those prepared with SPDP. Similar levels of exoprotein A antibodies were elicited by both conjugates. Higher levels of diphtheria toxoid antibodies were elicited by the conjugate prepared with SPDP than by the one prepared with ADH. The basis for the differences in the immunogenicities of these two pairs of S. aureus type 8 CP conjugates is discussed. PMID:1730492

  6. Development of covalent inhibitors that can overcome resistance to first-generation FGFR kinase inhibitors.

    PubMed

    Tan, Li; Wang, Jun; Tanizaki, Junko; Huang, Zhifeng; Aref, Amir R; Rusan, Maria; Zhu, Su-Jie; Zhang, Yiyun; Ercan, Dalia; Liao, Rachel G; Capelletti, Marzia; Zhou, Wenjun; Hur, Wooyoung; Kim, NamDoo; Sim, Taebo; Gaudet, Suzanne; Barbie, David A; Yeh, Jing-Ruey Joanna; Yun, Cai-Hong; Hammerman, Peter S; Mohammadi, Moosa; Jänne, Pasi A; Gray, Nathanael S

    2014-11-11

    The human FGF receptors (FGFRs) play critical roles in various human cancers, and several FGFR inhibitors are currently under clinical investigation. Resistance usually results from selection for mutant kinases that are impervious to the action of the drug or from up-regulation of compensatory signaling pathways. Preclinical studies have demonstrated that resistance to FGFR inhibitors can be acquired through mutations in the FGFR gatekeeper residue, as clinically observed for FGFR4 in embryonal rhabdomyosarcoma and neuroendocrine breast carcinomas. Here we report on the use of a structure-based drug design to develop two selective, next-generation covalent FGFR inhibitors, the FGFR irreversible inhibitors 2 (FIIN-2) and 3 (FIIN-3). To our knowledge, FIIN-2 and FIIN-3 are the first inhibitors that can potently inhibit the proliferation of cells dependent upon the gatekeeper mutants of FGFR1 or FGFR2, which confer resistance to first-generation clinical FGFR inhibitors such as NVP-BGJ398 and AZD4547. Because of the conformational flexibility of the reactive acrylamide substituent, FIIN-3 has the unprecedented ability to inhibit both the EGF receptor (EGFR) and FGFR covalently by targeting two distinct cysteine residues. We report the cocrystal structure of FGFR4 with FIIN-2, which unexpectedly exhibits a "DFG-out" covalent binding mode. The structural basis for dual FGFR and EGFR targeting by FIIN3 also is illustrated by crystal structures of FIIN-3 bound with FGFR4 V550L and EGFR L858R. These results have important implications for the design of covalent FGFR inhibitors that can overcome clinical resistance and provide the first example, to our knowledge, of a kinase inhibitor that covalently targets cysteines located in different positions within the ATP-binding pocket. PMID:25349422

  7. Development of covalent inhibitors that can overcome resistance to first-generation FGFR kinase inhibitors

    PubMed Central

    Tan, Li; Wang, Jun; Tanizaki, Junko; Huang, Zhifeng; Aref, Amir R.; Rusan, Maria; Zhu, Su-Jie; Zhang, Yiyun; Ercan, Dalia; Liao, Rachel G.; Capelletti, Marzia; Zhou, Wenjun; Hur, Wooyoung; Kim, NamDoo; Sim, Taebo; Gaudet, Suzanne; Barbie, David A.; Yeh, Jing-Ruey Joanna; Yun, Cai-Hong; Hammerman, Peter S.; Mohammadi, Moosa; Jänne, Pasi A.; Gray, Nathanael S.

    2014-01-01

    The human FGF receptors (FGFRs) play critical roles in various human cancers, and several FGFR inhibitors are currently under clinical investigation. Resistance usually results from selection for mutant kinases that are impervious to the action of the drug or from up-regulation of compensatory signaling pathways. Preclinical studies have demonstrated that resistance to FGFR inhibitors can be acquired through mutations in the FGFR gatekeeper residue, as clinically observed for FGFR4 in embryonal rhabdomyosarcoma and neuroendocrine breast carcinomas. Here we report on the use of a structure-based drug design to develop two selective, next-generation covalent FGFR inhibitors, the FGFR irreversible inhibitors 2 (FIIN-2) and 3 (FIIN-3). To our knowledge, FIIN-2 and FIIN-3 are the first inhibitors that can potently inhibit the proliferation of cells dependent upon the gatekeeper mutants of FGFR1 or FGFR2, which confer resistance to first-generation clinical FGFR inhibitors such as NVP-BGJ398 and AZD4547. Because of the conformational flexibility of the reactive acrylamide substituent, FIIN-3 has the unprecedented ability to inhibit both the EGF receptor (EGFR) and FGFR covalently by targeting two distinct cysteine residues. We report the cocrystal structure of FGFR4 with FIIN-2, which unexpectedly exhibits a “DFG-out” covalent binding mode. The structural basis for dual FGFR and EGFR targeting by FIIN3 also is illustrated by crystal structures of FIIN-3 bound with FGFR4 V550L and EGFR L858R. These results have important implications for the design of covalent FGFR inhibitors that can overcome clinical resistance and provide the first example, to our knowledge, of a kinase inhibitor that covalently targets cysteines located in different positions within the ATP-binding pocket. PMID:25349422

  8. Agaricus meleagris pyranose dehydrogenase: Influence of covalent FAD linkage on catalysis and stability

    PubMed Central

    Krondorfer, Iris; Brugger, Dagmar; Paukner, Regina; Scheiblbrandner, Stefan; Pirker, Katharina F.; Hofbauer, Stefan; Furtmüller, Paul G.; Obinger, Christian; Haltrich, Dietmar; Peterbauer, Clemens K.

    2014-01-01

    Pyranose dehydrogenase (PDH) is a monomeric flavoprotein belonging to the glucose–methanol–choline (GMC) family of oxidoreductases. It catalyzes the oxidation of free, non-phosphorylated sugars to the corresponding keto sugars. The enzyme harbors an FAD cofactor that is covalently attached to histidine 103 via an 8α-N(3) histidyl linkage. Our previous work showed that variant H103Y was still able to bind FAD (non-covalently) and perform catalysis but steady-state kinetic parameters for several substrates were negatively affected. In order to investigate the impact of the covalent FAD attachment in Agaricus meleagris PDH in more detail, pre-steady-state kinetics, reduction potential and stability of the variant H103Y in comparison to the wild-type enzyme were probed. Stopped-flow analysis revealed that the mutation slowed down the reductive half-reaction by around three orders of magnitude whereas the oxidative half-reaction was affected only to a minor degree. This was reflected by a decrease in the standard reduction potential of variant H103Y compared to the wild-type protein. The existence of an anionic semiquinone radical in the resting state of both the wild-type and variant H103Y was demonstrated using electron paramagnetic resonance (EPR) spectroscopy and suggested a higher mobility of the cofactor in the variant H103Y. Unfolding studies showed significant negative effects of the disruption of the covalent bond on thermal and conformational stability. The results are discussed with respect to the role of covalently bound FAD in catalysis and stability. PMID:25043975

  9. Physical Uncertainty Bounds (PUB)

    SciTech Connect

    Vaughan, Diane Elizabeth; Preston, Dean L.

    2015-03-19

    This paper introduces and motivates the need for a new methodology for determining upper bounds on the uncertainties in simulations of engineered systems due to limited fidelity in the composite continuum-level physics models needed to simulate the systems. We show that traditional uncertainty quantification methods provide, at best, a lower bound on this uncertainty. We propose to obtain bounds on the simulation uncertainties by first determining bounds on the physical quantities or processes relevant to system performance. By bounding these physics processes, as opposed to carrying out statistical analyses of the parameter sets of specific physics models or simply switching out the available physics models, one can obtain upper bounds on the uncertainties in simulated quantities of interest.

  10. Asymptotic entropy bounds

    NASA Astrophysics Data System (ADS)

    Bousso, Raphael

    2016-07-01

    We show that known entropy bounds constrain the information carried off by radiation to null infinity. We consider distant, planar null hypersurfaces in asymptotically flat spacetime. Their focusing and area loss can be computed perturbatively on a Minkowski background, yielding entropy bounds in terms of the energy flux of the outgoing radiation. In the asymptotic limit, we obtain boundary versions of the quantum null energy condition, of the generalized Second Law, and of the quantum Bousso bound.

  11. Covalent organic frameworks formed with two types of covalent bonds based on orthogonal reactions.

    PubMed

    Zeng, Yongfei; Zou, Ruyi; Luo, Zhong; Zhang, Huacheng; Yao, Xin; Ma, Xing; Zou, Ruqiang; Zhao, Yanli

    2015-01-28

    Covalent organic frameworks (COFs) are excellent candidates for various applications. So far, successful methods for the constructions of COFs have been limited to a few condensation reactions based on only one type of covalent bond formation. Thus, the exploration of a new judicious synthetic strategy is a crucial and emergent task for the development of this promising class of porous materials. Here, we report a new orthogonal reaction strategy to construct COFs by reversible formations of two types of covalent bonds. The obtained COFs consisting of multiple components show high surface area and high H2 adsorption capacity. The strategy is a general protocol applicable to construct not only binary COFs but also more complicated systems in which employing regular synthetic methods did not work. PMID:25581488

  12. Purification and characterization of a 14-kilodalton protein that is bound to the surface of polyhydroxyalkanoic acid granules in Rhodococcus ruber.

    PubMed Central

    Pieper-Fürst, U.; Madkour, M. H.; Mayer, F.; Steinbüchel, A.

    1994-01-01

    The N-terminal amino acid sequence of the polyhydroxyalkanoic acid (PHA) granule-associated M(r)-15,500 protein of Rhodococcus ruber (the GA14 protein) was analyzed. The sequence revealed that the corresponding structural gene is represented by open reading frame 3, encoding a protein with a calculated M(r) of 14,175 which was recently localized downstream of the PHA synthase gene (U. Pieper and A. Steinbüchel, FEMS Microbiol. Lett. 96:73-80, 1992). A recombinant strain of Escherichia coli XL1-Blue carrying the hybrid plasmid (pSKXA10*) with open reading frame 3 overexpressed the GA14 protein. The GA14 protein was subsequently purified in a three-step procedure including chromatography on DEAE-Sephacel, phenyl-Sepharose CL-4B, and Superose 12. Determination of the molecular weight by gel filtration as well as electron microscopic studies indicates that a tetrameric structure of the recombinant, native GA14 protein is most likely. Immunoelectron microscopy demonstrated a localization of the GA14 protein at the periphery of PHA granules as well as close to the cell membrane in R. ruber. Investigations of PHA-leaky and PHA-negative mutants of R. ruber indicated that expression of the GA14 protein depended strongly on PHA synthesis. Images PMID:8021220

  13. Targeting to cells of fluorescent liposomes covalently coupled with monoclonal antibody or protein A

    NASA Astrophysics Data System (ADS)

    Leserman, Lee D.; Barbet, Jacques; Kourilsky, François; Weinstein, John N.

    1980-12-01

    Many applications envisioned for liposomes in cell biology and chemotherapy require their direction to specific cellular targets1-3. The ability to use antibody as a means of conferring specificity to liposomes would markedly increase their usefulness. We report here a method for covalently coupling soluble proteins, including monoclonal antibody and Staphylococcus aureus protein A (ref. 4), to small sonicated liposomes, by using the heterobifunctional cross-linking reagent N-hydroxysuccinimidyl 3-(2-pyridyldithio)propionate (SPDP, Pharmacia). Liposomes bearing covalently coupled mouse monoclonal antibody against human β2-microglobulin [antibody B1.1G6 (IgG2a, κ) (B. Malissen et al., in preparation)] bound specifically to human, but not to mouse cells. Liposomes bearing protein A became bound to human cells previously incubated with the B1.1G6 antibody, but not to cells incubated without antibody. The coupling method results in efficient binding of protein to the liposomes without aggregation and without denaturation of the coupled ligand; at least 60% of liposomes bound functional protein. Further, liposomes did not leak encapsulated carboxyfluorescein (CF) as a consequence of the reaction.

  14. In vitro covalent binding of 3-(/sup 14/C)methylindole metabolites in goat tissues

    SciTech Connect

    Bray, T.M.; Carlson, J.R.; Nocerini, M.R.

    1984-05-01

    Covalent binding of 3-(/sup 14/C)methylindole (3(/sup 14/C)MI) in crude microsomal preparations of goat lung, liver, and kidney was measured to determine if a reactive intermediate was formed during the in vitro metabolism of 3-methylindole (3MI). The bound radioactivity was highest in lung compared to liver and kidney. The amount of bound radioactivity per nanomole of cytochrome P-450 was approximately 10 times higher in the lung compared to the liver. No detectable bound radioactivity was found when 3-(/sup 3/H)methyloxindole was used as the substrate. Cofactor requirements and the effects of inhibitors indicate that a mixed function oxidase (MFO) system is involved in formation of a reactive intermediate. Inhibitors and conjugating agents that are known to reduce the severity of 3MI-induced lung injury such as piperonyl butoxide (MFO inhibitor) and glutathione (conjugating agent) significantly decreased the in vitro binding of 3(/sup 14/C)MI. The results indicate that a reactive intermediate is produced during the metabolism of 3MI by the MFO system. The organ specificity in binding suggests that covalent binding by lung microsomes may be related to the mechanism of 3MI-induced lung injury.

  15. Single-step DNA immobilization on antifouling self-assembled monolayers covalently bound to silicon (111).

    PubMed

    Böcking, Till; Kilian, Kristopher A; Gaus, Katharina; Gooding, J Justin

    2006-04-11

    Hydrosilylation of alkenes with epoxide-terminated tri(ethylene oxide) moieties on Si-H surfaces yields homogeneous monolayers for the efficient coupling of biomolecules. The wetting properties of the epoxide-functionalized surface allow for the spotting of solutions of biomolecules, making the surface amenable to microarraying. Immobilization of thiolated DNA was achieved in a single step to fabricate biorecognition interfaces showing the hybridization of complementary DNA at low concentrations and negligible binding of noncomplementary DNA. PMID:16584219

  16. Semiconductor nanocrystals covalently bound to solid inorganic surfaces using self-assembled monolayers

    DOEpatents

    Alivisatos, A. Paul; Colvin, Vicki L.

    1998-01-01

    Methods are described for attaching semiconductor nanocrystals to solid inorganic surfaces, using self-assembled bifunctional organic monolayers as bridge compounds. Two different techniques are presented. One relies on the formation of self-assembled monolayers on these surfaces. When exposed to solutions of nanocrystals, these bridge compounds bind the crystals and anchor them to the surface. The second technique attaches nanocrystals already coated with bridge compounds to the surfaces. Analyses indicate the presence of quantum confined clusters on the surfaces at the nanolayer level. These materials allow electron spectroscopies to be completed on condensed phase clusters, and represent a first step towards synthesis of an organized assembly of clusters. These new products are also disclosed.

  17. Semiconductor nanocrystals covalently bound to solid inorganic surfaces using self-assembled monolayers

    DOEpatents

    Alivisatos, A.P.; Colvin, V.L.

    1998-05-12

    Methods are described for attaching semiconductor nanocrystals to solid inorganic surfaces, using self-assembled bifunctional organic monolayers as bridge compounds. Two different techniques are presented. One relies on the formation of self-assembled monolayers on these surfaces. When exposed to solutions of nanocrystals, these bridge compounds bind the crystals and anchor them to the surface. The second technique attaches nanocrystals already coated with bridge compounds to the surfaces. Analyses indicate the presence of quantum confined clusters on the surfaces at the nanolayer level. These materials allow electron spectroscopies to be completed on condensed phase clusters, and represent a first step towards synthesis of an organized assembly of clusters. These new products are also disclosed. 10 figs.

  18. Cellular uptake and covalent binding of nitroso-chloramphenicol

    SciTech Connect

    Murray, T.; Yunis, A.A.

    1981-09-01

    A comparative study of the cellular transport of CAP and its nitroso derivative (NO-CAP) was carried out in Raji cells, a transformed human lymphoblastoid cell line. Both agents were concentrated by the cells by a factor of 3 (cellular/extracellular concentration ratio). The cellular uptake of NO-CAP, like that of CAP, was found to be rapid and temperature-independent. Thus the greater cytotoxicity of NO-CAP is apparently not due to an enhanced uptake of the nitroso derivative relative to CAP. In contrast to the similarity of uptake, NO-CAP becomes covalently bound to both Raji cells and freshly isolated human bone marrow cells to a much higher extent (15-fold). Also, cells previously loaded with CAP or NO-CAP retain three times as much of the nitroso compound during a 24 hr dialysis against a drug-free isotonic solution. The increased binding of NO-CAP to human hematopoietic cells attests to the greater reactivity of the p-substituted aromatic nitroso group and is consistent with the postulate that reduction products of the nitro group of CAP may be responsible for CAP-induced aplastic anemia.

  19. Dielectrophoretic micropatterning with microparticle monolayers covalently linked to glass surfaces.

    PubMed

    Suzuki, Masato; Yasukawa, Tomoyuki; Mase, Yoshiaki; Oyamatsu, Daisuke; Shiku, Hitoshi; Matsue, Tomokazu

    2004-12-01

    Two-dimensional micropatterns of microparticles were fabricated on glass substrates with negative dielectrophoretic force, and the patterned microparticles were covalently bound on the substrate via cross-linking agents. The line and grid patterns of microparticles were prepared using the repulsive force of negative dielectrophoresis (n-DEP). The template interdigitated microband array (IDA) electrodes (width and gap 50 mum) were incorporated into the dielectrophoretic patterning cell with a fluidic channel. The microstructures on the glass substrates with amino or sulfhydryl groups were immobilized with the cross-linking agents disuccinimidyl suberate (DSS) and m-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS). Diaphorase (Dp), a flavoenzyme, was selectively attached on the patterned microparticles using the maleimide groups of MBS. The enzyme activity on the patterned particles was electrochemically characterized with a scanning electrochemical microscope (SECM) in the presence of NADH and ferrocenylmethanol as a redox mediator. The SECM images proved that Dp was selectively immobilized onto the surface of microparticles to maintain its catalytic activity. PMID:15568852

  20. Nuclear Clusters and Covalent Molecules on the femto-scale

    SciTech Connect

    Oertzen, Wolfram von

    2010-04-30

    Nuclear clusters like alpha-particles light N = Z nuclei are the building blocks of nuclear molecules. With additional 'valence' neutrons, which find there place in quantum mechanical two-center orbits a variety of covalently bound states in nuclei have been established in the last decade: in isotopes of {sup 9-12}Be, {sup 13-14}C and {sup 21}Ne. More recently we have studied molecular states in {sup 18,19,20}O-isotopes using the ({sup 7}Li,p) reaction on {sup 12,13,14}C targets at E{sub lab}({sup 7}Li) = 44 MeV. The systematics of the energies and cross sections show rotational bands with high moments of inertia. These are characteristic of large deformations or molecular structures where the clusters are well separated. Generally the large scale shell model calculations are unable to reproduce these cluster bands. With two clusters of different size (e.g. ({sup 14}C x {sup 4}He), or ({sup 16}O x {sup 4}He)) intrinsically reflection asymmetric shapes arise. The molecular structures appear as rotational bands split into parity inversion doublets.

  1. Bulk modulus for polar covalent crystals

    PubMed Central

    Xu, Bo; Wang, Qianqian; Tian, Yongjun

    2013-01-01

    A microscopic empirical model of bulk modulus based on atomic-scale parameters is proposed. These parameters include the bond length, the effective bonded valence electron (EBVE) number, and the coordination number product of two bonded atoms, etc. The estimated bulk moduli from our model are in good agreement with experimental values for various polar covalent crystals including ionic crystals. Our current work sheds lights on the nature of bulk modulus, provides useful clues for design of crystals with low compressibility, and is applicable to complex crystals such as minerals of geophysical importance. PMID:24166098

  2. Floating electron states in covalent semiconductors.

    PubMed

    Matsushita, Yu-ichiro; Furuya, Shinnosuke; Oshiyama, Atsushi

    2012-06-15

    We report first-principles electronic-structure calculations that clarify the floating nature of electron states in covalent semiconductors. It is found that wave functions of several conduction- and valence-band states, including the conduction-band minima, do not distribute near atomic sites, as was taken for granted, but float in interstitial channels in most semiconductors. The directions and shapes of the interstitial channels depend on the crystal symmetry so that mysterious variation of the energy gaps in SiC polymorphs is naturally explained by considering the floating nature. PMID:23004300

  3. The covalent bond in Particle Spectroscopy

    SciTech Connect

    Bugg, D. V.

    2010-08-05

    Meson resonances are linear combinations of qq-bar and meson-meson (MM) baryon resonances are combinations of qqq and meson-baryon. Mixing between these combinations arises via decays of confined states to meson-meson or meson-baryon. There is a useful analogy with the covalent bond in molecular physics. One eigenstate is lowered by the mixing. Cusps arise at thresholds. At sharp thresholds due to S-wave 2-particle decays, these cusps play a conspicuous role in many sets of data.

  4. Protocol for rational design of covalently interacting inhibitors.

    PubMed

    Schmidt, Thomas C; Welker, Armin; Rieger, Max; Sahu, Prabhat K; Sotriffer, Christoph A; Schirmeister, Tanja; Engels, Bernd

    2014-10-20

    The inhibition potencies of covalent inhibitors mainly result from the formation of a covalent bond to the enzyme during the inhibition mechanism. This class of inhibitors has essentially been ignored in previous target-directed drug discovery projects because of concerns about possible side effects. However, their advantages, such as higher binding energies and longer drug-target residence times moved them into the focus of recent investigations. While the rational design of non-covalent inhibitors became standard the corresponding design of covalent inhibitors is still in its early stages. Potent covalent inhibitors can be retrieved from large compound libraries by covalent docking approaches but protocols are missing that can reliably predict the influence of variations in the substitution pattern on the affinity and/or reactivity of a given covalent inhibitor. Hence, the wanted property profile can only be obtained from trial-and-error proceedings. This paper presents an appropriate protocol which is able to predict improved covalent inhibitors. It uses hybrid approaches, which mix quantum mechanical (QM) and molecular mechanical (MM) methods to predict variations in the reactivity of the inhibitor. They are also used to compute the required information about the non-covalent enzyme-inhibitor complex. Docking tools are employed to improve the inhibitor with respect to the non-covalent interactions formed in the binding site. PMID:25251382

  5. Smart Organic Two-Dimensional Materials Based on a Rational Combination of Non-covalent Interactions.

    PubMed

    Bai, Wei; Jiang, Ziwen; Ribbe, Alexander E; Thayumanavan, S

    2016-08-26

    Rational design of organic 2D (O2D) materials has made some progress, but it is still in its infancy. A class of self-assembling small molecules is presented that form nano/microscale supramolecular 2D materials in aqueous media. A judicial combination of four different intermolecular interactions forms the basis for the robust formation of these ultrathin assemblies. These assemblies can be programmed to disassemble in response to a specific protein and release its non-covalently bound guest molecules. PMID:27490155

  6. Bounding Species Distribution Models

    NASA Technical Reports Server (NTRS)

    Stohlgren, Thomas J.; Jarnevich, Cahterine S.; Morisette, Jeffrey T.; Esaias, Wayne E.

    2011-01-01

    Species distribution models are increasing in popularity for mapping suitable habitat for species of management concern. Many investigators now recognize that extrapolations of these models with geographic information systems (GIS) might be sensitive to the environmental bounds of the data used in their development, yet there is no recommended best practice for "clamping" model extrapolations. We relied on two commonly used modeling approaches: classification and regression tree (CART) and maximum entropy (Maxent) models, and we tested a simple alteration of the model extrapolations, bounding extrapolations to the maximum and minimum values of primary environmental predictors, to provide a more realistic map of suitable habitat of hybridized Africanized honey bees in the southwestern United States. Findings suggest that multiple models of bounding, and the most conservative bounding of species distribution models, like those presented here, should probably replace the unbounded or loosely bounded techniques currently used [Current Zoology 57 (5): 642-647, 2011].

  7. Bounding species distribution models

    USGS Publications Warehouse

    Stohlgren, T.J.; Jarnevich, C.S.; Esaias, W.E.; Morisette, J.T.

    2011-01-01

    Species distribution models are increasing in popularity for mapping suitable habitat for species of management concern. Many investigators now recognize that extrapolations of these models with geographic information systems (GIS) might be sensitive to the environmental bounds of the data used in their development, yet there is no recommended best practice for "clamping" model extrapolations. We relied on two commonly used modeling approaches: classification and regression tree (CART) and maximum entropy (Maxent) models, and we tested a simple alteration of the model extrapolations, bounding extrapolations to the maximum and minimum values of primary environmental predictors, to provide a more realistic map of suitable habitat of hybridized Africanized honey bees in the southwestern United States. Findings suggest that multiple models of bounding, and the most conservative bounding of species distribution models, like those presented here, should probably replace the unbounded or loosely bounded techniques currently used. ?? 2011 Current Zoology.

  8. Causality and Tsirelson's bounds

    SciTech Connect

    Buhrman, H.; Massar, S.

    2005-11-15

    We study the properties of no-signaling correlations that cannot be reproduced by local measurements on entangled quantum states. We say that such correlations violate Tsirelson bounds. We show that if these correlations are obtained by some reversible unitary quantum evolution U, then U cannot be written in the product form U{sub A}xU{sub B}. This implies that U can be used for signaling and for entanglement generation. This result is completely general and in fact can be viewed as a characterization of Tsirelson bounds. We then show how this result can be used as a tool to study Tsirelson bounds and we illustrate this by rederiving the Tsirelson bound of 2{radical}(2) for the Clauser-Horn-Shimony-Holt inequality, and by deriving a new Tsirelson bound for qutrits.

  9. Chromatographic separation of proteins on metal immobilized iminodiacetic acid-bound molded monolithic rods of macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate).

    PubMed

    Luo, Q; Zou, H; Xiao, X; Guo, Z; Kong, L; Mao, X

    2001-08-17

    Continuous rod of macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) was prepared by a free radical polymerization within the confines of a stainless-steel column. The epoxide groups of the rod were modified by a reaction with iminodiacetic acid (IDA) that affords the active site to form metal IDA chelates used for immobilized metal affinity chromatography (IMAC). The efficiency of coupling of IDA to the epoxide-contained matrix was studied as a function of reaction time and temperature. High-performance separation of proteins, based on immobilized different metals on the column, were described. The influence of pH on the adsorption capacity of bovine serum albumin on the Cu2+-IDA continuous rod column was investigated in the range from 5.0 to 9.0. Purification of lysozyme from egg white and human serum albumin (HSA) on the commercially available HSA solution were performed on the naked IDA and Cu2+-IDA continuous rod columns, respectively; and the purity of the obtained fractions was detected by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. PMID:11556331

  10. Photoinduced electron transfer and fluorescence mechanisms in covalently linked polynuclear aromatic-nucleotide complexes

    SciTech Connect

    Geacintov, N.E.; Mao, Bing; Zhao, Rushen; Chen, Junxin; Liu, Tong Ming; Ya, Nai-Qi; France, L.L.; Sutherland, J.D.

    1992-04-01

    The fluorescence of polycyclic aromatic hydrocarbon-nucleic acid complexes is quenched by photoinduced electron transfer mechanisms in aqueous solutions at ambient temperatures. These effects are illustrated with the biologically important compound benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), a mutagenic and carcinogenic metabolite of the environmental pollutant benzo[a]pyrene, which forms covalent mutagenic lesions with 2{prime}-deoxyguanosine (dG) residues in DNA. The dependence of the fluroescence yeild and fluorescence decay times of the covalent model adduct (+)-trans-BPDE-N{sup 2}-dG as a function of temperature and methanol/water composition are described. Because of the sensitivity of the fluorescence of the pyrenyl residue to the polarity of the microenvironment, the magnitude of the fluorescence yield can be used to distinguish between highly hydrophobic (e.g. intercalation) and other more solvent-exposed BPDE-nucleic acid binding sites.

  11. Photoinduced electron transfer and fluorescence mechanisms in covalently linked polynuclear aromatic-nucleotide complexes

    SciTech Connect

    Geacintov, N.E.; Mao, Bing; Zhao, Rushen; Chen, Junxin; Liu, Tong Ming; Ya, Nai-Qi . Dept. of Chemistry); France, L.L.; Sutherland, J.D. )

    1992-01-01

    The fluorescence of polycyclic aromatic hydrocarbon-nucleic acid complexes is quenched by photoinduced electron transfer mechanisms in aqueous solutions at ambient temperatures. These effects are illustrated with the biologically important compound benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE), a mutagenic and carcinogenic metabolite of the environmental pollutant benzo(a)pyrene, which forms covalent mutagenic lesions with 2{prime}-deoxyguanosine (dG) residues in DNA. The dependence of the fluroescence yeild and fluorescence decay times of the covalent model adduct (+)-trans-BPDE-N{sup 2}-dG as a function of temperature and methanol/water composition are described. Because of the sensitivity of the fluorescence of the pyrenyl residue to the polarity of the microenvironment, the magnitude of the fluorescence yield can be used to distinguish between highly hydrophobic (e.g. intercalation) and other more solvent-exposed BPDE-nucleic acid binding sites.

  12. Colorful surface architectures with three different types of dynamic covalent bonds: integration of anthocyanins, tritylium ions and flavins.

    PubMed

    Zhang, Kang-Da; Sakai, Naomi; Matile, Stefan

    2015-08-28

    Although they combine the best of covalent and non-covalent bonds, dynamic covalent bonds are usually not used together. Building on pioneering examples for functional systems with two orthogonal dynamic covalent bonds, we herein elaborate on multicomponent surface architectures that operate with three different types of dynamic covalent bonds. Disulfide exchange under basic conditions is used to grow single π stacks directly on oxide surfaces, hydrazone exchange under acidic conditions to add a second string or stack, and boronic-ester exchange under neutral conditions to build the third one. In this study, we show that this synthetic approach to complex systems provides access to emergent properties, as exemplified with ordered stacks of anthocyanins, pyrocatchol violet and riboflavins. The integration of anthocyanins, the central component of the pigments of plant flowers, is interesting to protect the blue flavylium cation against deprotonation, deplanarization and degradation. The integration of pyrocatchol violet is of interest to stabilize the blue, disfavored tritylium cation. The red riboflavin stacks are attractive because they generate high photocurrent. These colorful examples hint at the potential of synthetic methods that use three different types of dynamic covalent bonds in concert to build complex systems with emergent properties. PMID:26179486

  13. Designing Covalently Linked Heterodimeric Four-Helix Bundles.

    PubMed

    Chino, M; Leone, L; Maglio, O; Lombardi, A

    2016-01-01

    De novo design has proven a powerful methodology for understanding protein folding and function, and for mimicking or even bettering the properties of natural proteins. Extensive progress has been made in the design of helical bundles, simple structural motifs that can be nowadays designed with a high degree of precision. Among helical bundles, the four-helix bundle is widespread in nature, and is involved in numerous and fundamental processes. Representative examples are the carboxylate bridged diiron proteins, which perform a variety of different functions, ranging from reversible dioxygen binding to catalysis of dioxygen-dependent reactions, including epoxidation, desaturation, monohydroxylation, and radical formation. The "Due Ferri" (two-irons; DF) family of proteins is the result of a de novo design approach, aimed to reproduce in minimal four-helix bundle models the properties of the more complex natural diiron proteins, and to address how the amino acid sequence modulates their functions. The results so far obtained point out that asymmetric metal environments are essential to reprogram functions, and to achieve the specificity and selectivity of the natural enzymes. Here, we describe a design method that allows constructing asymmetric four-helix bundles through the covalent heterodimerization of two different α-helical harpins. In particular, starting from the homodimeric DF3 structure, we developed a protocol for covalently linking the two α2 monomers by using the Cu(I) catalyzed azide-alkyne cycloaddition. The protocol was then generalized, in order to include the construction of several linkers, in different protein positions. Our method is fast, low cost, and in principle can be applied to any couple of peptides/proteins we desire to link. PMID:27586346

  14. Sharing in covalent and hydrogen bonds

    NASA Astrophysics Data System (ADS)

    Perhacs, Pablo

    1998-11-01

    The sharing of a single electron between two spatial and spin coordinates ζ and ζsp/prime in a many electron system is discussed in terms of the single particle sharing amplitude, Covalent bonding is distinguished from non-bonding and anti- bonding. Molecules studied are the diatomics of seven of the first nine elements and the hydrides of the first row of eight elements. Analysis is extended to the complex of methane and hydrogen fluoride and to pairs of hydrogen fluoride, water, and ammonia. The behavior of covalent bonding. The ammonia dimer is shown not to be hydrogen bonded.

  15. Covalent Organic Frameworks for CO2 Capture.

    PubMed

    Zeng, Yongfei; Zou, Ruqiang; Zhao, Yanli

    2016-04-01

    As an emerging class of porous crystalline materials, covalent organic frameworks (COFs) are excellent candidates for various applications. In particular, they can serve as ideal platforms for capturing CO2 to mitigate the dilemma caused by the greenhouse effect. Recent research achievements using COFs for CO2 capture are highlighted. A background overview is provided, consisting of a brief statement on the current CO2 issue, a summary of representative materials utilized for CO2 capture, and an introduction to COFs. Research progresses on: i) experimental CO2 capture using different COFs synthesized based on different covalent bond formations, and ii) computational simulation results of such porous materials on CO2 capture are summarized. Based on these experimental and theoretical studies, careful analyses and discussions in terms of the COF stability, low- and high-pressure CO2 uptake, CO2 selectivity, breakthrough performance, and CO2 capture conditions are provided. Finally, a perspective and conclusion section of COFs for CO2 capture is presented. Recent advancements in the field are highlighted and the strategies and principals involved are discussed. PMID:26924720

  16. Non-covalent and covalent modifications modulate the reactivity of monomeric mammalian globins.

    PubMed

    Ascenzi, Paolo; Marino, Maria; Polticelli, Fabio; Coletta, Massimo; Gioia, Magda; Marini, Stefano; Pesce, Alessandra; Nardini, Marco; Bolognesi, Martino; Reeder, Brandon J; Wilson, Michael T

    2013-09-01

    Multimeric globins (e.g., hemoglobin) are considered to be the prototypes of allosteric enzymes, whereas monomeric globins (e.g., myoglobin; Mb) usually are assumed to be non-allosteric. However, the modulation of the functional properties of monomeric globins by non-covalent (or allosteric) and covalent modifications casts doubts on this general assumption. Here, we report examples referable to these two extreme mechanisms modulating the reactivity of three mammalian monomeric globins. Sperm whale Mb, which acts as a reserve supply of O2 and facilitates the O2 flux within a myocyte, displays the allosteric modulation of the O2 affinity on lactate, an obligatory product of glycolysis under anaerobic conditions, thus facilitating O2 diffusion to the mitochondria in supporting oxidative phosphorylation. Human neuroglobin (NGB), which appears to protect neurons from hypoxia in vitro and in vivo, undergoes hypoxia-dependent phosphorylation (i.e., covalent modulation) affecting the coordination equilibrium of the heme-Fe atom and, in turn, the heme-protein reactivity. This facilitates heme-Fe-ligand binding and enhances the rate of anaerobic nitrite reduction to form NO, thus contributing to cellular adaptation to hypoxia. The reactivity of human cytoglobin (CYGB), which has been postulated to protect cells against oxidative stress, depends on both non-covalent and covalent mechanisms. In fact, the heme reactivity of CYGB depends on the lipid, such as oleate, binding which stabilizes the penta-coordination geometry of the heme-Fe atom. Lastly, the reactivity of NGB and CYGB is modulated by the redox state of the intramolecular CysCD7/CysD5 and CysB2/CysE9 residue pairs, respectively, affecting the heme-Fe atom coordination state. In conclusion, the modulation of monomeric globins reactivity by non-covalent and covalent modifications appears a very widespread phenomenon, opening new perspectives in cell survival and protection. This article is part of a Special Issue

  17. Assessing the effects of surface-bound humic acid on the phototoxicity of anatase and rutile TiO2 nanoparticles in vitro.

    PubMed

    He, Xiaojia; Sanders, Sabrieon; Aker, Winfred G; Lin, Yunfeng; Douglas, Jessica; Hwang, Huey-Min

    2016-04-01

    In this study, the cytotoxicity of two different crystal phases of TiO2 nanoparticles, with surface modification by humic acid (HA), to Escherichia coli, was assessed. The physicochemical properties of TiO2 nanoparticles were thoroughly characterized. Three different initial concentrations, namely 50, 100, and 200ppm, of HA were used for synthesis of HA coated TiO2 nanoparticles (denoted as A/RHA50, A/RHA100, and A/RHA200, respectively). Results indicate that rutile (LC50 (concentration that causes 50% mortality compared the control group)=6.5) was more toxic than anatase (LC50=278.8) under simulated sunlight (SSL) irradiation, possibly due to an extremely narrow band gap. It is noted that HA coating increased the toxicity of anatase, but decreased that of rutile. Additionally, AHA50 and RHA50 had the biggest differences compared to uncoated anatase and rutile with LC50 of 201.9 and 21.6, respectively. We then investigated the formation of reactive oxygen species (ROS) by TiO2 nanoparticles in terms of hydroxyl radicals (OH) and superoxide anions (O2(-)). Data suggested that O2(-) was the main ROS that accounted for the higher toxicity of rutile upon SSL irradiation. We also observed that HA coating decreased the generation of OH and O2(-) on rutile, but increased O2(-) formation on anatase. Results from TEM analysis also indicated that HA coated rutile tended to be attached to the surface of E. coli more than anatase. PMID:27090694

  18. Self-templated chemically stable hollow spherical covalent organic framework.

    PubMed

    Kandambeth, Sharath; Venkatesh, V; Shinde, Digambar B; Kumari, Sushma; Halder, Arjun; Verma, Sandeep; Banerjee, Rahul

    2015-01-01

    Covalent organic frameworks are a family of crystalline porous materials with promising applications. Although active research on the design and synthesis of covalent organic frameworks has been ongoing for almost a decade, the mechanisms of formation of covalent organic frameworks crystallites remain poorly understood. Here we report the synthesis of a hollow spherical covalent organic framework with mesoporous walls in a single-step template-free method. A detailed time-dependent study of hollow sphere formation reveals that an inside-out Ostwald ripening process is responsible for the hollow sphere formation. The synthesized covalent organic framework hollow spheres are highly porous (surface area ∼1,500 m(2 )g(-1)), crystalline and chemically stable, due to the presence of strong intramolecular hydrogen bonding. These mesoporous hollow sphere covalent organic frameworks are used for a trypsin immobilization study, which shows an uptake of 15.5 μmol g(-1) of trypsin. PMID:25858416

  19. Self-templated chemically stable hollow spherical covalent organic framework

    NASA Astrophysics Data System (ADS)

    Kandambeth, Sharath; Venkatesh, V.; Shinde, Digambar B.; Kumari, Sushma; Halder, Arjun; Verma, Sandeep; Banerjee, Rahul

    2015-04-01

    Covalent organic frameworks are a family of crystalline porous materials with promising applications. Although active research on the design and synthesis of covalent organic frameworks has been ongoing for almost a decade, the mechanisms of formation of covalent organic frameworks crystallites remain poorly understood. Here we report the synthesis of a hollow spherical covalent organic framework with mesoporous walls in a single-step template-free method. A detailed time-dependent study of hollow sphere formation reveals that an inside-out Ostwald ripening process is responsible for the hollow sphere formation. The synthesized covalent organic framework hollow spheres are highly porous (surface area ~1,500 m2 g-1), crystalline and chemically stable, due to the presence of strong intramolecular hydrogen bonding. These mesoporous hollow sphere covalent organic frameworks are used for a trypsin immobilization study, which shows an uptake of 15.5 μmol g-1 of trypsin.

  20. Covalent Polyisobutylene-Paclitaxel Conjugates for Controlled Release from Potential Vascular Stent Coatings.

    PubMed

    Trant, John F; McEachran, Matthew J; Sran, Inderpreet; Turowec, Bethany A; de Bruyn, John R; Gillies, Elizabeth R

    2015-07-01

    The development of covalent polyisobutylene (PIB)-paclitaxel (PTX) conjugates as a potential approach to controlling drug release from vascular stent coatings is described. PIB-PTX materials containing ∼24 and ∼48 wt % PTX, conjugated via ester linkages, were prepared. The PTX release profiles were compared with those of physical mixtures of PTX with carboxylic acid-functionalized PIB and with the triblock copolymer polystyrene-b-PIB-b-polystyrene (SIBS). Covalent conjugation led to significantly slower drug release. Atomic force microscopy imaging of coatings of the materials suggested that the physical mixtures exhibited multiple domains corresponding to phase separation, whereas the materials in which PTX was covalently conjugated appeared homogeneous. Coatings of the conjugated materials on stainless steel surfaces suffered less surface erosion than the physically mixed materials, remained intact, and adhered well to the surface throughout the thirty-five day study. Tensile testing and rheological studies suggested that the incorporation of PTX into the polymer introduces similar physical changes to the PIB as the incorporation of a glassy polystyrene block does in SIBS. Cytotoxicity assays showed that the coatings did not release toxic levels of PTX or other species into a cell culture medium over a 24 h period, yet the levels of PTX in the materials were sufficient to prevent C2C12 cells from adhering to and proliferating on them. Overall, these results indicate that covalent PIB-PTX conjugates have promise as coatings for vascular stents. PMID:26066902

  1. Covalently functionalized carbon nanostructures and methods for their separation

    DOEpatents

    Wang, YuHuang; Brozena, Alexandra H; Deng, Shunliu; Zhang, Yin

    2015-03-17

    The present invention is directed to carbon nanostructures, e.g., carbon nanotubes, methods of covalently functionalizing carbon nanostructures, and methods of separating and isolating covalently functionalized carbon. In some embodiments, carbon nanotubes are reacted with alkylating agents to provide water soluble covalently functionalized carbon nanotubes. In other embodiments, carbon nanotubes are reacted with a thermally-responsive agent and exposed to light in order to separate carbon nanotubes of a specific chirality from a mixture of carbon nanotubes.

  2. Bound infragravity waves

    NASA Astrophysics Data System (ADS)

    Okihiro, Michele; Guza, R. T.; Seymour, R. J.

    1992-07-01

    Model predictions of bound (i.e., nonlinearly forced by and coupled to wave groups) infragravity wave energy are compared with about 2 years of observations in 8- to 13-m depths at Imperial Beach, California, and Barbers Point, Hawaii. Frequency-directional spectra of free waves at sea and swell frequencies, estimated with a small array of four pressure sensors, are used to predict the bound wave spectra below 0.04 Hz. The predicted total bound wave energy is always less than the observed infragravity energy, and the underprediction increases with increasing water depth and especially with decreasing swell energy. At most half, and usually much less, of the observed infragravity energy is bound. Bound wave spectra are also predicted with data from a single wave gage in 183-m depth at Point Conception, California, and the assumption of unidirectional sea and swell. Even with energetic swell, less than 10% of the total observed infragravity energy in 183-m depth is bound. Free waves, either leaky or edge waves, are more energetic than bound waves at both the shallow and deep sites. The low level of infragravity energy observed in 183-m depth compared with 8- to 13-m depths, with similarly moderate sea and swell energy, suggests that leaky (and very high-mode edge) waves contribute less than 10% of the infragravity energy in 8-13 m. Most of the free infragravity energy in shallow water is refractively trapped and does not reach deep water.

  3. Surface engineering of stainless steel materials by covalent collagen immobilization to improve implant biocompatibility.

    PubMed

    Müller, Rainer; Abke, Jochen; Schnell, Edith; Macionczyk, Frank; Gbureck, Uwe; Mehrl, Robert; Ruszczak, Zbigniev; Kujat, Richard; Englert, Carsten; Nerlich, Michael; Angele, Peter

    2005-12-01

    It was shown recently that the deposition of thin films of tantalum and tantalum oxide enhanced the long-term biocompatibility of stainless steel biomaterials due to an increase in their corrosion resistance. In this study, we used this tantalum oxide coating as a basis for covalent immobilization of a collagen layer, which should result in a further improvement of implant tissue integration. Because of the high degradation rate of natural collagen in vivo, covalent immobilization as well as carbodiimide induced cross-linking of the protein was performed. It was found that the combination of the silane-coupling agent aminopropyl triethoxysilane and the linker molecule N,N'-disulphosuccinimidyl suberate was a very effective system for collagen immobilizing. Mechanical and enzymatic stability testing revealed a higher stability of covalent bound collagen layers compared to physically adsorbed collagen layers. The biological response induced by the surface modifications was evaluated by in vitro cell culture with human mesenchymal stem cells as well as by in vivo subcutaneous implantation into nude mice. The presence of collagen clearly improved the cytocompatibility of the stainless steel implants which, nevertheless, significantly depended on the cross-linking degree of the collagen layer. PMID:15967497

  4. Spontaneous formation of organic helical architectures through dynamic covalent chemistry.

    PubMed

    Li, Wenfang; Dong, Zeyuan; Zhu, Junyan; Luo, Quan; Liu, Junqiu

    2014-12-01

    The spontaneous formation of organic helical structures, accompanied with an amplification of chirality, by dynamic covalent bonds between achiral and chiral building blocks is reported. PMID:25325888

  5. Grade-12 Students' Misconceptions of Covalent Bonding and Structure.

    ERIC Educational Resources Information Center

    Peterson, Raymond F.; Treagust, David F.

    1989-01-01

    Describes a multiple choice, pencil and paper, diagnostic instrument used to measure student understanding of covalent bonding and structure concepts. Reports evidence of seven commonly held misconceptions. (MVL)

  6. Thermochemistry of Non-Covalent Ion–Molecule Interactions

    PubMed Central

    Armentrout, P. B.; Rodgers, M. T.

    2013-01-01

    The thermochemistry of non-covalent ion–molecule complexes has been examined by measuring quantitative bond dissociation energies using threshold collision-induced dissociation in guided ion beam tandem mass spectrometers (GIBMS). The methods used are briefly reviewed and several examples of the types of information and insight that can be obtained from such thermodynamic information are discussed. The hydration of metal cations, both singly and doubly charged, is reviewed and the trends elucidated, mainly on the basis of electrostatic contributions. The binding of alkali metal cations to amino acids has been examined for a range of systems, with both the overall polarizability of the amino acid and the local dipole moment of heteroatomic side-chains shown to be important contributors. The gas-phase interactions of the 12-crown-4 (12C4) polyether with alkali metal cations, classic molecular recognition systems in solution, have been newly compared to previous GIBMS work. These results validate the previous hypothesis that excited conformers were present for Rb+(12C4) and Cs+(12C4) and offer clues as to how and why they are formed. PMID:24349924

  7. Oriented Thin Films of a Benzodithiophene Covalent Organic Framework

    PubMed Central

    2014-01-01

    A mesoporous electron-donor covalent organic framework based on a benzodithiophene core, BDT-COF, was obtained through condensation of a benzodithiophene-containing diboronic acid and hexahydroxytriphenylene (HHTP). BDT-COF is a highly porous, crystalline, and thermally stable material, which can be handled in air. Highly porous, crystalline oriented thin BDT-COF films were synthesized from solution on different polycrystalline surfaces, indicating the generality of the synthetic strategy. The favorable orientation, crystallinity, porosity, and the growth mode of the thin BDT-COF films were studied by means of X-ray diffraction (XRD), 2D grazing incidence diffraction (GID), transmission and scanning electron microscopy (TEM, SEM), and krypton sorption. The highly porous thin BDT-COF films were infiltrated with soluble fullerene derivatives, such as [6,6]-phenyl C61 butyric acid methyl ester (PCBM), to obtain an interpenetrated electron-donor/acceptor host–guest system. Light-induced charge transfer from the BDT-framework to PCBM acceptor molecules was indicated by efficient photoluminescence quenching. Moreover, we monitored the dynamics of photogenerated hole-polarons via transient absorption spectroscopy. This work represents a combined study of the structural and optical properties of highly oriented mesoporous thin COF films serving as host for the generation of periodic interpenetrated electron-donor and electron-acceptor systems. PMID:24559375

  8. Evidence for covalent binding of acyl glucuronides to serum albumin via an imine mechanism as revealed by tandem mass spectrometry.

    PubMed Central

    Ding, A; Ojingwa, J C; McDonagh, A F; Burlingame, A L; Benet, L Z

    1993-01-01

    Acyl glucuronide metabolites of bilirubin and many drugs can react with serum albumin in vivo to form covalent adducts. Such adducts may be responsible for some toxic effects of carboxylic nonsteroidal antiinflammatory agents. The mechanism of formation of the adducts and their chemical structures are unknown. In this paper we describe the use of tandem mass spectrometry to locate binding sites and elucidate the binding mechanism involved in the formation of covalent adducts from tolmetin glucuronide and albumin in vitro. Human serum albumin and excess tolmetin glucuronide were coincubated in the presence of sodium cyanoborohydride to trap imine intermediates. The total protein product was reduced, carboxymethylated, and digested with trypsin. Six tolmetin-containing peptides (indicated by absorbance at 313 nm) were isolated by high-pressure liquid chromatography and analyzed by liquid secondary-ion mass spectrometry and collision-induced dissociation, using a four-sector tandem mass spectrometer. All six peptides contained tolmetin linked covalently via a glucuronic acid to protein lysine groups. Major attachment sites on the protein were Lys-195, -199, and -525; minor sites were identified as Lys-137, -351, and -541. Our results show unambiguously that the glucuronic acid moiety of acyl glucuronides can be retained within the structure when these reactive metabolites bind covalently to proteins, and they suggest that acyl migration followed by Schiff base (imine) formation is a credible mechanism for the generation of covalent adducts in vivo. PMID:8483897

  9. Complete ligand loss in electron ionization of the weakly bound organometallic tungsten hexacarbonyl dimer.

    PubMed

    Neustetter, Michael; Mauracher, Andreas; Limão-Vieira, Paulo; Denifl, Stephan

    2016-04-21

    We observed the bare W2(+) metal cation upon electron ionization of the weakly bound W(CO)6 dimer. This metal cation can be only observed due to the fast conversion of the weak cluster bond into a strong covalent bond between the metal moieties. PMID:27006205

  10. Covalent Polymers Containing Discrete Heterocyclic Anion Receptors

    PubMed Central

    Rambo, Brett M.; Silver, Eric S.; Bielawski, Christopher W.; Sessler, Jonathan L.

    2010-01-01

    This chapter covers recent advances in the development of polymeric materials containing discrete heterocyclic anion receptors, and focuses on advances in anion binding and chemosensor chemistry. The development of polymers specific for anionic species is a relatively new and flourishing area of materials chemistry. The incorporation of heterocyclic receptors capable of complexing anions through non-covalent interactions (e.g., hydrogen bonding and electrostatic interactions) provides a route to not only sensitive but also selective polymer materials. Furthermore, these systems have been utilized in the development of polymers capable of extracting anionic species from aqueous environments. These latter materials may lead to advances in water purification and treatment of diseases resulting from surplus ions. PMID:20871791

  11. Cell Signalling Through Covalent Modification and Allostery

    NASA Astrophysics Data System (ADS)

    Johnson, Louise N.

    Phosphorylation plays essential roles in nearly every aspect of cell life. Protein kinases catalyze the transfer of the γ-phosphate of ATP to a serine, threonine or tyrosine residue in protein substrates. This covalent modification allows activation or inhibition of enzyme activity, creates recognition sites for other proteins and promotes order/disorder or disorder/order transitions. These properties regulate ­signalling pathways and cellular processes that mediate metabolism, transcription, cell cycle progression, differentiation, cytoskeleton arrangement and cell movement, apoptosis, intercellular communication, and neuronal and immunological functions. In this lecture I shall review the structural consequences of protein phosphorylation using our work on glycogen phosphorylase and the cell cycle cyclin dependent protein kinases as illustrations. Regulation of protein phosphorylation may be disrupted in the diseased state and protein kinases have become high profile targets for drug development. To date there are 11 compounds that have been approved for clinical use in the treatment of cancer.

  12. A Photoresponsive Smart Covalent Organic Framework.

    PubMed

    Huang, Ning; Ding, Xuesong; Kim, Jangbae; Ihee, Hyotcherl; Jiang, Donglin

    2015-07-20

    Ordered π-columnar structures found in covalent organic frameworks (COFs) render them attractive as smart materials. However, external-stimuli-responsive COFs have not been explored. Here we report the design and synthesis of a photoresponsive COF with anthracene units as the photoresponsive π-building blocks. The COF is switchable upon photoirradiation to yield a concavo-convex polygon skeleton through the interlayer [4π+4π] cycloaddition of anthracene units stacked in the π-columns. This cycloaddition reaction is thermally reversible; heating resets the anthracene layers and regenerates the COF. These external-stimuli-induced structural transformations are accompanied by profound changes in properties, including gas adsorption, π-electronic function, and luminescence. The results suggest that COFs are useful for designing smart porous materials with properties that are controllable by external stimuli. PMID:26095503

  13. A Photoresponsive Smart Covalent Organic Framework**

    PubMed Central

    Huang, Ning; Ding, Xuesong; Kim, Jangbae; Ihee, Hyotcherl; Jiang, Donglin

    2015-01-01

    Ordered π-columnar structures found in covalent organic frameworks (COFs) render them attractive as smart materials. However, external-stimuli-responsive COFs have not been explored. Here we report the design and synthesis of a photoresponsive COF with anthracene units as the photoresponsive π-building blocks. The COF is switchable upon photoirradiation to yield a concavo-convex polygon skeleton through the interlayer [4π+4π] cycloaddition of anthracene units stacked in the π-columns. This cycloaddition reaction is thermally reversible; heating resets the anthracene layers and regenerates the COF. These external-stimuli-induced structural transformations are accompanied by profound changes in properties, including gas adsorption, π-electronic function, and luminescence. The results suggest that COFs are useful for designing smart porous materials with properties that are controllable by external stimuli. PMID:26095503

  14. DNA Linked To Single Wall Carbon Nanotubes: Covalent Versus Non-Covalent Approach

    NASA Astrophysics Data System (ADS)

    Chung, C.-L.; Nguyen, K.; Lyonnais, S.; Streiff, S.; Campidelli, S.; Goux-Capes, L.; Bourgoin, J.-P.; Filoramo, A.

    2008-10-01

    Nanometer-scale structures represent a novel and intriguing field, where scientists and engineers manipulate materials at the atomic and molecular scale levels to produce innovative materials. Carbon nanotubes constitute a relatively new class of materials exhibiting exceptional mechanical and electronic properties and were found to be promising candidates for molecular electronics, sensing or biomedical applications. Considering the bottom-up strategy in nanotechnology, the combination of the recognition properties of DNA with the electronic properties of single walled carbon nanotubes (SWNTs) seems to be a promising approach for the future of electronics. With the aim to assemble DNA with SWNTs, two complementary strategies have been envisioned: the covalent linkage of DNA on carboxylic groups of SWNTs under classical coupling condition and the non-covalent approach based on biotin-streptavidin molecular recognition properties. Here, we present and compare the results that we obtained with these two different methods; we want to objectively show the advantages and disadvantages of each approach.

  15. Fatty acids of Treponema pallidum and Borrelia burgdorferi lipoproteins.

    PubMed Central

    Belisle, J T; Brandt, M E; Radolf, J D; Norgard, M V

    1994-01-01

    A fundamental ultrastructural feature shared by the spirochetal pathogens Treponema pallidum subsp. pallidum (T. pallidum) and Borrelia burgdorferi, the etiological agents of venereal syphilis and Lyme disease, respectively, is that their most abundant membrane proteins contain covalently attached fatty acids. In this study, we identified the fatty acids covalently bound to lipoproteins of B. burgdorferi and T. pallidum and examined potential acyl donors to these molecules. Palmitate was the predominant fatty acid of both B. burgdorferi and T. pallidum lipoproteins. T. pallidum lipoproteins also contained substantial amounts of stearate, a fatty acid not typically prevalent in prokaryotic lipoproteins. In both spirochetes, the fatty acids of cellular lipids differed from those of their respective lipoproteins. To characterize phospholipids in these organisms, spirochetes were metabolically labeled with [3H]palmitate or [3H]oleate; B. burgdorferi contained only phosphatidylglycerol and phosphatidylcholine, while T. pallidum contained phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and cardiolipin. Although palmitate predominated in the lipoproteins, there were no apparent differences in the incorporation of these two fatty acids into phospholipids (putative acyl donors). Phospholipase A1 and A2 digestion of phosphatidylcholine from B. burgdorferi and T. pallidum labeled with either [3H]palmitate or [3H]oleate also revealed that neither fatty acid was incorporated preferentially into the 1 and 2 positions (potential acyl donor sites) of the glycerol backbone. The combined findings suggest that fatty acid utilization during lipoprotein synthesis is determined largely by the fatty acid specificities of the lipoprotein acyl transferases. These findings also provide the basis for ongoing efforts to elucidate the relationship between lipoprotein acylation and the physiological functions and inflammatory

  16. Covalent Docking Predicts Substrates for Haloalkanoate Dehalogenase Superfamily Phosphatases

    PubMed Central

    2015-01-01

    Enzyme function prediction remains an important open problem. Though structure-based modeling, such as metabolite docking, can identify substrates of some enzymes, it is ill-suited to reactions that progress through a covalent intermediate. Here we investigated the ability of covalent docking to identify substrates that pass through such a covalent intermediate, focusing particularly on the haloalkanoate dehalogenase superfamily. In retrospective assessments, covalent docking recapitulated substrate binding modes of known cocrystal structures and identified experimental substrates from a set of putative phosphorylated metabolites. In comparison, noncovalent docking of high-energy intermediates yielded nonproductive poses. In prospective predictions against seven enzymes, a substrate was identified for five. For one of those cases, a covalent docking prediction, confirmed by empirical screening, and combined with genomic context analysis, suggested the identity of the enzyme that catalyzes the orphan phosphatase reaction in the riboflavin biosynthetic pathway of Bacteroides. PMID:25513739

  17. A Photoresponsive Surface Covalent Organic Framework: Surface-Confined Synthesis, Isomerization, and Controlled Guest Capture and Release.

    PubMed

    Liu, Chunhua; Zhang, Wei; Zeng, Qingdao; Lei, Shengbin

    2016-05-10

    By introducing an azobenzene group to the backbone of diboronic acid, we have obtained a surface-confined, photoresponsive single-layer covalent organic framework with long-range order and almost entire surface coverage. Scanning tunneling microscopic characterization indicates that though the covalent linkage provides a significant locking effect, isomerization can still happen under UV irradiation, which causes destruction of the surface COF. Furthermore, the decomposed surface COF can recover upon annealing. This photoinduced decomposition provides a facile approach for the controlled capture and release of targeted objects using these nanoporous surface COFs as a host, which has been demonstrated in this work using copper phthalocyanine as a model guest. PMID:26990558

  18. Ab initio study of chemical bond interactions between covalently functionalized carbon nanotubes via amide, ester and anhydride linkages

    NASA Astrophysics Data System (ADS)

    Ben Doudou, Bessem; Chen, Jun; Vivet, Alexandre; Poilâne, Christophe

    2016-03-01

    In this paper, we have investigated the chemical bond interactions between covalently functionalized zigzag (5,0) and (8,0) SWCNT-SWCNT via various covalent linkages. Side-to-side junctions connected via amide, ester and anhydride linkages were particularly studied. The geometries and energy of the forming reaction were investigated using first-principles density functional theory. Furthermore, the band structures and the total density of states (DOS) of the junctions have also been analyzed. Our results show that several promising structures could be obtained by using chemical connection strategy and particularly the junctions formed by coupling amino functionalized SWCNT and carboxylic acid functionalized SWCNT was more favorable.

  19. Structural and mechanistic analysis of trans-3-chloroacrylic acid dehalogenase activity

    SciTech Connect

    Pegan, Scott D.; Serrano, Hector; Whitman, Christian P.; Mesecar, Andrew D.

    2008-12-01

    The X-ray structure of a noncovalently modified trans-3-chloroacrylic acid dehalogenase with a substrate-homolog acetate bound in the active site has been determined to 1.7 Å resolution. Elucidation of catalytically important water is reported and multiple conformations of the catalytic residue αGlu52 are observed. Trans-3-chloroacrylic acid dehalogenase (CaaD) is a critical enzyme in the trans-1, 3-dichloropropene (DCP) degradation pathway in Pseudomonas pavonaceae 170. This enzyme allows bacteria to use trans-DCP, a common component in commercially produced fumigants, as a carbon source. CaaD specifically catalyzes the fourth step of the pathway by cofactor-independent dehalogenation of a vinyl carbon–halogen bond. Previous studies have reported an X-ray structure of CaaD under acidic conditions with a covalent modification of the catalytic βPro1 residue. Here, the 1.7 Å resolution X-ray structure of CaaD under neutral (pH 6.5) conditions is reported without the presence of the covalent adduct. In this new structure, a substrate-like acetate molecule is bound within the active site in a position analogous to the putative substrate-binding site. Additionally, a catalytically important water molecule was identified, consistent with previously proposed reaction schemes. Finally, flexibility of the catalytically relevant side chain αGlu52 is observed in the structure, supporting its role in the catalytic mechanism.

  20. Mass Spectrometric and Spectrofluorometric Studies of the Interaction of Aristolochic Acids with Proteins

    PubMed Central

    Li, Weiwei; Hu, Qin; Chan, Wan

    2015-01-01

    Aristolochic acid (AA) is a potent carcinogen and nephrotoxin and is associated with the development of “Chinese herb nephropathy” and Balkan endemic nephropathy. Despite decades of research, the specific mechanism of the observed nephrotoxicity has remained elusive and the potential effects on proteins due to the observed toxicity of AA are not well-understood. To better understand the pharmacotoxicological features of AA, we investigated the non-covalent interactions of AA with proteins. The protein-binding properties of AA with bovine serum albumin (BSA) and lysozyme were characterized using spectrofluorometric and mass spectrometric (MS) techniques. Moreover, the protein-AA complexes were clearly identified by high-resolution MS analyses. To the best of our knowledge, this is the first direct evidence of non-covalently bound protein-AA complexes. An analysis of the spectrofluorometric data by a modified Stern−Volmer plot model also revealed that both aristolochic acid I (AAI) and aristolochic acid II (AAII) were bound to BSA and lysozyme in 1:1 stoichiometries. A significantly stronger protein binding property was observed in AAII than in AAI as evidenced by the spectrofluorometric and MS analyses, which may explain the observed higher mutagenicity of AAII. PMID:26471474

  1. Mass Spectrometric and Spectrofluorometric Studies of the Interaction of Aristolochic Acids with Proteins

    NASA Astrophysics Data System (ADS)

    Li, Weiwei; Hu, Qin; Chan, Wan

    2015-10-01

    Aristolochic acid (AA) is a potent carcinogen and nephrotoxin and is associated with the development of “Chinese herb nephropathy” and Balkan endemic nephropathy. Despite decades of research, the specific mechanism of the observed nephrotoxicity has remained elusive and the potential effects on proteins due to the observed toxicity of AA are not well-understood. To better understand the pharmacotoxicological features of AA, we investigated the non-covalent interactions of AA with proteins. The protein-binding properties of AA with bovine serum albumin (BSA) and lysozyme were characterized using spectrofluorometric and mass spectrometric (MS) techniques. Moreover, the protein-AA complexes were clearly identified by high-resolution MS analyses. To the best of our knowledge, this is the first direct evidence of non-covalently bound protein-AA complexes. An analysis of the spectrofluorometric data by a modified Stern-Volmer plot model also revealed that both aristolochic acid I (AAI) and aristolochic acid II (AAII) were bound to BSA and lysozyme in 1:1 stoichiometries. A significantly stronger protein binding property was observed in AAII than in AAI as evidenced by the spectrofluorometric and MS analyses, which may explain the observed higher mutagenicity of AAII.

  2. Bounding the Bogoliubov coefficients

    SciTech Connect

    Boonserm, Petarpa; Visser, Matt

    2008-11-15

    While over the last century or more considerable effort has been put into the problem of finding approximate solutions for wave equations in general, and quantum mechanical problems in particular, it appears that as yet relatively little work seems to have been put into the complementary problem of establishing rigourous bounds on the exact solutions. We have in mind either bounds on parametric amplification and the related quantum phenomenon of particle production (as encoded in the Bogoliubov coefficients), or bounds on transmission and reflection coefficients. Modifying and streamlining an approach developed by one of the present authors [M. Visser, Phys. Rev. A 59 (1999) 427-438, (arXiv:quant-ph/9901030)], we investigate this question by developing a formal but exact solution for the appropriate second-order linear ODE in terms of a time-ordered exponential of 2x2 matrices, then relating the Bogoliubov coefficients to certain invariants of this matrix. By bounding the matrix in an appropriate manner, we can thereby bound the Bogoliubov coefficients.

  3. Modeling the role of covalent enzyme modification in Escherichia coli nitrogen metabolism

    NASA Astrophysics Data System (ADS)

    Kidd, Philip B.; Wingreen, Ned S.

    2010-03-01

    In the bacterium Escherichia coli, the enzyme glutamine synthetase (GS) converts ammonium into the amino acid glutamine. GS is principally active when the cell is experiencing nitrogen limitation, and its activity is regulated by a bicyclic covalent modification cascade. The advantages of this bicyclic-cascade architecture are poorly understood. We analyze a simple model of the GS cascade in comparison to other regulatory schemes and conclude that the bicyclic cascade is suboptimal for maintaining metabolic homeostasis of the free glutamine pool. Instead, we argue that the lag inherent in the covalent modification of GS slows the response to an ammonium shock and thereby allows GS to transiently detoxify the cell, while maintaining homeostasis over longer times.

  4. Second-Generation Non-Covalent NAAA Inhibitors are Protective in a Model of Multiple Sclerosis.

    PubMed

    Migliore, Marco; Pontis, Silvia; Fuentes de Arriba, Angel Luis; Realini, Natalia; Torrente, Esther; Armirotti, Andrea; Romeo, Elisa; Di Martino, Simona; Russo, Debora; Pizzirani, Daniela; Summa, Maria; Lanfranco, Massimiliano; Ottonello, Giuliana; Busquet, Perrine; Jung, Kwang-Mook; Garcia-Guzman, Miguel; Heim, Roger; Scarpelli, Rita; Piomelli, Daniele

    2016-09-01

    Palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) are endogenous lipid mediators that suppress inflammation. Their actions are terminated by the intracellular cysteine amidase, N-acylethanolamine acid amidase (NAAA). Even though NAAA may offer a new target for anti-inflammatory therapy, the lipid-like structures and reactive warheads of current NAAA inhibitors limit the use of these agents as oral drugs. A series of novel benzothiazole-piperazine derivatives that inhibit NAAA in a potent and selective manner by a non-covalent mechanism are described. A prototype member of this class (8) displays high oral bioavailability, access to the central nervous system (CNS), and strong activity in a mouse model of multiple sclerosis (MS). This compound exemplifies a second generation of non-covalent NAAA inhibitors that may be useful in the treatment of MS and other chronic CNS disorders. PMID:27404798

  5. Covalent Immobilization of Oriented Photosystem II on a Nanostructured Electrode for Solar Water Oxidation

    PubMed Central

    2013-01-01

    Photosystem II (PSII) offers a biological and sustainable route of photochemical water oxidation to O2 and can provide protons and electrons for the generation of solar fuels, such as H2. We present a rational strategy to electrostatically improve the orientation of PSII from a thermophilic cyanobacterium, Thermosynechococcus elongatus, on a nanostructured indium tin oxide (ITO) electrode and to covalently immobilize PSII on the electrode. The ITO electrode was modified with a self-assembled monolayer (SAM) of phosphonic acid ITO linkers with a dangling carboxylate moiety. The negatively charged carboxylate attracts the positive dipole on the electron acceptor side of PSII via Coulomb interactions. Covalent attachment of PSII in its electrostatically improved orientation to the SAM-modified ITO electrode was accomplished via an amide bond to further enhance red-light-driven, direct electron transfer and stability of the PSII hybrid photoelectrode. PMID:23829513

  6. Covalent immobilization of oriented photosystem II on a nanostructured electrode for solar water oxidation.

    PubMed

    Kato, Masaru; Cardona, Tanai; Rutherford, A William; Reisner, Erwin

    2013-07-24

    Photosystem II (PSII) offers a biological and sustainable route of photochemical water oxidation to O2 and can provide protons and electrons for the generation of solar fuels, such as H2. We present a rational strategy to electrostatically improve the orientation of PSII from a thermophilic cyanobacterium, Thermosynechococcus elongatus , on a nanostructured indium tin oxide (ITO) electrode and to covalently immobilize PSII on the electrode. The ITO electrode was modified with a self-assembled monolayer (SAM) of phosphonic acid ITO linkers with a dangling carboxylate moiety. The negatively charged carboxylate attracts the positive dipole on the electron acceptor side of PSII via Coulomb interactions. Covalent attachment of PSII in its electrostatically improved orientation to the SAM-modified ITO electrode was accomplished via an amide bond to further enhance red-light-driven, direct electron transfer and stability of the PSII hybrid photoelectrode. PMID:23829513

  7. Modeling the role of covalent enzyme modification in Escherichia coli nitrogen metabolism

    PubMed Central

    Kidd, Philip B

    2013-01-01

    In the bacterium Escherichia coli, the enzyme glutamine synthetase (GS) converts ammonium into the amino acid glutamine. GS is principally active when the cell is experiencing nitrogen limitation, and its activity is regulated by a bicyclic covalent modification cascade. The advantages of this bicyclic-cascade architecture are poorly understood. We analyze a simple model of the GS cascade in comparison to other regulatory schemes and conclude that the bicyclic cascade is suboptimal for maintaining metabolic homeostasis of the free glutamine pool. Instead, we argue that the lag inherent in the covalent modification of GS slows the response to an ammonium shock and thereby allows GS to transiently detoxify the cell, while maintaining homeostasis over longer times. PMID:20057006

  8. Validation of EMP bounds

    SciTech Connect

    Warne, L.K.; Merewether, K.O.; Chen, K.C.; Jorgenson, R.E.; Morris, M.E.; Solberg, J.E.; Lewis, J.G.; Derr, W.

    1996-07-01

    Test data on canonical weapon-like fixtures are used to validate previously developed analytical bounding results. The test fixtures were constructed to simulate (but be slightly worse than) weapon ports of entry but have known geometries (and electrical points of contact). The exterior of the test fixtures exhibited exterior resonant enhancement of the incident fields at the ports of entry with magnitudes equal to those of weapon geometries. The interior consisted of loaded transmission lines adjusted to maximize received energy or voltage but incorporating practical weapon geometrical constraints. New analytical results are also presented for bounding the energies associated with multiple bolt joints and for bounding the exterior resonant enhancement of the exciting fields.

  9. A novel tyrosine-heme C−O covalent linkage in F43Y myoglobin: a new post-translational modification of heme proteins.

    PubMed

    Yan, Dao-Jing; Li, Wei; Xiang, Yu; Wen, Ge-Bo; Lin, Ying-Wu; Tan, Xiangshi

    2015-01-01

    Heme post-translational modification plays a key role in tuning the structure and function of heme proteins. We herein report a novel tyrosine-heme covalent C−O bond in an artificially produced sperm whale myoglobin (Mb) mutant, F43Y Mb, which formed spontaneously in vivo between the Tyr43 hydroxy group and the heme 4-vinyl group. This highlights the diverse chemistry of heme post-translational modifications, and lays groundwork for further investigation of the structural and functional diversity of covalently-bound heme proteins. PMID:25392956

  10. Antioxidative activity of bound-form phenolics in potato peel.

    PubMed

    Nara, Kazuhiro; Miyoshi, Takayuki; Honma, Tamaki; Koga, Hidenori

    2006-06-01

    Free and bound-form phenolics were isolated from potato (cv. Toyoshiro) flesh and peel. The free and bound-form phenolics in the peel showed high DPPH radical scavenging activity, while those in the flesh showed low activity. The total amount of chlorogenic acid and caffeic acid in the free-form phenolics from the peel was highly correlated with the DPPH radical scavenging activity. Ferulic acid was identified as the active radical scavenging compound in the bound-form phenolics from the peel. The potato peel may therefore offer an effective source of an antioxidative. PMID:16794331

  11. O2 reduction reaction by biologically relevant anionic ligand bound iron porphyrin complexes.

    PubMed

    Samanta, Subhra; Das, Pradip Kumar; Chatterjee, Sudipta; Sengupta, Kushal; Mondal, Biswajit; Dey, Abhishek

    2013-11-18

    Iron porphyrin complex with a covalently attached thiolate ligand and another with a covalently attached phenolate ligand has been synthesized. The thiolate bound complex shows spectroscopic features characteristic of P450, including the hallmark absorption spectrum of the CO adduct. Electrocatalytic O2 reduction by this complex, which bears a terminal alkyne group, is investigated by both physiabsorbing on graphite surfaces (fast electron transfer rates) and covalent attachment to azide terminated self-assembled monolayer (physiologically relevant electron transfer rates) using the terminal alkyne group. Analysis of the steady state electrochemical kinetics reveals that this catalyst can selectively reduce O2 to H2O with a second-order k(cat.) ~10(7) M(-1 )s(-1) at pH 7. The analogous phenolate bound iron porphyrin complex reduces O2 with a second-order rate constant of 10(5) M(-1) s(-1) under the same conditions. The anionic ligand bound iron porphyrin complexes catalyze oxygen reduction reactions faster than any known synthetic heme porphyrin analogues. The kinetic parameters of O2 reduction of the synthetic thiolate bound complex, which is devoid of any second sphere effects present in protein active sites, provide fundamental insight into the role of the protein environment in tuning the reactivity of thiolate bound iron porphyrin containing metalloenzymes. PMID:24171513

  12. Ionic-covalent character of metal and nonmetal oxides.

    PubMed

    Duffy, J A

    2006-12-14

    The acid-base properties of oxidic media are quantified in terms of the optical basicity concept, which serves to correlate many properties with chemical constitution. Optical basicity values, Lambda, have been assigned to 25 oxides such that they relate to Lambda for crystalline CaO being taken as unity. Since Lambda for an oxide is proportional to the degree of negative charge borne by the oxide-(-II) atom or ion, it follows that optical basicity should go hand-in-hand with the ionic/covalent nature of the cation-oxide-(-II) bonding. Unfortunately, this assumption produces many anomalies and trends that do not fit normal inorganic trends. The problem is resolved by adjusting the influence of ionic forms to the bonding by taking into account the heats of formation. In contrast to the (Pauling) electronegativity treatment of oxides, this procedure allows assignment of percentage ionicity to the bonding, and the trends in these in the Periodic Table are as expected for inorganic oxides. PMID:17149841

  13. Porous silicon: electrochemical microstructuring, photoluminescence, and covalent modificaiton

    NASA Astrophysics Data System (ADS)

    Prigozhin, Maxim B.; Shiwsankar, Pauline; Algar, W. Russ; Krull, Ulrich J.

    2008-06-01

    Interest in porous silicon (PS) has increased dramatically over the past two decades due to aspects such as photoluminescence due to quantum confinement, large surface area, and micro/nanoscale architecture. In this work, <111> p-type silicon wafers have been electrochemically etched with ethanolic solutions of hydrofluoric acid. Discrete surface domains showing luminescence were observed. The domains were typically many tens of micrometers in size and had a height of about 6-8 μm. Interestingly, central round wells of 10-30 μm diameter were observed to form within domains. Investigation of luminescence in depth profile of the wells was done using confocal fluorescence microscopy, and the results indicated that the domains were fully porous and luminescent throughout the entire depth. Spectrally, the peak fluorescence emission was in the range of 550-750 nm and the spectra had an average FWHM equal to about 150 nm. Covalent attachment of organic monolayers to the porous silicon surfaces was done to try and passivate against oxidation, and also to explore the possibilities of bioconjugation and tuning of the photoluminescence wavelength. A reaction of hydrogen terminated silicon with ω-undecylenyl alcohol was done using irradiation by a UV source, and successful derivatization was confirmed with IR spectroscopy. Bulk electrochemical etching of silicon provided a method to generate distributed luminescent structures suitable for compartmentalization of reactions within wells of micrometer dimensions without the use of spatially resolved fabrication methodologies such as epitaxial deposition, lithography, or ion beam technologies.

  14. Covalency-reinforced oxygen evolution reaction catalyst

    PubMed Central

    Yagi, Shunsuke; Yamada, Ikuya; Tsukasaki, Hirofumi; Seno, Akihiro; Murakami, Makoto; Fujii, Hiroshi; Chen, Hungru; Umezawa, Naoto; Abe, Hideki; Nishiyama, Norimasa; Mori, Shigeo

    2015-01-01

    The oxygen evolution reaction that occurs during water oxidation is of considerable importance as an essential energy conversion reaction for rechargeable metal–air batteries and direct solar water splitting. Cost-efficient ABO3 perovskites have been studied extensively because of their high activity for the oxygen evolution reaction; however, they lack stability, and an effective solution to this problem has not yet been demonstrated. Here we report that the Fe4+-based quadruple perovskite CaCu3Fe4O12 has high activity, which is comparable to or exceeding those of state-of-the-art catalysts such as Ba0.5Sr0.5Co0.8Fe0.2O3−δ and the gold standard RuO2. The covalent bonding network incorporating multiple Cu2+ and Fe4+ transition metal ions significantly enhances the structural stability of CaCu3Fe4O12, which is key to achieving highly active long-life catalysts. PMID:26354832

  15. Covalency-reinforced oxygen evolution reaction catalyst.

    PubMed

    Yagi, Shunsuke; Yamada, Ikuya; Tsukasaki, Hirofumi; Seno, Akihiro; Murakami, Makoto; Fujii, Hiroshi; Chen, Hungru; Umezawa, Naoto; Abe, Hideki; Nishiyama, Norimasa; Mori, Shigeo

    2015-01-01

    The oxygen evolution reaction that occurs during water oxidation is of considerable importance as an essential energy conversion reaction for rechargeable metal-air batteries and direct solar water splitting. Cost-efficient ABO3 perovskites have been studied extensively because of their high activity for the oxygen evolution reaction; however, they lack stability, and an effective solution to this problem has not yet been demonstrated. Here we report that the Fe(4+)-based quadruple perovskite CaCu3Fe4O12 has high activity, which is comparable to or exceeding those of state-of-the-art catalysts such as Ba(0.5)Sr(0.5)Co(0.8)Fe(0.2)O(3-δ) and the gold standard RuO2. The covalent bonding network incorporating multiple Cu(2+) and Fe(4+) transition metal ions significantly enhances the structural stability of CaCu3Fe4O12, which is key to achieving highly active long-life catalysts. PMID:26354832

  16. Covalency-reinforced oxygen evolution reaction catalyst

    NASA Astrophysics Data System (ADS)

    Yagi, Shunsuke; Yamada, Ikuya; Tsukasaki, Hirofumi; Seno, Akihiro; Murakami, Makoto; Fujii, Hiroshi; Chen, Hungru; Umezawa, Naoto; Abe, Hideki; Nishiyama, Norimasa; Mori, Shigeo

    2015-09-01

    The oxygen evolution reaction that occurs during water oxidation is of considerable importance as an essential energy conversion reaction for rechargeable metal-air batteries and direct solar water splitting. Cost-efficient ABO3 perovskites have been studied extensively because of their high activity for the oxygen evolution reaction; however, they lack stability, and an effective solution to this problem has not yet been demonstrated. Here we report that the Fe4+-based quadruple perovskite CaCu3Fe4O12 has high activity, which is comparable to or exceeding those of state-of-the-art catalysts such as Ba0.5Sr0.5Co0.8Fe0.2O3-δ and the gold standard RuO2. The covalent bonding network incorporating multiple Cu2+ and Fe4+ transition metal ions significantly enhances the structural stability of CaCu3Fe4O12, which is key to achieving highly active long-life catalysts.

  17. Ionic Covalent Organic Frameworks with Spiroborate Linkage.

    PubMed

    Du, Ya; Yang, Haishen; Whiteley, Justin Michael; Wan, Shun; Jin, Yinghua; Lee, Se-Hee; Zhang, Wei

    2016-01-26

    A novel type of ionic covalent organic framework (ICOF), which contains sp(3)  hybridized boron anionic centers and tunable countercations, was constructed by formation of spiroborate linkages. These ICOFs exhibit high BET surface areas up to 1259 m(2)  g(-1) and adsorb a significant amount of H2 (up to 3.11 wt %, 77 K, 1 bar) and CH4 (up to 4.62 wt %, 273 K, 1 bar). Importantly, the materials show good thermal stabilities and excellent resistance to hydrolysis, remaining nearly intact when immersed in water or basic solution for two days. The presence of permanently immobilized ion centers in ICOFs enables the transportation of lithium ions with room-temperature lithium-ion conductivity of 3.05×10(-5)  S cm(-1) and an average Li(+) transference number value of 0.80±0.02. Our approach thus provides a convenient route to highly stable COFs with ionic linkages, which can potentially serve as absorbents for alternative energy sources such as H2, CH4, and also as solid lithium electrolytes/separators for the next-generation lithium batteries. PMID:26696304

  18. COVALENT BINDING OF REDUCED METABOLITES OF [15N3] TNT TO SOIL ORGANIC MATTER DURING A BIOREMEDIATION PROCESS ANALYZED BY 15N NMR SPECTROSCOPY. (R826646)

    EPA Science Inventory

    Evidence is presented for the covalent binding of
    biologically reduced metabolites of 2,4,6-15N3-trinitrotoluene
    (TNT) to different soil fractions (humic acids, fulvic
    acids, and humin) using liquid 15N NMR spectroscopy. A
    silylation p...

  19. Computing Graphical Confidence Bounds

    NASA Technical Reports Server (NTRS)

    Mezzacappa, M. A.

    1983-01-01

    Approximation for graphical confidence bounds is simple enough to run on programmable calculator. Approximation is used in lieu of numerical tables not always available, and exact calculations, which often require rather sizable computer resources. Approximation verified for collection of up to 50 data points. Method used to analyze tile-strength data on Space Shuttle thermal-protection system.

  20. Chloramphenicol Biosynthesis: The Structure of CmlS, a Flavin-Dependent Halogenase Shwing a Covalent Flavin-Aspartate Bond

    SciTech Connect

    Podzelinska, K.; Latimer, R; Bhattacharya, A; Vining, L; Zechel, D; Jia, Z

    2010-01-01

    Chloramphenicol is a halogenated natural product bearing an unusual dichloroacetyl moiety that is critical for its antibiotic activity. The operon for chloramphenicol biosynthesis in Streptomyces venezuelae encodes the chloramphenicol halogenase CmlS, which belongs to the large and diverse family of flavin-dependent halogenases (FDH's). CmlS was previously shown to be essential for the formation of the dichloroacetyl group. Here we report the X-ray crystal structure of CmlS determined at 2.2 {angstrom} resolution, revealing a flavin monooxygenase domain shared by all FDHs, but also a unique 'winged-helix' C-terminal domain that creates a T-shaped tunnel leading to the halogenation active site. Intriguingly, the C-terminal tail of this domain blocks access to the halogenation active site, suggesting a structurally dynamic role during catalysis. The halogenation active site is notably nonpolar and shares nearly identical residues with Chondromyces crocatus tyrosyl halogenase (CndH), including the conserved Lys (K71) that forms the reactive chloramine intermediate. The exception is Y350, which could be used to stabilize enolate formation during substrate halogenation. The strictly conserved residue E44, located near the isoalloxazine ring of the bound flavin adenine dinucleotide (FAD) cofactor, is optimally positioned to function as a remote general acid, through a water-mediated proton relay, which could accelerate the reaction of the chloramine intermediate during substrate halogenation, or the oxidation of chloride by the FAD(C4{alpha})-OOH intermediate. Strikingly, the 8{alpha} carbon of the FAD cofactor is observed to be covalently attached to D277 of CmlS, a residue that is highly conserved in the FDH family. In addition to representing a new type of flavin modification, this has intriguing implications for the mechanism of FDHs. Based on the crystal structure and in analogy to known halogenases, we propose a reaction mechanism for CmlS.

  1. Synthesis and Photophysical Study of a [NiFe] Hydrogenase Biomimetic Compound Covalently Linked to a Re-diimine Photosensitizer

    PubMed Central

    2015-01-01

    The synthesis, photophysics, and photochemistry of a linked dyad ([Re]-[NiFe2]) containing an analogue ([NiFe2]) of the active site of [NiFe] hydrogenase, covalently bound to a Re-diimine photosensitizer ([Re]), are described. Following excitation, the mechanisms of electron transfer involving the [Re] and [NiFe2] centers and the resulting decomposition were investigated. Excitation of the [Re] center results in the population of a diimine-based metal-to-ligand charge transfer excited state. Reductive quenching by NEt3 produces the radically reduced form of [Re], [Re]− (kq = 1.4 ± 0.1 × 107 M–1 s–1). Once formed, [Re]− reduces the [NiFe2] center to [NiFe2]−, and this reduction was followed using time-resolved infrared spectroscopy. The concentration dependence of the electron transfer rate constants suggests that both inter- and intramolecular electron transfer pathways are involved, and the rate constants for these processes have been estimated (kinter = 5.9 ± 0.7 × 108 M–1 s–1, kintra = 1.5 ± 0.1 × 105 s–1). For the analogous bimolecular system, only intermolecular electron transfer could be observed (kinter = 3.8 ± 0.5 × 109 M–1 s–1). Fourier transform infrared spectroscopic studies confirms that decomposition of the dyad occurs upon prolonged photolysis, and this appears to be a major factor for the low activity of the system toward H2 production in acidic conditions. PMID:26605700

  2. Covalent adduction of nitrogen mustards to model protein nucleophiles.

    PubMed

    Thompson, Vanessa R; DeCaprio, Anthony P

    2013-08-19

    Protein adducts have the potential to serve as unique biomarkers of exposure to compounds of interest. Many xenobiotics (or their metabolites) are electrophilic and therefore reactive with nucleophilic amino acid residues on proteins. Nitrogen mustards are reactive xenobiotics with potential use as chemical warfare agents (CWA) or agents of terrorist attack, in addition to being employed as chemotherapeutic agents. The present study utilized cysteine-, lysine-, and histidine-containing model peptides to characterize in vitro adduction of the nitrogen mustards mechloroethamine (HN-2) and tris-(2-chlorethyl)amine (HN-3) to these nucleophilic amino acid residues by means of liquid chromatography-tandem mass spectrometry. The study assessed the structure of adducts formed, the time course of adduct formation, concentration-response relationships, and temporal stability of adducts. Adduction was hypothesized to occur on all three model peptides via initial formation of a reactive aziridinium intermediate for both mechloroethamine and tris-(2-chlorethyl)amine, followed by covalent adduction to nucleophilic residues. While adduction was found to occur most readily with cysteine, it was also observed at lysine and histidine, demonstrating that adduction by mechloroethamine and tris-(2-chlorethyl)amine is possible at multiple nucleophilic sites. Following solid phase extraction cleanup, adducts formed with mechloroethamine were stable for up to three weeks. Adducts formed with tris-(2-chlorethyl)amine were less stable; however, hydrolyzed secondary adducts were observed throughout the three week period. This study demonstrates that the nitrogen mustards mechloroethamine and tris-(2-chlorethyl)amine form stable adducts with reactive protein nucleophiles other than cysteine. PMID:23859065

  3. Capillary electrophoretic focusing of covalently derivatized protein induced by surfactant.

    PubMed

    Oukacine, Farid; Quirino, Joselito P; Mesbah, Kiarach; Taverna, Myriam

    2016-05-01

    In this communication, we present a very simple strategy to focus covalently derivatized proteins for high sensitivity CE analysis by LIF detection. We demonstrated that the covalently tagged protein can be focused just by adding SDS at a concentration above the CMC in the derivatized sample. Under specific injection conditions, SDS concentration below the CMC is also sufficient to induce the focusing of the tagged protein. This method allows the quantification and detection of the covalently tagged protein in a narrow zone with an efficiency approaching 220 000 plates/m. Very good linearity was obtained for the ubiquitin in a concentration range of 2-25 μM. PMID:26940436

  4. Non-covalent and covalent functionalization of graphene for device applications

    NASA Astrophysics Data System (ADS)

    Jandhyala, Srikar

    In order to continue improving the performance of electronic devices and also to increase functionality, incorporation of alternative channel materials into the current silicon based technology is inevitable. Graphene is one such material which is being heavily investigated owing to its high carrier mobility, one atom thickness, and other electronic as well as physical attributes. There are various architectures proposed for graphene based devices. This dissertation focuses on one of the challenges in integrating graphene based devices, i.e. gate dielectrics. For the more common device architectures utilizing electric field-effect in graphene, a thin and high dielectric constant (high-kappa) material is desired for gate dielectric applications. Although, atomic layer deposition (ALD) is the most suitable technique for depositing such dielectrics on various substrates, it is difficult to initiate dielectric growth using ALD on graphene because it lacks out-of-plane bonds owing to the sp2 hybridization of carbon atoms. An approach involving non-covalent functionalization of graphene surface with ozone (O3) at room temperature is studied for depositing high-kappa oxides with ALD. A scheme was developed for in-situ electrical monitoring of transport properties of back-gate graphene field-effect transistors (GFETs) during the ALD process. It was established that O 3 is mostly physisorbed on (high-quality) graphene at room temperature and its effects on graphene properties are reversed upon introduction of metal precursor which reacts with the adsorbed O3 molecules resulting in oxide deposition. Utilizing this knowledge, a high-pressure O3 functionalization approach was developed for depositing oxide gate dielectrics on graphene using ALD and top-gate GFETs with dielectric thickness below 5 nm were demonstrated. A low-kappa tunnel dielectric is the proposed requirement for certain graphene based devices. Covalent functionalization of graphene with fluorine through plasma

  5. Cell wall-bound cationic and anionic class III isoperoxidases of pea root: biochemical characterization and function in root growth

    PubMed Central

    Lüthje, Sabine

    2012-01-01

    Cell wall isolated from pea roots was used to separate and characterize two fractions possessing class III peroxidase activity: (i) ionically bound proteins and (ii) covalently bound proteins. Modified SDS–PAGE separated peroxidase isoforms by their apparent molecular weights: four bands of 56, 46, 44, and 41kDa were found in the ionically bound fraction (iPOD) and one band (70kDa) was resolved after treatment of the cell wall with cellulase and pectinase (cPOD). Isoelectric focusing (IEF) patterns for iPODs and cPODs were significantly different: five iPODs with highly cationic pI (9.5–9.2) were detected, whereas the nine cPODs were anionic with pI values between pH 3.7 and 5. iPODs and cPODs showed rather specific substrate affinity and different sensitivity to inhibitors, heat, and deglycosylation treatments. Peroxidase and oxidase activities and their IEF patterns for both fractions were determined in different zones along the root and in roots of different ages. New iPODs with pI 9.34 and 9.5 were induced with root growth, while the activity of cPODs was more related to the formation of the cell wall in non-elongating tissue. Treatment with auxin that inhibits root growth led to suppression of iPOD and induction of cPOD. A similar effect was obtained with the widely used elicitor, chitosan, which also induced cPODs with pI 5.3 and 5.7, which may be specifically related to pathogen defence. The differences reported here between biochemical properties of cPOD and iPOD and their differential induction during development and under specific treatments implicate that they are involved in specific and different physiological processes. Abbreviations:cPODcovalently bound peroxidaseDAB3,3'-diaminobenzidineDEPMPOspin-trap (5-diethoxy-phosphoryl-5-methyl-1-pyrroline-n-oxide)EPRelectron paramagnetic resonanceHRPhorseradish peroxidaseIAAindole-3-acetic acidHRPhorseradish peroxidaseIEFisoelectric focusingiPODionically bound peroxidaseNAAnaphthalene acetic acid

  6. Progress in the Experimental Observation of Thiamin Diphosphate-bound Intermediates on Enzymes and Mechanistic Information Derived from these Observations

    PubMed Central

    Jordan, Frank; Nemeria, Natalia S.

    2014-01-01

    Thiamin diphosphate (ThDP), the vitamin B1 coenzyme is an excellent representative of coenzymes, which carry out electrophilic catalysis by forming a covalent complex with their substrates. The function of ThDP is to greatly increase the acidity of two carbon acids by stabilizing their conjugate bases, the ylide/carbene/C2-carbanion of the thiazolium ring and the C2α-carbanion/enamine, once the substrate binds to ThDP. In recent years, several ThDP-bound intermediates on such pathways have been characterized by both solution and solid-state methods. Prominent among these advances are X-ray crystallographic results identifying both oxidative and non-oxidative intermediates, rapid chemical quench followed by NMR detection of a several intermediates which are stable under acidic conditions, solid-state NMR and circular dichroism detection of the states of ionization and tautomerization of the 4′-aminopyrimidine moiety of ThDP in some of the intermediates. These methods also enabled in some cases determination of the rate-limiting step in the complex series of steps. This review is an update of a review with the same title published by the authors in 2005 in this Journal. Much progress has been made in the intervening decade in the identification of the intermediates and their application to gain additional mechanistic insight. PMID:15888311

  7. Lipid Bilayers Covalently Anchored to Carbon Nanotubes

    PubMed Central

    Dayani, Yasaman; Malmstadt, Noah

    2012-01-01

    The unique physical and electrical properties of carbon nanotubes make them an exciting material for applications in various fields such as bioelectronics and biosensing. Due to the poor water solubility of carbon nanotubes, functionalization for such applications has been a challenge. Of particular need are functionalization methods for integrating carbon nanotubes with biomolecules and constructing novel hybrid nanostructures for bionanoelectronic applications. We present a novel method for the fabrication of dispersible, biocompatible carbon nanotube-based materials. Multi-walled carbon nanotubes (MWCNTs) are covalently modified with primary amine-bearing phospholipids in a carbodiimide-activated reaction. These modified carbon nanotubes have good dispersibility in nonpolar solvents. Fourier transform infrared (FTIR) spectroscopy shows peaks attributable to the formation of amide bonds between lipids and the nanotube surface. Simple sonication of lipid-modified nanotubes with other lipid molecules leads to the formation of a uniform lipid bilayer coating the nanotubes. These bilayer-coated nanotubes are highly dispersible and stable in aqueous solution. Confocal fluorescence microscopy shows labeled lipids on the surface of bilayer-modified nanotubes. Transmission electron microscopy (TEM) shows the morphology of dispersed bilayer-coated MWCNTs. Fluorescence quenching of lipid-coated MWCNTs confirms the bilayer configuration of the lipids on the nanotube surface and fluorescence anisotropy measurements show that the bilayer is fluid above the gel-to-liquid transition temperature. The membrane protein α-hemolysin spontaneously inserts into the MWCNT-supported bilayer, confirming the biomimetic membrane structure. These biomimetic nanostructures are a promising platform for the integration of carbon nanotube-based materials with biomolecules. PMID:22568448

  8. X-ray Crystallography Reveals a Reduced Substrate Complex of UDP-Galactopyranose Mutase Poised for Covalent Catalysis by Flavin

    SciTech Connect

    Gruber, Todd D.; Westler, William M.; Kiessling, Laura L.; Forest, Katrina T.

    2009-11-04

    The flavoenzyme uridine 5'-diphosphate galactopyranose mutase (UGM or Glf) catalyzes the interconversion of UDP-galactopyranose and UDP-galactofuranose. The latter is a key building block for cell wall construction in numerous pathogens, including Mycobacterium tuberculosis. Mechanistic studies of UGM suggested a novel role for the flavin, and we previously provided evidence that the catalytic mechanism proceeds through a covalent flavin-galactose iminium. Here, we describe 2.3 and 2.5 {angstrom} resolution X-ray crystal structures of the substrate-bound enzyme in oxidized and reduced forms, respectively. In the latter, C1 of the substrate is 3.6 {angstrom} from the nucleophilic flavin N5 position. This orientation is consistent with covalent catalysis by flavin.

  9. The single NqrB and NqrC subunits in the Na(+)-translocating NADH: quinone oxidoreductase (Na(+)-NQR) from Vibrio cholerae each carry one covalently attached FMN.

    PubMed

    Casutt, Marco S; Schlosser, Andreas; Buckel, Wolfgang; Steuber, Julia

    2012-10-01

    The Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) is the prototype of a novel class of flavoproteins carrying a riboflavin phosphate bound to serine or threonine by a phosphodiester bond to the ribityl side chain. This membrane-bound, respiratory complex also contains one non-covalently bound FAD, one non-covalently bound riboflavin, ubiquinone-8 and a [2Fe-2S] cluster. Here, we report the quantitative analysis of the full set of flavin cofactors in the Na(+)-NQR and characterize the mode of linkage of the riboflavin phosphate to the membrane-bound NqrB and NqrC subunits. Release of the flavin by β-elimination and analysis of the cofactor demonstrates that the phosphate group is attached at the 5'-position of the ribityl as in authentic FMN and that the Na(+)-NQR contains approximately 1.7mol covalently bound FMN per mol non-covalently bound FAD. Therefore, each of the single NqrB and NqrC subunits in the Na(+)-NQR carries a single FMN. Elimination of the phosphodiester bond yields a dehydro-2-aminobutyrate residue, which is modified with β-mercaptoethanol by Michael addition. Proteolytic digestion followed by mass determination of peptide fragments reveals exclusive modification of threonine residues, which carry FMN in the native enzyme. The described reactions allow quantification and localization of the covalently attached FMNs in the Na(+)-NQR and in related proteins belonging to the Rhodobacter nitrogen fixation (RNF) family of enzymes. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012). PMID:22366169

  10. Can in vitro metabolism-dependent covalent binding data in liver microsomes distinguish hepatotoxic from nonhepatotoxic drugs? An analysis of 18 drugs with consideration of intrinsic clearance and daily dose.

    PubMed

    Obach, R Scott; Kalgutkar, Amit S; Soglia, John R; Zhao, Sabrina X

    2008-09-01

    In vitro covalent binding assessments of drugs have been useful in providing retrospective insights into the association between drug metabolism and a resulting toxicological response. On the basis of these studies, it has been advocated that in vitro covalent binding to liver microsomal proteins in the presence and the absence of NADPH be used routinely to screen drug candidates. However, the utility of this approach in predicting toxicities of drug candidates accurately remains an unanswered question. Importantly, the years of research that have been invested in understanding metabolic bioactivation and covalent binding and its potential role in toxicity have focused only on those compounds that demonstrate toxicity. Investigations have not frequently queried whether in vitro covalent binding could be observed with drugs with good safety records. Eighteen drugs (nine hepatotoxins and nine nonhepatotoxins in humans) were assessed for in vitro covalent binding in NADPH-supplemented human liver microsomes. Of the two sets of nine drugs, seven in each set were shown to undergo some degree of covalent binding. Among hepatotoxic drugs, acetaminophen, carbamazepine, diclofenac, indomethacin, nefazodone, sudoxicam, and tienilic acid demonstrated covalent binding, while benoxaprofen and felbamate did not. Of the nonhepatotoxic drugs evaluated, buspirone, diphenhydramine, meloxicam, paroxetine, propranolol, raloxifene, and simvastatin demonstrated covalent binding, while ibuprofen and theophylline did not. A quantitative comparison of covalent binding in vitro intrinsic clearance did not separate the two groups of compounds, and in fact, paroxetine, a nonhepatotoxin, showed the greatest amount of covalent binding in microsomes. Including factors such as the fraction of total metabolism comprised by covalent binding and the total daily dose of each drug improved the discrimination between hepatotoxic and nontoxic drugs based on in vitro covalent binding data; however, the

  11. Supramolecular motifs in dynamic covalent PEG-hemiaminal organogels

    PubMed Central

    Fox, Courtney H.; ter Hurrne, Gijs M.; Wojtecki, Rudy J.; Jones, Gavin O.; Horn, Hans W.; Meijer, E. W.; Frank, Curtis W.; Hedrick, James L.; García, Jeannette M.

    2015-01-01

    Dynamic covalent materials are stable materials that possess reversible behaviour triggered by stimuli such as light, redox conditions or temperature; whereas supramolecular crosslinks depend on the equilibrium constant and relative concentrations of crosslinks as a function of temperature. The combination of these two reversible chemistries can allow access to materials with unique properties. Here, we show that this combination of dynamic covalent and supramolecular chemistry can be used to prepare organogels comprising distinct networks. Two materials containing hemiaminal crosslink junctions were synthesized; one material is comprised of dynamic covalent junctions and the other contains hydrogen-bonding bis-hemiaminal moieties. Under specific network synthesis conditions, these materials exhibited self-healing behaviour. This work reports on both the molecular-level detail of hemiaminal crosslink junction formation as well as the macroscopic behaviour of hemiaminal dynamic covalent network (HDCN) elastomeric organogels. These materials have potential applications as elastomeric components in printable materials, cargo carriers and adhesives. PMID:26174864

  12. Petawatt laser absorption bounded

    PubMed Central

    Levy, Matthew C.; Wilks, Scott C.; Tabak, Max; Libby, Stephen B.; Baring, Matthew G.

    2014-01-01

    The interaction of petawatt (1015 W) lasers with solid matter forms the basis for advanced scientific applications such as table-top particle accelerators, ultrafast imaging systems and laser fusion. Key metrics for these applications relate to absorption, yet conditions in this regime are so nonlinear that it is often impossible to know the fraction of absorbed light f, and even the range of f is unknown. Here using a relativistic Rankine-Hugoniot-like analysis, we show for the first time that f exhibits a theoretical maximum and minimum. These bounds constrain nonlinear absorption mechanisms across the petawatt regime, forbidding high absorption values at low laser power and low absorption values at high laser power. For applications needing to circumvent the absorption bounds, these results will accelerate a shift from solid targets, towards structured and multilayer targets, and lead the development of new materials. PMID:24938656

  13. Petawatt laser absorption bounded

    NASA Astrophysics Data System (ADS)

    Levy, Matthew C.; Wilks, Scott C.; Tabak, Max; Libby, Stephen B.; Baring, Matthew G.

    2014-06-01

    The interaction of petawatt (1015 W) lasers with solid matter forms the basis for advanced scientific applications such as table-top particle accelerators, ultrafast imaging systems and laser fusion. Key metrics for these applications relate to absorption, yet conditions in this regime are so nonlinear that it is often impossible to know the fraction of absorbed light f, and even the range of f is unknown. Here using a relativistic Rankine-Hugoniot-like analysis, we show for the first time that f exhibits a theoretical maximum and minimum. These bounds constrain nonlinear absorption mechanisms across the petawatt regime, forbidding high absorption values at low laser power and low absorption values at high laser power. For applications needing to circumvent the absorption bounds, these results will accelerate a shift from solid targets, towards structured and multilayer targets, and lead the development of new materials.

  14. Inactivation of the Mycobacterium tuberculosis antigen 85 complex by covalent, allosteric inhibitors.

    PubMed

    Favrot, Lorenza; Lajiness, Daniel H; Ronning, Donald R

    2014-09-01

    The rise of multidrug-resistant and totally drug-resistant tuberculosis and the association with an increasing number of HIV-positive patients developing tuberculosis emphasize the necessity to find new antitubercular targets and drugs. The antigen 85 (Ag85) complex from Mycobacterium tuberculosis plays important roles in the biosynthesis of major components of the mycobacterial cell envelope. For this reason, Ag85 has emerged as an attractive drug target. Recently, ebselen was identified as an effective inhibitor of the Ag85 complex through covalent modification of a cysteine residue proximal to the Ag85 active site and is therefore a covalent, allosteric inhibitor. To expand the understanding of this process, we have solved the x-ray crystal structures of Ag85C covalently modified with ebselen and other thiol-reactive compounds, p-chloromercuribenzoic acid and iodoacetamide, as well as the structure of a cysteine to glycine mutant. All four structures confirm that chemical modification or mutation at this particular cysteine residue leads to the disruption of the active site hydrogen-bonded network essential for Ag85 catalysis. We also describe x-ray crystal structures of Ag85C single mutants within the catalytic triad and show that a mutation of any one of these three residues promotes the same conformational change observed in the cysteine-modified forms. These results provide evidence for active site dynamics that may afford new strategies for the development of selective and potent Ag85 inhibitors. PMID:25028518

  15. Inactivation of the Mycobacterium tuberculosis Antigen 85 Complex by Covalent, Allosteric Inhibitors*

    PubMed Central

    Favrot, Lorenza; Lajiness, Daniel H.; Ronning, Donald R.

    2014-01-01

    The rise of multidrug-resistant and totally drug-resistant tuberculosis and the association with an increasing number of HIV-positive patients developing tuberculosis emphasize the necessity to find new antitubercular targets and drugs. The antigen 85 (Ag85) complex from Mycobacterium tuberculosis plays important roles in the biosynthesis of major components of the mycobacterial cell envelope. For this reason, Ag85 has emerged as an attractive drug target. Recently, ebselen was identified as an effective inhibitor of the Ag85 complex through covalent modification of a cysteine residue proximal to the Ag85 active site and is therefore a covalent, allosteric inhibitor. To expand the understanding of this process, we have solved the x-ray crystal structures of Ag85C covalently modified with ebselen and other thiol-reactive compounds, p-chloromercuribenzoic acid and iodoacetamide, as well as the structure of a cysteine to glycine mutant. All four structures confirm that chemical modification or mutation at this particular cysteine residue leads to the disruption of the active site hydrogen-bonded network essential for Ag85 catalysis. We also describe x-ray crystal structures of Ag85C single mutants within the catalytic triad and show that a mutation of any one of these three residues promotes the same conformational change observed in the cysteine-modified forms. These results provide evidence for active site dynamics that may afford new strategies for the development of selective and potent Ag85 inhibitors. PMID:25028518

  16. Rapid LC-TOFMS method for identification of binding sites of covalent acylglucuronide-albumin complexes.

    PubMed

    Ohkawa, T; Norikura, R; Yoshikawa, T

    2003-04-10

    A method for rapid identification of binding sites of covalent adducts was developed using delta bilirubin as a model compound. Delta bilirubin, containing intact human serum albumin (HSA), was digested with trypsin and the peptide fragments were monitored at 436 nm, but no predominant peaks were detected indicating the instability of the digested peptides containing bilirubin-related compounds. Therefore, the high-performance liquid chromatography time-of-flight mass spectrometer (LC-TOFMS) data of digested fragments of delta bilirubin were compared with those of control digests of HSA, revealing a characteristic peptide in the digest mixture of delta bilirubin. This peptide was sequenced by high-performance liquid chromatography time-of-flight tandem mass spectrometry (LC-TOFMS/MS) and identified as LDELRDEGKASSAK (Leu182 to Lys195) with a modification of a 178 Da increase at Lys190. This indicated the Lys190 to be a predominant covalent binding site of BGs on HSA via the imine mechanism and the binding between the bilirubin moiety and the glucuronic acid moiety to be unstable to digestion with trypsin. The method of comparing LC-TOFMS data requires no specific detection such as fluorescence or radioactivity for every compound. This should accelerate the structure elucidation of covalent adducts and be helpful for studying the relationship between the structure of ligands and specific binding sites. PMID:12667932

  17. Absolute quantitation of NAPQI-modified rat serum albumin by LC-MS/MS: monitoring acetaminophen covalent binding in vivo.

    PubMed

    LeBlanc, André; Shiao, Tze Chieh; Roy, René; Sleno, Lekha

    2014-09-15

    Acetaminophen is known to cause hepatoxicity via the formation of a reactive metabolite, N-acetyl p-benzoquinone imine (NAPQI), as a result of covalent binding to liver proteins. Serum albumin (SA) is known to be covalently modified by NAPQI and is present at high concentrations in the bloodstream and is therefore a potential biomarker to assess the levels of protein modification by NAPQI. A newly developed method for the absolute quantitation of serum albumin containing NAPQI covalently bound to its active site cysteine (Cys34) is described. This optimized assay represents the first absolute quantitation of a modified protein, with very low stoichiometric abundance, using a protein-level standard combined with isotope dilution. The LC-MS/MS assay is based on a protein standard modified with a custom-designed reagent, yielding a surrogate peptide (following digestion) that is a positional isomer to the target peptide modified by NAPQI. To illustrate the potential of this approach, the method was applied to quantify NAPQI-modified SA in plasma from rats dosed with acetaminophen. The resulting method is highly sensitive (capable of quantifying down to 0.0006% of total RSA in its NAPQI-modified form) and yields excellent precision and accuracy statistics. A time-course pharmacokinetic study was performed to test the usefulness of this method for following acetaminophen-induced covalent binding at four dosing levels (75-600 mg/kg IP), showing the viability of this approach to directly monitor in vivo samples. This approach can reliably quantify NAPQI-modified albumin, allowing direct monitoring of acetaminophen-related covalent binding. PMID:25168029

  18. Non-covalent interactions between carbon nanotubes and conjugated polymers

    NASA Astrophysics Data System (ADS)

    Tuncel, Dönüs

    2011-09-01

    Carbon nanotubes (CNTs) are interest to many different disciplines including chemistry, physics, biology, material science and engineering because of their unique properties and potential applications in various areas spanning from optoelectronics to biotechnology. However, one of the drawbacks associated with these materials is their insolubility which limits their wide accessibility for many applications. Various approaches have been adopted to circumvent this problem including modification of carbon nanotube surfaces by non-covalent and covalent attachments of solubilizing groups. Covalent approach modification may alter the intrinsic properties of carbon nanotubes and, in turn make them undesirable for many applications. On the other hand, a non-covalent approach helps to improve the solubility of CNTs while preserving their intrinsic properties. Among many non-covalent modifiers of CNTs, conjugated polymers are receiving increasing attention and highly appealing because of a number of reasons. To this end, the aim of this feature article is to review the recent results on the conjugated polymer-based non-covalent functionalization of CNTs with an emphasis on the effect of conjugated polymers in the dispersibility/solubility, optical, thermal and mechanical properties of carbon nanotubes as well as their usage in the purification and isolation of a specific single-walled nanotube from the mixture of the various tubes.

  19. Immunodetection of human topoisomerase I-DNA covalent complexes

    PubMed Central

    Patel, Anand G.; Flatten, Karen S.; Peterson, Kevin L.; Beito, Thomas G.; Schneider, Paula A.; Perkins, Angela L.; Harki, Daniel A.; Kaufmann, Scott H.

    2016-01-01

    A number of established and investigational anticancer drugs slow the religation step of DNA topoisomerase I (topo I). These agents induce cytotoxicity by stabilizing topo I-DNA covalent complexes, which in turn interact with advancing replication forks or transcription complexes to generate lethal lesions. Despite the importance of topo I-DNA covalent complexes, it has been difficult to detect these lesions within intact cells and tumors. Here, we report development of a monoclonal antibody that specifically recognizes covalent topo I-DNA complexes, but not free topo I or DNA, by immunoblotting, immunofluorescence or flow cytometry. Utilizing this antibody, we demonstrate readily detectable topo I-DNA covalent complexes after treatment with camptothecins, indenoisoquinolines and cisplatin but not nucleoside analogues. Topotecan-induced topo I-DNA complexes peak at 15–30 min after drug addition and then decrease, whereas indotecan-induced complexes persist for at least 4 h. Interestingly, simultaneous staining for covalent topo I-DNA complexes, phospho-H2AX and Rad51 suggests that topotecan-induced DNA double-strand breaks occur at sites distinct from stabilized topo I-DNA covalent complexes. These studies not only provide new insight into the action of topo I-directed agents, but also illustrate a strategy that can be applied to study additional topoisomerases and their inhibitors in vitro and in vivo. PMID:26917015

  20. Immunodetection of human topoisomerase I-DNA covalent complexes.

    PubMed

    Patel, Anand G; Flatten, Karen S; Peterson, Kevin L; Beito, Thomas G; Schneider, Paula A; Perkins, Angela L; Harki, Daniel A; Kaufmann, Scott H

    2016-04-01

    A number of established and investigational anticancer drugs slow the religation step of DNA topoisomerase I (topo I). These agents induce cytotoxicity by stabilizing topo I-DNA covalent complexes, which in turn interact with advancing replication forks or transcription complexes to generate lethal lesions. Despite the importance of topo I-DNA covalent complexes, it has been difficult to detect these lesions within intact cells and tumors. Here, we report development of a monoclonal antibody that specifically recognizes covalent topo I-DNA complexes, but not free topo I or DNA, by immunoblotting, immunofluorescence or flow cytometry. Utilizing this antibody, we demonstrate readily detectable topo I-DNA covalent complexes after treatment with camptothecins, indenoisoquinolines and cisplatin but not nucleoside analogues. Topotecan-induced topo I-DNA complexes peak at 15-30 min after drug addition and then decrease, whereas indotecan-induced complexes persist for at least 4 h. Interestingly, simultaneous staining for covalent topo I-DNA complexes, phospho-H2AX and Rad51 suggests that topotecan-induced DNA double-strand breaks occur at sites distinct from stabilized topo I-DNA covalent complexes. These studies not only provide new insight into the action of topo I-directed agents, but also illustrate a strategy that can be applied to study additional topoisomerases and their inhibitorsin vitroandin vivo. PMID:26917015

  1. Covalent Functionalization of NiTi Surfaces with Bioactive Peptide Amphiphile Nanofibers

    PubMed Central

    Sargeant, Timothy D.; Rao, Mukti S.; Koh, Chung-Yan

    2009-01-01

    Surface modification enables the creation of bioactive implants using traditional material substrates without altering the mechanical properties of the bulk material. For applications such as bone plates and stents, it is desirable to modify the surface of metal alloy substrates to facilitate cellular attachment, proliferation, and possibly differentiation. In this work we present a general strategy for altering the surface chemistry of nickel-titanium shape memory alloy (NiTi) in order to covalently attach self-assembled peptide amphiphile (PA) nanofibers with bioactive functions. Bioactivity in the systems studied here includes biological adhesion and proliferation of osteoblast and endothelial cell types. The optimized surface treatment creates a uniform TiO2 layer with low levels of Ni on the NiTi surface, which is subsequently covered with an aminopropylsilane coating using a novel, lower temperature vapor deposition method. This method produces an aminated surface suitable for covalent attachment of PA molecules containing terminal carboxylic acid groups. The functionalized NiTi surfaces have been characterized by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectroscopy (ToF-SIMS), and atomic force microscopy (AFM). These techniques offer evidence that the treated metal surfaces consist primarily of TiO2 with very little Ni, and also confirm the presence of the aminopropylsilane overlayer. Self-assembled PA nanofibers presenting the biological peptide adhesion sequence Arg-Gly-Asp-Ser are capable of covalently anchoring to the treated substrate, as demonstrated by spectrofluorimetry and AFM. Cell culture and scanning electron microscopy (SEM) demonstrate cellular adhesion, spreading, and proliferation on these functionalized metal surfaces. Furthermore, these experiments demonstrate that covalent attachment is crucial for creating robust PA nanofiber coatings, leading to confluent cell monolayers. PMID:18083225

  2. Localization and Function of the Membrane-bound Riboflavin in the Na+-translocating NADH:Quinone Oxidoreductase (Na+-NQR) from Vibrio cholerae*

    PubMed Central

    Casutt, Marco S.; Huber, Tamara; Brunisholz, René; Tao, Minli; Fritz, Günter; Steuber, Julia

    2010-01-01

    The sodium ion-translocating NADH:quinone oxidoreductase (Na+-NQR) from the human pathogen Vibrio cholerae is a respiratory membrane protein complex that couples the oxidation of NADH to the transport of Na+ across the bacterial membrane. The Na+-NQR comprises the six subunits NqrABCDEF, but the stoichiometry and arrangement of these subunits are unknown. Redox-active cofactors are FAD and a 2Fe-2S cluster on NqrF, covalently attached FMNs on NqrB and NqrC, and riboflavin and ubiquinone-8 with unknown localization in the complex. By analyzing the cofactor content and NADH oxidation activity of subcomplexes of the Na+-NQR lacking individual subunits, the riboflavin cofactor was unequivocally assigned to the membrane-bound NqrB subunit. Quantitative analysis of the N-terminal amino acids of the holo-complex revealed that NqrB is present in a single copy in the holo-complex. It is concluded that the hydrophobic NqrB harbors one riboflavin in addition to its covalently attached FMN. The catalytic role of two flavins in subunit NqrB during the reduction of ubiquinone to ubiquinol by the Na+-NQR is discussed. PMID:20558724

  3. Bound anionic states of adenine

    SciTech Connect

    Haranczyk, Maciej; Gutowski, Maciej S; Li, Xiang; Bowen, Kit H

    2007-03-20

    Anionic states of nucleic acid bases are involved in DNA damage by low-energy electrons and in charge transfer through DNA. Previous gas phase studies of free, unsolvated nucleic acid base parent anions probed only dipole-bound states, which are not present in condensed phase environments, but did not observe valence anionic states, which for purine bases, are thought to be adiabatically unbound. Contrary to this expectation, we have demonstrated that some thus far ignored tautomers of adenine, which result from enamine-imine transformations, support valence anionic states with electron vertical detachment energies as large as 2.2 eV, and at least one of these anionic tautomers is adiabatically bound. Moreover, we predict that the new anionic tautomers should also dominate in solutions and should be characterized by larger values of electron vertical detachment energy than the canonical valence anion. All of the new-found anionic tautomers might be formed in the course of dissociative electron attachment followed by a hydrogen atom attachment to a carbon atom, and they might affect the structure and properties of DNA and RNA exposed to low-energy electrons. The discovery of these valence anionic states of adenine was facilitated by the development of: (i) a new experimental method for preparing parent anions of nucleic acid bases for photoelectron experiments, and (ii) a new combinatorial/ quantum chemical approach for identification of the most stable tautomers of organic molecules. The computational portion of this work was supported by the: (i) Polish State Committee for Scientific Research (KBN) Grants: DS/8000-4-0140-7 (M.G.) and N204 127 31/2963 (M.H.), (ii) European Social Funds (EFS) ZPORR/2.22/II/2.6/ARP/U/2/05 (M.H.), and (iii) US DOE Office of Biological and Environmental Research, Low Dose Radiation Research Program (M.G.). M.H. holds the Foundation for Polish Science (FNP) award for young scientists. The calculations were performed at the Academic

  4. Universal bounds on current fluctuations

    NASA Astrophysics Data System (ADS)

    Pietzonka, Patrick; Barato, Andre C.; Seifert, Udo

    2016-05-01

    For current fluctuations in nonequilibrium steady states of Markovian processes, we derive four different universal bounds valid beyond the Gaussian regime. Different variants of these bounds apply to either the entropy change or any individual current, e.g., the rate of substrate consumption in a chemical reaction or the electron current in an electronic device. The bounds vary with respect to their degree of universality and tightness. A universal parabolic bound on the generating function of an arbitrary current depends solely on the average entropy production. A second, stronger bound requires knowledge both of the thermodynamic forces that drive the system and of the topology of the network of states. These two bounds are conjectures based on extensive numerics. An exponential bound that depends only on the average entropy production and the average number of transitions per time is rigorously proved. This bound has no obvious relation to the parabolic bound but it is typically tighter further away from equilibrium. An asymptotic bound that depends on the specific transition rates and becomes tight for large fluctuations is also derived. This bound allows for the prediction of the asymptotic growth of the generating function. Even though our results are restricted to networks with a finite number of states, we show that the parabolic bound is also valid for three paradigmatic examples of driven diffusive systems for which the generating function can be calculated using the additivity principle. Our bounds provide a general class of constraints for nonequilibrium systems.

  5. Crystalline fibres of a covalent organic framework through bottom-up microfluidic synthesis.

    PubMed

    Rodríguez-San-Miguel, David; Abrishamkar, Afshin; Navarro, Jorge A R; Rodriguez-Trujillo, Romen; Amabilino, David B; Mas-Ballesté, Ruben; Zamora, Félix; Puigmartí-Luis, Josep

    2016-07-28

    A microfluidic chip has been used to prepare fibres of a porous polymer with high structural order, setting a precedent for the generation of a wide variety of materials using this reagent mixing approach that provides unique materials not accessible easily through bulk processes. The reaction between 1,3,5-tris(4-aminophenyl)benzene and 1,3,5-benzenetricarbaldehyde in acetic acid under continuous microfluidic flow conditions leads to the formation of a highly crystalline and porous covalent organic framework (hereafter denoted as MF-COF-1), consisting of fibrillar micro-structures, which have mechanical stability that allows for direct drawing of objects on a surface. PMID:27321768

  6. Covalent assembly of a soluble T cell receptor-peptide-major histocompatibility class I complex.

    PubMed Central

    Grégoire, C; Lin, S Y; Mazza, G; Rebai, N; Luescher, I F; Malissen, B

    1996-01-01

    We used stepwise photochemical cross-linking for specifically assembling soluble and covalent complexes made of a T-cell antigen receptor (TCR) and a class I molecule of the major histocompatibility complex (MHC) bound to an antigenic peptide. For that purpose, we have produced in myeloma cells a single-chain Fv construct of a TCR specific for a photoreactive H-2Kd-peptide complex. Photochemical cross-linking of this TCR single-chain Fv with a soluble form of the photoreactive H-2Kd-peptide ligand resulted in the formation of a ternary covalent complex. We have characterized the soluble ternary complex and showed that it reacted with antibodies specific for epitopes located either on the native TCR or on the Kd molecules. By preventing the fast dissociation kinetics observed with most T cell receptors, this approach provides a means of preparing soluble TCR-peptide-MHC complexes on large-scale levels. Images Fig. 3 Fig. 4 PMID:8692966

  7. Aromatic sulfonyl fluorides covalently kinetically stabilize transthyretin to prevent amyloidogenesis while affording a fluorescent conjugate.

    PubMed

    Grimster, Neil P; Connelly, Stephen; Baranczak, Aleksandra; Dong, Jiajia; Krasnova, Larissa B; Sharpless, K Barry; Powers, Evan T; Wilson, Ian A; Kelly, Jeffery W

    2013-04-17

    Molecules that bind selectively to a given protein and then undergo a rapid chemoselective reaction to form a covalent conjugate have utility in drug development. Herein a library of 1,3,4-oxadiazoles substituted at the 2 position with an aryl sulfonyl fluoride and at the 5 position with a substituted aryl known to have high affinity for the inner thyroxine binding subsite of transthyretin (TTR) was conceived of by structure-based design principles and was chemically synthesized. When bound in the thyroxine binding site, most of the aryl sulfonyl fluorides react rapidly and chemoselectively with the pKa-perturbed K15 residue, kinetically stabilizing TTR and thus preventing amyloid fibril formation, known to cause polyneuropathy. Conjugation t50s range from 1 to 4 min, ~1400 times faster than the hydrolysis reaction outside the thyroxine binding site. X-ray crystallography confirms the anticipated binding orientation and sheds light on the sulfonyl fluoride activation leading to the sulfonamide linkage to TTR. A few of the aryl sulfonyl fluorides efficiently form conjugates with TTR in plasma. Eleven of the TTR covalent kinetic stabilizers synthesized exhibit fluorescence upon conjugation and therefore could have imaging applications as a consequence of the environment sensitive fluorescence of the chromophore. PMID:23350654

  8. Covalent modification of mushroom tyrosinase with different amphiphic polymers for pharmaceutical and biocatalysis applications

    SciTech Connect

    Morpurgo, M.; Schiavon, O.; Caliceti, P.

    1996-01-01

    Two different poly(ethylene glycol) derivatives (linear, mol wt 5000 and a branched form, mol wt 10000) and a new polymer (poly-[acryloylmorfoline], mol wt 5500) were covalently bound to the enzyme tyrosinase. The polymer-protein conjugates were studied with a view to their potential pharmaceutical application and to their use for the bioconversion of phenolic substrates in organic solvents. V{sub max} and K{sub m} for the dopa-dopaquinone conversion, thermostability, stability toward inactivation by dopa oxidation products, half-life in blood circulation, and behavior in organic solvents for the different adducts were investigated. Arrhenius plots for the dopa-dopaquinone conversion were also obtained in order to study the effects of temperature on the different enzyme forms. Covalent attachment of the polymers increased enzyme stability in aqueous solution and the solubility in organic solvents. However, organic solvent solubilization brought about loss of enzyme conformation as assessed by CD measurements, which is accompanied by a nonreversible loss of catalytic activity. 30 refs., 4 figs., 4 tabs.

  9. Surface functionalization of silica-coated magnetic nanoparticles for covalent attachment of cholesterol oxidase

    NASA Astrophysics Data System (ADS)

    Šulek, Franja; Drofenik, Miha; Habulin, Maja; Knez, Željko

    2010-01-01

    A systematic approach towards the fabrication of highly functionalized silica shell magnetic nanoparticles, presently used for enzyme immobilization, is herein fully presented. The synthesis of bare maghemite (γ-Fe 2O 3) nanoparticles was accomplished by thermal co-precipitation of iron ions in ammonia alkaline solution at harsh reaction conditions, respectively. Primary surface engineering of maghemite nanoparticles was successfully performed by the proper deposition of silica onto nanoparticles surface under strictly regulated reaction conditions. Next, the secondary surface functionalization of the particles was achieved by coating the particles with organosilane followed by glutaraldehyde activation in order to enhance protein immobilization. Covalent immobilization of cholesterol oxidase was attempted afterwards. The structural and magnetic properties of magnetic silica nanocomposites were characterized by TEM and vibrating sample magnetometer (VSM) instruments. X-ray diffraction measurements confirmed the spinel structure and average size of uncoated maghemite nanoparticles to be around 20 nm in diameter. SEM-EDS spectra indicated a strong signal for Si, implying the coating procedure of silica onto the particles surface to be successfully accomplished. Fourier transform infrared (FT-IR) spectra analysis confirmed the binding of amino silane molecules onto the surface of the maghemite nanoparticles mediated Si-O-Si chemical bonds. Compared to the free enzyme, the covalently bound cholesterol oxidase retained 50% of its activity. Binding of enzyme onto chemically modified magnetic nanoparticles via glutaraldehyde activation is a promising method for developing biosensing components in biomedicine.

  10. Aromatic Sulfonyl Fluorides Covalently Kinetically Stabilize Transthyretin to Prevent Amyloidogenesis while Affording a Fluorescent Conjugate

    PubMed Central

    Grimster, Neil P.; Connelly, Stephen; Baranczak, Aleksandra; Dong, Jiajia; Krasnova, Larissa B.; Sharpless, K. Barry; Powers, Evan T.; Wilson, Ian A.; Kelly, Jeffery W.

    2013-01-01

    Molecules that bind selectively to a given protein and then undergo a rapid chemoselective reaction to form a covalent conjugate have utility in drug development. Herein a library of 1,3,4-oxadiazoles substituted at the 2 position with an aryl sulfonyl fluoride and at the 5 position with a substituted aryl known to have high affinity for the inner thyroxine binding subsite of transthyretin (TTR) were conceived of by structure-based design principles and were chemically synthesized. When bound in the thyroxine binding site, most of the aryl sulfonyl fluorides react rapidly and chemoselectively with the pKa-perturbed K15 residue, kinetically stabilizing TTR and thus preventing amyloid fibril formation, known to cause polyneuropathy. Conjugation t50s range from 1 to 4 min, ~ 1400 times faster than the hydrolysis reaction outside the thyroxine binding site. X-ray crystallography confirms the anticipated binding orientation and sheds light on the sulfonyl fluoride activation leading to the sulfonamide linkage to TTR. A few of the aryl sulfonyl fluorides efficiently form conjugates with TTR in plasma. A few of the TTR covalent kinetic stabilizers synthesized exhibit fluorescence upon conjugation and therefore could have imaging applications as a consequence of the environment sensitive fluorescence of the chromophore. PMID:23350654

  11. Covalent linkage of nanodiamond-paclitaxel for drug delivery and cancer therapy

    NASA Astrophysics Data System (ADS)

    Liu, Kuang-Kai; Zheng, Wen-Wei; Wang, Chi-Ching; Chiu, Yu-Chung; Cheng, Chia-Liang; Lo, Yu-Shiu; Chen, Chinpiao; Chao, Jui-I.

    2010-08-01

    A nanoparticle-conjugated cancer drug provides a novel strategy for cancer therapy. In this study, we manipulated nanodiamond (ND), a carbon nanomaterial, to covalently link paclitaxel for cancer drug delivery and therapy. Paclitaxel was bound to the surface of 3-5 nm sized ND through a succession of chemical modifications. The ND-paclitaxel conjugation was measured by atomic force microscope and nuclear magnetic resonance spectroscopy, and confirmed with infrared spectroscopy by the detection of deuterated paclitaxel. Treatment with 0.1-50 µg ml - 1 ND-paclitaxel for 48 h significantly reduced the cell viability in the A549 human lung carcinoma cells. ND-paclitaxel induced both mitotic arrest and apoptosis in A549 cells. However, ND alone or denatured ND-paclitaxel (after treatment with strong alkaline solution, 1 M NaOH) did not induce the damage effects on A549 cells. ND-paclitaxel was taken into lung cancer cells in a concentration-dependent manner using flow cytometer analysis. The ND-paclitaxel particles were located in the microtubules and cytoplasm of A549 cells observed by confocal microscopy. Furthermore, ND-paclitaxel markedly blocked the tumor growth and formation of lung cancer cells in xenograft SCID mice. Together, we provide a functional covalent conjugation of ND-paclitaxel, which can be delivered into lung carcinoma cells and preserves the anticancer activities on the induction of mitotic blockage, apoptosis and anti-tumorigenesis.

  12. Stimulus-responsive Controlled Release System by Covalent Immobilization of an Enzyme into Mesoporous Silica Nanoparticles

    PubMed Central

    Méndez, Jessica; Monteagudo, Alina; Griebenow, Kai

    2012-01-01

    Mesoporous silica nanoparticles (MSN) have emerged as an attractive class of drug delivery carriers for therapeutic agents. Herein, we explored the covalent immobilization of proteins into MSN to generate a stimulus-responsive controlled release system. First, MSN were functionalized with thiol groups using (mercaptopropyl)-trimethoxysilane (MPTMS). Functionalization was verified by X-ray photoelectron spectroscopy (XP), Fourier-transform infrared (FTIR) spectroscopy, and dynamic light scattering. The model enzyme carbonic anhydrase (CA) was coupled to sulfosuccinimidyl 6-[3'(2-pyridyldithio)-propionamido]hexanoate (Sulfo-LC-SPDP) at a low ratio of 1:1 to prevent enzyme inactivation and subsequently covalently immobilized into MSN via thiol-disulfide interchange. The enzyme could be released from MSN with 10 mM glutathione which represents intra-cellular redox conditions while it remained bound to the MSN at extra-cellular redox conditions represented by 1 μM glutathione. The activity of the released enzyme was >80% demonstrating that the enzyme was still largely functional and active after immobilization and release. Human cervical cancer (HeLa) cells were incubated with the MSN-CA bioconjugates at various concentrations for 24 h and the data show good biocompatibility. In summary, we demonstrate the potential of MSN as potential drug delivery systems for proteins. PMID:22375899

  13. Characterizing the Covalent Targets of a Small Molecule Inhibitor of the Lysine Acetyltransferase P300.

    PubMed

    Shrimp, Jonathan H; Sorum, Alexander W; Garlick, Julie M; Guasch, Laura; Nicklaus, Marc C; Meier, Jordan L

    2016-02-11

    C646 inhibits the lysine acetyltransferases (KATs) p300 and CBP and represents the most potent and selective small molecule KAT inhibitor identified to date. To gain insights into the cellular activity of this epigenetic probe, we applied chemoproteomics to identify covalent targets of the C646 chemotype. Modeling and synthetic derivatization was used to develop a clickable analogue (C646-yne) that inhibits p300 similarly to the parent compound and enables enrichment of bound proteins. LC-MS/MS identified the major covalent targets of C646-yne as highly abundant cysteine-containing proteins, and follow-up studies found that C646 can inhibit tubulin polymerization in vitro. Finally, we provide evidence that thiol reactivity of C646 may limit its ability to antagonize acetylation in cells. These findings should enable a more precise interpretation of studies utilizing C646 as a chemical probe of KAT activity and suggest that an underappreciated liability of electrophile-containing inhibitors is a reduction in their cellular potency due to consumption by abundant protein and metabolite thiol sinks. PMID:26985290

  14. Covalent thiol adducts arising from reactive intermediates of cocaine biotransformation.

    PubMed

    Schneider, Kevin J; DeCaprio, Anthony P

    2013-11-18

    Exposure to cocaine results in the depletion of hepatocellular glutathione and macromolecular protein binding in humans. Such cocaine-induced responses have generally been attributed to oxidative stress and reactive metabolites resulting from oxidative activation of the cocaine tropane nitrogen. However, little conclusive data exists on the mechanistic pathways leading to protein modification or the structure and specificity of cocaine-derived adduction products. We now report a previously uncharacterized route of cocaine bioactivation leading to the covalent adduction of biological thiols, including cysteine and glutathione. Incubation of cocaine with biological nucleophiles in an in vitro biotransformation system containing human liver microsomes identified a monooxygenase-mediated event leading to the oxidation of, and subsequent sulfhydryl addition to, the cocaine aryl moiety. Adduct structures were confirmed using ultra-high performance liquid chromatography coupled to high resolution, high mass accuracy mass spectrometry. Examination of assays containing transgenic bactosomes expressing single human cytochrome P450 isoforms determined the role of P450s 1A2, 2C19, and 2D6 in the oxidation process resulting in adduct formation. P450-catalyzed aryl epoxide formation and subsequent attack by free nucleophilic moieties is consistent with the resulting adduct structures, mechanisms of formation, and the empirical observation of multiple structural and stereo isomers. Analogous adduction mechanisms were maintained across all sulfhydryl-containing nucleophile models examined; N-acetylcysteine, glutathione, and a synthetic cysteine-containing hexapeptide. Predictive in silico calculations of molecular reactivity and electrophilicity/nucleophilicity were compared to the results of in vitro assay incubations in order to better understand the adduction process using the principles of hard and soft acid and base (HSAB) theory. This study elucidated a novel metabolic

  15. Bound charges and currents

    NASA Astrophysics Data System (ADS)

    Herczyński, Andrzej

    2013-03-01

    Bound charges and currents are among the conceptually challenging topics in advanced courses on electricity and magnetism. It may be tempting for students to believe that they are merely computational tools for calculating electric and magnetic fields in matter, particularly because they are usually introduced through abstract manipulation of integral identities, with the physical interpretation provided a posteriori. Yet these charges and currents are no less real than free charges and currents and can be measured experimentally. A simpler and more direct approach to introducing this topic, suggested by the ideas in the classic book by Purcell and emphasizing the physical origin of these phenomena, is proposed.

  16. Lipoic acid - biological activity and therapeutic potential.

    PubMed

    Gorąca, Anna; Huk-Kolega, Halina; Piechota, Aleksandra; Kleniewska, Paulina; Ciejka, Elżbieta; Skibska, Beata

    2011-01-01

    α-Lipoic acid (LA; 5-(1,2-dithiolan-3-yl)pentanoic acid) was originally isolated from bovine liver by Reed et al. in 1951. LA was once considered a vitamin. Subsequently, it was found that LA is not a vitamin and is synthesized by plants and animals. LA is covalently bound to the ε-amino group of lysine residues and functions as a cofactor for mitochondrial enzymes by catalyzing the oxidative decarboxylation of pyruvate, α-ketoglutarate and branched-chain α-keto acids. LA and its reduced form - dihydrolipoic acid (DHLA), meet all the criteria for an ideal antioxidant because they can easily quench radicals, can chelate metals, have an amphiphlic character and they do not exhibit any serious side effects. They interact with other antioxidants and can regenerate them. For this reason, LA is called an antioxidant of antioxidants. LA has an influence on the second messenger nuclear factor κB (NF-κB) and attenuates the release of free radicals and cytotoxic cytokines. The therapeutic action of LA is based on its antioxidant properties. Current studies support its use in the ancillary treatment of many diseases, such as diabetes, cardiovascular, neurodegenerative, autoimmune diseases, cancer and AIDS. This review was undertaken to gather the most recent information regarding the therapeutic properties of LA and its possible utility in disease treatment. PMID:22001972

  17. Synthesis and anti-cancer activity of covalent conjugates of artemisinin and a transferrin-receptor targeting peptide.

    PubMed

    Oh, Steve; Kim, Byung Ju; Singh, Narendra P; Lai, Henry; Sasaki, Tomikazu

    2009-02-01

    Artemisinin, a natural product isolated from Artemisia annua L., shows a unique anti-cancer activity by an iron dependent mechanism. Artemisinin was covalently conjugated to a transferrin-receptor targeting peptide, HAIYPRH that binds to a cavity on the surface of transferrin receptor. This enables artemisinin to be co-internalized with receptor-bound transferrin. The iron released from transferrin can activate artemisinin to generate toxic radical species to kill cells. The artemisinin-peptide conjugates showed potent anti-cancer activity against Molt-4 leukemia cells with a significantly improved cancer/normal cells selectivity. PMID:18838215

  18. Evidence for specific and non-covalent binding of lipids to natural and recombinant Mycobacterium bovis BCG hsp60 proteins, and to the Escherichia coli homologue GroEL.

    PubMed

    De Bruyn, J; Soetaert, K; Buyssens, P; Calonne, I; De Coene, J L; Gallet, X; Brasseur, R; Wattiez, R; Falmagne, P; Montrozier, H; Lanéelle, M A; Daffé, M

    2000-07-01

    Heat-shock proteins (Hsps) from various origins are known to share a conserved structure and are assumed to be key partners in the biogenesis of proteins. Fractionation of the mycobacterial Hsp60, a 65 kDa protein also called Cpn60, from Mycobacterium bovis BCG zinc-deficient culture filtrate on phenyl-Sepharose followed by Western blotting revealed the existence of four Hsp60-1 and Hsp60-2 forms, based on their hydrophobicity behaviour. Hsp60-2 species were further purified by ion-exchange chromatography and partial amino acid sequences of cyanogen bromide (CNBr) peptides of purified Hsp60-2 species showed identity with the amino acid sequence deduced from the hsp60-2 gene, indicating that the various Hsp60-2 forms are encoded by the same gene. In addition, the mycobacterial Hsp60-2 was overexpressed in E. coli using the pRR3Hsp60-2 plasmid and analysed on phenyl-Sepharose. The elution pattern of the recombinant Hsp60-2, as well as that of Escherichia coli GroEL, was similar to that of the native Hsp60-2 from the culture filtrate of M. bovis BCG and entirely different from that of the mycobacterial antigen 85. Extraction of mycobacterial Hsp60-2 forms, recombinant BCG Hsp60-2 and E. coli GroEL with organic solvents releases various amounts of non-covalently bound lipids. The presence of lipids on Hsp60-2 was confirmed by labelling M. bovis BCG with radioactive palmitate. The radioactivity was specifically associated with Hsp60 in the aqueous phase and the 19 and 38 kDa lipoproteins in the Triton X-114 phase. Analysis of the lipids extracted from purified Hsp60-2, recombinant BCG Hsp60-2 and E. coli GroEL by TLC showed the same pattern for all the samples. Acid methanolysis of the lipids followed by GC analysis led to the identification of C(16:0), C(18:0) and C(18:1) as the major fatty acyl constituents, and of methylglycoside in these proteins. Altogether, these data demonstrate that lipids are non-covalently bound to Hsp60-2 and homologous proteins. PMID

  19. Covalent Conjugation of a Peptide Triazole to HIV-1 gp120 Enables Intramolecular Binding Site Occupancy

    PubMed Central

    2015-01-01

    The HIV-1 gp120 glycoprotein is the main viral surface protein responsible for initiation of the entry process and, as such, can be targeted for the development of entry inhibitors. We previously identified a class of broadly active peptide triazole (PT) dual antagonists that inhibit gp120 interactions at both its target receptor and coreceptor binding sites, induce shedding of gp120 from virus particles prior to host–cell encounter, and consequently can prevent viral entry and infection. However, our understanding of the conformational alterations in gp120 by which PT elicits its dual receptor antagonism and virus inactivation functions is limited. Here, we used a recently developed computational model of the PT–gp120 complex as a blueprint to design a covalently conjugated PT–gp120 recombinant protein. Initially, a single-cysteine gp120 mutant, E275CYU-2, was expressed and characterized. This variant retains excellent binding affinity for peptide triazoles, for sCD4 and other CD4 binding site (CD4bs) ligands, and for a CD4-induced (CD4i) ligand that binds the coreceptor recognition site. In parallel, we synthesized a PEGylated and biotinylated peptide triazole variant that retained gp120 binding activity. An N-terminally maleimido variant of this PEGylated PT, denoted AE21, was conjugated to E275C gp120 to produce the AE21–E275C covalent conjugate. Surface plasmon resonance interaction analysis revealed that the PT–gp120 conjugate exhibited suppressed binding of sCD4 and 17b to gp120, signatures of a PT-bound state of envelope protein. Similar to the noncovalent PT–gp120 complex, the covalent conjugate was able to bind the conformationally dependent mAb 2G12. The results argue that the PT–gp120 conjugate is structurally organized, with an intramolecular interaction between the PT and gp120 domains, and that this structured state embodies a conformationally entrapped gp120 with an altered bridging sheet but intact 2G12 epitope. The similarities of

  20. Covalent agonists for studying G protein-coupled receptor activation

    PubMed Central

    Weichert, Dietmar; Kruse, Andrew C.; Manglik, Aashish; Hiller, Christine; Zhang, Cheng; Hübner, Harald; Kobilka, Brian K.; Gmeiner, Peter

    2014-01-01

    Structural studies on G protein-coupled receptors (GPCRs) provide important insights into the architecture and function of these important drug targets. However, the crystallization of GPCRs in active states is particularly challenging, requiring the formation of stable and conformationally homogeneous ligand-receptor complexes. Native hormones, neurotransmitters, and synthetic agonists that bind with low affinity are ineffective at stabilizing an active state for crystallogenesis. To promote structural studies on the pharmacologically highly relevant class of aminergic GPCRs, we here present the development of covalently binding molecular tools activating Gs-, Gi-, and Gq-coupled receptors. The covalent agonists are derived from the monoamine neurotransmitters noradrenaline, dopamine, serotonin, and histamine, and they were accessed using a general and versatile synthetic strategy. We demonstrate that the tool compounds presented herein display an efficient covalent binding mode and that the respective covalent ligand-receptor complexes activate G proteins comparable to the natural neurotransmitters. A crystal structure of the β2-adrenoreceptor in complex with a covalent noradrenaline analog and a conformationally selective antibody (nanobody) verified that these agonists can be used to facilitate crystallogenesis. PMID:25006259

  1. Increased Protein Structural Resolution from Diethylpyrocarbonate-based Covalent Labeling and Mass Spectrometric Detection

    PubMed Central

    Zhou, Yuping; Vachet, Richard W.

    2012-01-01

    Covalent labeling and mass spectrometry are seeing increased used together as a way to obtain insight into the 3-dimensional structure of proteins and protein complexes. Several amino acid specific (e.g. diethylpyrocarbonate) and non-specific (e.g. hydroxyl radicals) labeling reagents are available for this purpose. Diethylpyrocarbonate (DEPC) is a promising labeling reagent because it can potentially probe up to 30% of the residues in the average protein and gives only one reaction product, thereby facilitating mass spectrometric analysis. It was recently reported, though, that DEPC modifications are labile for some amino acids. Here, we show that label loss is more significant and widespread than previously thought, especially for Ser, Thr, Tyr, and His residues, when relatively long protein digestion times are used. Such label loss ultimately decreases the amount of protein structural information that is obtainable with this reagent. We find, however, that the number of DEPC modified residues, and thus protein structural information, can be significantly increased by decreasing the time between the covalent labeling reaction and the mass spectrometric analysis. This is most effectively accomplished using short (e.g. 2 h) proteolytic digestions with enzymes such as immobilized chymotrypsin or Glu-C rather than using methods (e.g. microwave or ultrasonic irradiation) that accelerate proteolysis in other ways. Using short digestion times, we show that the percentage of solvent accessible residues that can be modified by DEPC increases from 44% to 67% for cytochrome c, 35% to 81% for myoglobin, and 76% to 95% for β-2-microglobulin. In effect, these increased numbers of modified residues improve the protein structural resolution available from this covalent labeling method. As compared to typical overnight digestion conditions, the short digestion times decrease the average distance between modified residues from 11 Å to 7 Å for myoglobin, 13 Å to 10 Å for

  2. Increased Protein Structural Resolution from Diethylpyrocarbonate-based Covalent Labeling and Mass Spectrometric Detection

    NASA Astrophysics Data System (ADS)

    Zhou, Yuping; Vachet, Richard W.

    2012-04-01

    Covalent labeling and mass spectrometry are seeing increased use together as a way to obtain insight into the 3-dimensional structure of proteins and protein complexes. Several amino acid specific (e.g., diethylpyrocarbonate) and non-specific (e.g., hydroxyl radicals) labeling reagents are available for this purpose. Diethylpyrocarbonate (DEPC) is a promising labeling reagent because it can potentially probe up to 30% of the residues in the average protein and gives only one reaction product, thereby facilitating mass spectrometric analysis. It was recently reported, though, that DEPC modifications are labile for some amino acids. Here, we show that label loss is more significant and widespread than previously thought, especially for Ser, Thr, Tyr, and His residues, when relatively long protein digestion times are used. Such label loss ultimately decreases the amount of protein structural information that is obtainable with this reagent. We find, however, that the number of DEPC modified residues and, thus, protein structural information, can be significantly increased by decreasing the time between the covalent labeling reaction and the mass spectrometric analysis. This is most effectively accomplished using short (e.g., 2 h) proteolytic digestions with enzymes such as immobilized chymotrypsin or Glu-C rather than using methods (e.g., microwave or ultrasonic irradiation) that accelerate proteolysis in other ways. Using short digestion times, we show that the percentage of solvent accessible residues that can be modified by DEPC increases from 44% to 67% for cytochrome c, 35% to 81% for myoglobin, and 76% to 95% for β-2-microglobulin. In effect, these increased numbers of modified residues improve the protein structural resolution available from this covalent labeling method. Compared with typical overnight digestion conditions, the short digestion times decrease the average distance between modified residues from 11 to 7 Å for myoglobin, 13 to 10 Å for

  3. Constructing covalent interface in rubber/clay nanocomposite by combining structural modification and interlamellar silylation of montmorillonite.

    PubMed

    Zha, Chao; Wang, Wencai; Lu, Yonglai; Zhang, Liqun

    2014-11-12

    Strong interfacial interaction and nanodispersion are necessary for polymer nanocomposites with expectations on mechanical performance. In this work, montmorillonite (MMT) was first structurally modified by acid treatment to produce more silanol groups on the layer surface. This was followed by chemical modification of γ-methacryloxy propyl trimethoxysilane molecule (KH570) through covalent grafting with the silanol groups. (29)Si and (27)Al magic angle spinning (MAS) NMR results revealed the microstructural changes of MMT after acid treatment and confirmed the increase of silanol groups on acid-treated MMT surfaces. Thermogravimetric analysis indicated an increase in the grafted amount of organosilane on the MMT surface. X-ray diffraction (XRD) showed that the functionalization process changed the highly ordered stacking structure of the MMT mineral into a highly disordered structure, indicating successful grafting of organosilane to the interlayer surface of the crystalline sheets. The styrene-butadiene rubber (SBR)/MMT nanocomposites were further prepared by co-coagulating with SBR latex and grafted-MMT aqueous suspension. During vulcanization, a covalent interface between modified MMT and rubber was established through peroxide-radical-initiated reactions, and layer aggregation was effectively prevented. The SBR/MMT nanocomposites had highly and uniformly dispersed MMT layers, and the covalent interfacial interaction was finally achieved and exhibited high performance. PMID:25322875

  4. Covalent functionalization of monolayered transition metal dichalcogenides by phase engineering.

    PubMed

    Voiry, Damien; Goswami, Anandarup; Kappera, Rajesh; e Silva, Cecilia de Carvalho Castro; Kaplan, Daniel; Fujita, Takeshi; Chen, Mingwei; Asefa, Tewodros; Chhowalla, Manish

    2015-01-01

    Chemical functionalization of low-dimensional materials such as nanotubes, nanowires and graphene leads to profound changes in their properties and is essential for solubilizing them in common solvents. Covalent attachment of functional groups is generally achieved at defect sites, which facilitate electron transfer. Here, we describe a simple and general method for covalent functionalization of two-dimensional transition metal dichalcogenide nanosheets (MoS₂, WS₂ and MoSe₂), which does not rely on defect engineering. The functionalization reaction is instead facilitated by electron transfer between the electron-rich metallic 1T phase and an organohalide reactant, resulting in functional groups that are covalently attached to the chalcogen atoms of the transition metal dichalcogenide. The attachment of functional groups leads to dramatic changes in the optoelectronic properties of the material. For example, we show that it renders the metallic 1T phase semiconducting, and gives it strong and tunable photoluminescence and gate modulation in field-effect transistors. PMID:25515889

  5. Electronic properties of two-dimensional covalent organic frameworks.

    PubMed

    Zhu, P; Meunier, V

    2012-12-28

    The electronic properties of a number of two-dimensional covalent organic frameworks are studied using a combination of density functional theory and quasiparticle theory calculations. The effect of composition and system size on the electronic band gap is systematically considered for a series of systems, using van der Waals corrected density functional theory calculations to determine the effect of a graphene substrate on deposited covalent frameworks. We predict that covalent organic frameworks' (COFs') electronic properties, such as their band gap can be fine tuned by appropriate modifications of their structures, specifically by increasing organic chain-links in the framework. The effect of strain on the electronic properties is also studied. The graphene substrate is shown to not significantly alter the properties of COFs, thereby indicating the robustness of COFs' intrinsic properties for practical applications. PMID:23277948

  6. Electronic properties of two-dimensional covalent organic frameworks

    NASA Astrophysics Data System (ADS)

    Zhu, P.; Meunier, V.

    2012-12-01

    The electronic properties of a number of two-dimensional covalent organic frameworks are studied using a combination of density functional theory and quasiparticle theory calculations. The effect of composition and system size on the electronic band gap is systematically considered for a series of systems, using van der Waals corrected density functional theory calculations to determine the effect of a graphene substrate on deposited covalent frameworks. We predict that covalent organic frameworks' (COFs') electronic properties, such as their band gap can be fine tuned by appropriate modifications of their structures, specifically by increasing organic chain-links in the framework. The effect of strain on the electronic properties is also studied. The graphene substrate is shown to not significantly alter the properties of COFs, thereby indicating the robustness of COFs' intrinsic properties for practical applications.

  7. Optical Detection of Enzymatic Activity and Inhibitors on Non-Covalently Functionalized Fluorescent Graphene Oxide.

    PubMed

    Kang, Tae Woog; Jeon, Su-Ji; Kim, Hye-In; Park, Jung Hyun; Yim, DaBin; Lee, Hye-Rim; Ju, Jong-Min; Kim, Man-Jin; Kim, Jong-Ho

    2016-05-24

    It has been of great interest to measure the activity of acetylcholinesterase (AChE) and its inhibitor, as AChE is known to accelerate the aggregation of the amyloid beta peptides that underlie Alzheimer's disease. Herein, we report the development of graphene oxide (GO) fluorescence-based biosensors for the detection of AChE activity and AChE inhibitors. To this end, GO was non-covalently functionalized with phenoxy-modified dextran (PhO-dex-GO) through hydrophobic interaction; the resulting GO showed excellent colloidal stability and intense fluorescence in various aqueous solutions as compared to pristine GO and the GO covalently functionalized with dextran. The fluorescence of PhO-dex-GO remarkably increased as AChE catalyzed the hydrolysis of acetylthiocholine (ATCh) to give thiocholine and acetic acid. It was found that the turn-on fluorescence response of PhO-dex-GO to AChE activity was induced by protonation of carboxyl groups on it from the product of the enzymatic hydrolysis reaction, acetic acid. On the basis of its turn-on fluorescence response, PhO-dex-GO was able to report kinetic and thermodynamic parameters involving a maximum velocity, a Michaelis constant, and an inhibition dissociation constant for AChE activity and inhibition. These parameters enable us to determine the activity of AChE and the efficiency of the inhibitor. PMID:27136042

  8. From Highly Crystalline to Outer Surface-Functionalized Covalent Organic Frameworks—A Modulation Approach

    PubMed Central

    2015-01-01

    Crystallinity and porosity are of central importance for many properties of covalent organic frameworks (COFs), including adsorption, diffusion, and electronic transport. We have developed a new method for strongly enhancing both aspects through the introduction of a modulating agent in the synthesis. This modulator competes with one of the building blocks during the solvothermal COF growth, resulting in highly crystalline frameworks with greatly increased domain sizes reaching several hundreds of nanometers. The obtained materials feature fully accessible pores with an internal surface area of over 2000 m2 g–1. Compositional analysis via NMR spectroscopy revealed that the COF-5 structure can form over a wide range of boronic acid-to-catechol ratios, thus producing frameworks with compositions ranging from highly boronic acid-deficient to networks with catechol voids. Visualization of an −SH-functionalized modulating agent via iridium staining revealed that the COF domains are terminated by the modulator. Using functionalized modulators, this synthetic approach thus also provides a new and facile method for the external surface functionalization of COF domains, providing accessible sites for post-synthetic modification reactions. We demonstrate the feasibility of this concept by covalently attaching fluorescent dyes and hydrophilic polymers to the COF surface. We anticipate that the realization of highly crystalline COFs with the option of additional surface functionality will render the modulation concept beneficial for a range of applications, including gas separations, catalysis, and optoelectronics. PMID:26694214

  9. From Highly Crystalline to Outer Surface-Functionalized Covalent Organic Frameworks--A Modulation Approach.

    PubMed

    Calik, Mona; Sick, Torben; Dogru, Mirjam; Döblinger, Markus; Datz, Stefan; Budde, Harald; Hartschuh, Achim; Auras, Florian; Bein, Thomas

    2016-02-01

    Crystallinity and porosity are of central importance for many properties of covalent organic frameworks (COFs), including adsorption, diffusion, and electronic transport. We have developed a new method for strongly enhancing both aspects through the introduction of a modulating agent in the synthesis. This modulator competes with one of the building blocks during the solvothermal COF growth, resulting in highly crystalline frameworks with greatly increased domain sizes reaching several hundreds of nanometers. The obtained materials feature fully accessible pores with an internal surface area of over 2000 m(2) g(-1). Compositional analysis via NMR spectroscopy revealed that the COF-5 structure can form over a wide range of boronic acid-to-catechol ratios, thus producing frameworks with compositions ranging from highly boronic acid-deficient to networks with catechol voids. Visualization of an -SH-functionalized modulating agent via iridium staining revealed that the COF domains are terminated by the modulator. Using functionalized modulators, this synthetic approach thus also provides a new and facile method for the external surface functionalization of COF domains, providing accessible sites for post-synthetic modification reactions. We demonstrate the feasibility of this concept by covalently attaching fluorescent dyes and hydrophilic polymers to the COF surface. We anticipate that the realization of highly crystalline COFs with the option of additional surface functionality will render the modulation concept beneficial for a range of applications, including gas separations, catalysis, and optoelectronics. PMID:26694214

  10. Evaluation of structural and functional properties of chitosan-chlorogenic acid complexes.

    PubMed

    Wei, Zihao; Gao, Yanxiang

    2016-05-01

    The objectives of the present study were to first synthesize chitosan-chlorogenic acid (CA) covalent complex and then compare structural and functional properties between chitosan-CA covalent complex and physical complex. First, chitosan-CA covalent complex was synthesized and its total phenolic content was as high as 276.5 ± 6.2 mg/g. Then structural and functional properties of chitosan-CA covalent and physical complexes were analyzed. The covalent reaction induced formation of both amide and ester bonds in chitosan. Data of X-ray diffraction (XRD) and scanning electron microscopy (SEM) indicated that the complexations of CA changed crystallinity and morphology of chitosan, and covalent complexation induced a larger change of physical structure than physical complexation. In terms of functional properties, chitosan-CA covalent complex exhibited better thermal stability than physical complex in terms of antioxidant activity, and the viscosity of chitosan was significantly increased by covalent modification. PMID:26820353

  11. Stability and electronic properties of 3D covalent organic frameworks.

    PubMed

    Lukose, Binit; Kuc, Agnieszka; Heine, Thomas

    2013-05-01

    Covalent organic frameworks (COFs) are a class of covalently linked crystalline nanoporous materials, versatile for nanoelectronic and storage applications. 3D COFs, in particular, have very large pores and low mass densities. Extensive theoretical studies of their energetic and mechanical stability, as well as their electronic properties, have been carried out for all known 3D COFs. COFs are energetically stable and their bulk modulus ranges from 3 to 20 GPa. Electronically, all COFs are semiconductors with band gaps corresponding to the HOMO-LUMO gaps of the building units. PMID:23212235

  12. On the road towards electroactive covalent organic frameworks.

    PubMed

    Dogru, Mirjam; Bein, Thomas

    2014-05-30

    Covalent organic frameworks (COFs) are a novel class of porous crystalline organic materials assembled from molecular building blocks. The construction principles of these materials allow for the design of precisely controllable structures since their chemical and physical properties can be easily tuned through the selection of the building blocks and the linkage motif. Their extraordinary and versatile properties impart functionality that is of great interest in areas such as gas storage, separation, catalysis and optoelectronics. This feature article discusses key aspects of the design of covalent organic frameworks with a focus on electroactive COFs for potential optoelectronic and photovoltaic applications. PMID:24667827

  13. In vitro degradation of covalently cross-linked arabinoxylan hydrogels by bifidobacteria.

    PubMed

    Martínez-López, Ana L; Carvajal-Millan, Elizabeth; Micard, Valérie; Rascón-Chu, Agustín; Brown-Bojorquez, Francisco; Sotelo-Cruz, Norberto; López-Franco, Yolanda L; Lizardi-Mendoza, Jaime

    2016-06-25

    Arabinoxylan gels with different cross-linking densities, swelling ratios, and rheological properties were obtained by increasing the concentration of arabinoxylan from 4 to 6% (w/v) during oxidative gelation by laccase. The degradation of these covalently cross-linked gels by a mixture of two Bifidobacterium strains (Bifidobacterium longum and Bifidobacterium adolescentis) was investigated. The kinetics of the evolution of structural morphology of the arabinoxylan gel, the carbohydrate utilization profiles and the bacterial production of short-acid fatty acid (SCFA) were measured. Scanning electron microscopy analysis of the degraded gels showed multiple cavity structures resulting from the bacterial action. The total SCFA decreased when the degree of cross-linking increased in the gels. A slower fermentation of arabinoxylan chains was obtained for arabinoxylan gels with more dense network structures. These results suggest that the differences in the structural features and properties studied in this work affect the degradation time of the arabinoxylan gels. PMID:27083795

  14. Mechanoassisted Synthesis of Sulfonated Covalent Organic Frameworks with High Intrinsic Proton Conductivity.

    PubMed

    Peng, Yongwu; Xu, Guodong; Hu, Zhigang; Cheng, Youdong; Chi, Chenglong; Yuan, Daqiang; Cheng, Hansong; Zhao, Dan

    2016-07-20

    It is challenging to introduce pendent sulfonic acid groups into modularly built crystalline porous frameworks for intrinsic proton conduction. Herein, we report the mechanoassisted synthesis of two sulfonated covalent organic frameworks (COFs) possessing one-dimensional nanoporous channels decorated with pendent sulfonic acid groups. These COFs exhibit high intrinsic proton conductivity as high as 3.96 × 10(-2) S cm(-1) with long-term stability at ambient temperature and 97% relative humidity (RH). In addition, they were blended with nonconductive polyvinylidene fluoride (PVDF) affording a series of mixed-matrix membranes (MMMs) with proton conductivity up to 1.58 × 10(-2) S cm(-1) and low activation energy of 0.21 eV suggesting the Grotthuss mechanism for proton conduction. Our study has demonstrated the high intrinsic proton conductivity of COFs shedding lights on their wide applications in proton exchange membranes. PMID:27385672

  15. Boronic acid-modified magnetic materials for antibody purification.

    PubMed

    Dhadge, Vijaykumar L; Hussain, Abid; Azevedo, Ana M; Aires-Barros, Raquel; Roque, Ana C A

    2014-02-01

    Aminophenyl boronic acids can form reversible covalent ester interactions with cis-diol-containing molecules, serving as a selective tool for binding glycoproteins as antibody molecules that possess oligosaccharides in both the Fv and Fc regions. In this study, amino phenyl boronic acid (APBA) magnetic particles (MPs) were applied for the magnetic separation of antibody molecules. Iron oxide MPs were firstly coated with dextran to avoid non-specific binding and then with 3-glycidyloxypropyl trimethoxysilane to allow further covalent coupling of APBA (APBA_MP). When contacted with pure protein solutions of human IgG (hIgG) and bovine serum albumin (BSA), APBA_MP bound 170 ± 10 mg hIgG g(-1) MP and eluted 160 ± 5 mg hIgG g(-1) MP, while binding only 15 ± 5 mg BSA g(-1) MP. The affinity constant for the interaction between hIgG and APBA_MP was estimated as 4.9 × 10(5) M(-1) (Ka) with a theoretical maximum capacity of 492 mg hIgG adsorbed g(-1) MP (Qmax), whereas control particles bound a negligible amount of hIgG and presented an estimated theoretical maximum capacity of 3.1 mg hIgG adsorbed g(-1) MP (Qmax). APBA_MPs were also tested for antibody purification directly from CHO cell supernatants. The particles were able to bind 98% of IgG loaded and to recover 95% of pure IgG (purity greater than 98%) at extremely mild conditions. PMID:24258155

  16. Petawatt laser absorption bounded

    NASA Astrophysics Data System (ADS)

    Levy, Matthew; Wilks, Scott; Tabak, Max; Libby, Stephen; Baring, Matthew

    2014-10-01

    The interaction of petawatt (1015 W) lasers with solid matter forms the basis for advanced scientific applications such as table-top relativistic particle accelerators, ultrafast charged particle imaging systems and fast ignition inertial confinement fusion. Key metrics for these applications relate to absorption, yet conditions in this regime are so nonlinear that it is often impossible to know the fraction of absorbed light f, and even the range of f is unknown. In this presentation, using a relativistic Rankine-Hugoniot-like analysis, we show how to derive the theoretical maximum and minimum of f. These boundaries constrain nonlinear absorption mechanisms across the petawatt regime, forbidding high absorption values at low laser power and low absorption values at high laser power. Close agreement is shown with several dozens of published experimental data points and simulation results, helping to confirm the theory. For applications needing to circumvent the absorption bounds, these results will accelerate a shift from solid targets, towards structured and multilayer targets, and lead the development of new materials.

  17. The mitochondrial acyl carrier protein (ACP) coordinates mitochondrial fatty acid synthesis with iron sulfur cluster biogenesis

    PubMed Central

    Van Vranken, Jonathan G; Jeong, Mi-Young; Wei, Peng; Chen, Yu-Chan; Gygi, Steven P; Winge, Dennis R; Rutter, Jared

    2016-01-01

    Mitochondrial fatty acid synthesis (FASII) and iron sulfur cluster (FeS) biogenesis are both vital biosynthetic processes within mitochondria. In this study, we demonstrate that the mitochondrial acyl carrier protein (ACP), which has a well-known role in FASII, plays an unexpected and evolutionarily conserved role in FeS biogenesis. ACP is a stable and essential subunit of the eukaryotic FeS biogenesis complex. In the absence of ACP, the complex is destabilized resulting in a profound depletion of FeS throughout the cell. This role of ACP depends upon its covalently bound 4’-phosphopantetheine (4-PP)-conjugated acyl chain to support maximal cysteine desulfurase activity. Thus, it is likely that ACP is not simply an obligate subunit but also exploits the 4-PP-conjugated acyl chain to coordinate mitochondrial fatty acid and FeS biogenesis. DOI: http://dx.doi.org/10.7554/eLife.17828.001 PMID:27540631

  18. The mitochondrial acyl carrier protein (ACP) coordinates mitochondrial fatty acid synthesis with iron sulfur cluster biogenesis.

    PubMed

    Van Vranken, Jonathan G; Jeong, Mi-Young; Wei, Peng; Chen, Yu-Chan; Gygi, Steven P; Winge, Dennis R; Rutter, Jared

    2016-01-01

    Mitochondrial fatty acid synthesis (FASII) and iron sulfur cluster (FeS) biogenesis are both vital biosynthetic processes within mitochondria. In this study, we demonstrate that the mitochondrial acyl carrier protein (ACP), which has a well-known role in FASII, plays an unexpected and evolutionarily conserved role in FeS biogenesis. ACP is a stable and essential subunit of the eukaryotic FeS biogenesis complex. In the absence of ACP, the complex is destabilized resulting in a profound depletion of FeS throughout the cell. This role of ACP depends upon its covalently bound 4'-phosphopantetheine (4-PP)-conjugated acyl chain to support maximal cysteine desulfurase activity. Thus, it is likely that ACP is not simply an obligate subunit but also exploits the 4-PP-conjugated acyl chain to coordinate mitochondrial fatty acid and FeS biogenesis. PMID:27540631

  19. Direct Covalent Biomolecule Immobilization on Plasma-Nanotextured Chemically Stable Substrates.

    PubMed

    Tsougeni, K; Petrou, P S; Awsiuk, K; Marzec, M M; Ioannidis, N; Petrouleas, V; Tserepi, A; Kakabakos, S E; Gogolides, E

    2015-07-15

    A new method for direct covalent immobilization of protein molecules (including antibodies) on organic polymers with plasma-induced random micronanoscale topography and stable-in-time chemical functionality is presented. This is achieved using a short (1-5 min) plasma etching and simultaneous micronanotexturing process, followed by a fast thermal annealing step, which induces accelerated hydrophobic recovery while preserving important chemical functionality created by the plasma. Surface-bound biomolecules resist harsh washing with sodium dodecyl sulfate and other detergents even at elevated temperatures, losing less than 40% of the biomolecules bound even at the harshest washing conditions. X-ray photoelectron spectroscopy, secondary-ion mass spectrometry, and electron paramagnetic resonance are used to unveil the chemical modification of the plasma-treated and stabilized surfaces. The nanotextured and chemically stabilized surfaces are used as substrates for the development of immunochemical assays for the sensitive detection of C-reactive protein and salmonella lipopolysaccharides through immobilization of the respective analyte-specific antibodies onto them. Such substrates are stable for a period of 1 year with ambient storage. PMID:26098201

  20. Studies on the metabolism of chlorotrianisene to a reactive intermediate and subsequent covalent binding to microsomal proteins

    SciTech Connect

    Juedes, M.J.

    1989-01-01

    The studies on chlorotrianisene were conducted to determine whether metabolism of chlorotrianisene occurs via the cytochrome P450 monooxygenase system and whether a reactive intermediate is being formed that is capable of binding covalently to microsomal proteins. ({sup 3}H)-chlorotrianisene was incubated with liver microsomes supplemented with NADPH. At the termination of the incubation, the protein was trapped on a glass filter and the unbound chlorotrianisene was removed by extensive washing of the protein with organic solvent. A dramatic stimulation of covalent binding was demonstrated in microsomes from rats treated with methylcholanthrene (60 fold increase) versus control or phenobarbital treatment. Verification of covalent binding was achieved by localization of radiolabeled bands following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the macromolecules in the incubation mixture. Further analysis of the radiolabeled macromolecules separated on SDS-PAGE revealed that these macromolecules were degraded by protease degradation indicating that the macromolecules were proteins. Further investigations were done to determine the cause of the dramatic stimulation of covalent binding detected in microsomes from methylcholanthrene treated rats versus control or phenobarbital treated rats. Further evidence for the participation of P-450c was obtained with a reconstituted cytochrome P-450 system. Incubations of chlorotrianisene with reconstituted P-450c and NADPH-cytochrome P-450 reductase exhibited covalent binding characteristics comparable to those seen in microsomal incubations. Investigations into the nature of the binding site and the reactive intermediate are currently being conducted. By analyzing the BSA adduct, the author intends to isolate the specific amino acid binding site(s).

  1. Covalent organic frameworks as pH responsive signaling scaffolds.

    PubMed

    Zhang, Yuwei; Shen, Xiaochen; Feng, Xiao; Xia, Hong; Mu, Ying; Liu, Xiaoming

    2016-09-25

    A β-ketoenamine based covalent organic framework, COF-JLU4, was synthesized by condensation of 2,5-dimethoxyterephthalohydrazide with triformylphloroglucinol under solvothermal conditions. This COF has strong crystallinity, good porosity, photoluminescence properties and wettability for water. It can serve as the first COF-based fluorescent pH sensor in aqueous solutions. PMID:27545686

  2. Valence, Covalence, Hypervalence, Oxidation State, and Coordination Number

    ERIC Educational Resources Information Center

    Smith, Derek W.

    2005-01-01

    Valence as a numerical measure of an atom's combining power, expressed by the number of bonds it forms in a molecular formulation of the compound in question, was unable to cope with coordination compounds. The covalence of an atom is the nearest model equivalent, but is subject to ambiguity since it often depends on which bonding model is being…

  3. Repeatable mechanochemical activation of dynamic covalent bonds in thermoplastic elastomers.

    PubMed

    Imato, Keiichi; Kanehara, Takeshi; Nojima, Shiki; Ohishi, Tomoyuki; Higaki, Yuji; Takahara, Atsushi; Otsuka, Hideyuki

    2016-08-18

    Repeated mechanical scission and recombination of dynamic covalent bonds incorporated in segmented polyurethane elastomers are demonstrated by utilizing a diarylbibenzofuranone-based mechanophore and by the design of the segmented polymer structures. The repeated mechanochemical reactions can accompany clear colouration and simultaneous fading. PMID:27424868

  4. Nanoscale covalent organic frameworks as smart carriers for drug delivery.

    PubMed

    Bai, Linyi; Phua, Soo Zeng Fiona; Lim, Wei Qi; Jana, Avijit; Luo, Zhong; Tham, Huijun Phoebe; Zhao, Lingzhi; Gao, Qiang; Zhao, Yanli

    2016-03-18

    Two porous covalent organic frameworks (COFs) with good biocompatibility were employed as drug nanocarriers, where three different drugs were loaded for subsequent drug release in vitro. The present work demonstrates that COFs are applicable in drug delivery for therapeutic applications. PMID:26877025

  5. Fabrication of bilayer tetrathiafulvalene integrated surface covalent organic frameworks.

    PubMed

    Dong, Wei-Long; Li, Shu-Ying; Yue, Jie-Yu; Wang, Cheng; Wang, Dong; Wan, Li-Jun

    2016-06-29

    A bilayer covalent organic framework (COF) of TTF-based building blocks was obtained by imine reaction between tetrathiafulvalene tetraaldehyde (4ATTF) and p-phenylenediamine (PPDA). Direct evidence for the eclipsed stacking of bilayer structure via π-π interaction between TTF units is provided by high resolution scanning tunneling microscopy. PMID:27314983

  6. Covalent binding to glutathione of the DNA-alkylating antitumor agent, S23906-1.

    PubMed

    David-Cordonnier, Marie-Hélène; Laine, William; Joubert, Alexandra; Tardy, Christelle; Goossens, Jean-François; Kouach, Mostafa; Briand, Gilbert; Thi Mai, Huong Doan; Michel, Sylvie; Tillequin, Francois; Koch, Michel; Leonce, Stéphane; Pierre, Alain; Bailly, Christian

    2003-07-01

    The benzoacronycine derivative, S23906-1, was characterized recently as a novel potent antitumor agent through alkylation of the N2 position of guanines in DNA. We show here that its reactivity towards DNA can be modulated by glutathione (GSH). The formation of covalent adducts between GSH and S23906-1 was evidenced by EI-MS, and the use of different GSH derivatives, amino acids and dipeptides revealed that the cysteine thiol group is absolutely required for complex formation because glutathione disulfide (GSSG) and other S-blocked derivatives failed to react covalently with S23906-1. Gel shift assays and fluorescence measurements indicated that the binding of S23906-1 to DNA and to GSH are mutually exclusive. Binding of S23906-1 to an excess of GSH prevents DNA alkylation. Additional EI-MS measurements performed with the mixed diester, S28053-1, showed that the acetate leaving group at the C1 position is the main reactive site in the drug: a reaction scheme common to GSH and guanines is presented. At the cellular level, the presence of GSH slightly reduces the cytotoxic potential of S23906-1 towards KB-3-1 epidermoid carcinoma cells. The GSH-induced threefold reduction of the cytotoxicity of S23906-1 is attributed to the reduced formation of lethal drug-DNA covalent complexes in cells. Treatment of the cells with buthionine sulfoximine, an inhibitor of GSH biosynthesis, facilitates the formation of drug-DNA adducts and promotes the cytotoxic activity. This study identifies GSH as a reactant for the antitumor drug, S23906-1, and illustrates a pathway by which GSH may modulate the cellular sensitivity to this DNA alkylating agent. The results presented here, using GSH as a biological nucleophile, fully support our initial hypothesis that DNA alkylation is the major mechanism of action of the promising anticancer drug S23906-1. PMID:12823555

  7. Model of early self-replication based on covalent complementarity for a copolymer of glycerate-3-phosphate and glycerol-3-phosphate

    NASA Astrophysics Data System (ADS)

    Weber, Arthur L.

    1989-03-01

    Glyceraldehyde-3-phosphate acts as the substrate in a model of early self-replication of a phosphodiester copolymer of glycerate-3-phosphate and glycerol-3-phosphate. This model of self-replication is based on covalent complementarity in which information transfer is mediated by a single covalent bond, in contrast to multiple weak interactions that establish complementarity in nucleic acid replication. This replication model is connected to contemporary biochemistry through its use of glyceraldehyde-3-phosphate, a central metabolite of glycolysis and photosynthesis.

  8. Model of early self-replication based on covalent complementarity for a copolymer of glycerate-3-phosphate and glycerol-3-phosphate

    NASA Technical Reports Server (NTRS)

    Weber, Arthur L.

    1989-01-01

    Glyceraldehyde-3-phosphate acts as the substrate in a model of early self-replication of a phosphodiester copolymer of glycerate-3-phosphate and glycerol-3-phosphate. This model of self-replication is based on covalent complementarity in which information transfer is mediated by a single covalent bond, in contrast to multiple weak interactions that establish complementarity in nucleic acid replication. This replication model is connected to contemporary biochemistry through its use of glyceraldehyde-3-phosphate, a central metabolite of glycolysis and photosynthesis.

  9. NCIPLOT: a program for plotting non-covalent interaction regions.

    PubMed

    Contreras-García, Julia; Johnson, Erin R; Keinan, Shahar; Chaudret, Robin; Piquemal, Jean-Philip; Beratan, David N; Yang, Weitao

    2011-03-01

    Non-covalent interactions hold the key to understanding many chemical, biological, and technological problems. Describing these non-covalent interactions accurately, including their positions in real space, constitutes a first step in the process of decoupling the complex balance of forces that define non-covalent interactions. Because of the size of macromolecules, the most common approach has been to assign van der Waals interactions (vdW), steric clashes (SC), and hydrogen bonds (HBs) based on pairwise distances between atoms according to their van der Waals radii. We recently developed an alternative perspective, derived from the electronic density: the Non-Covalent Interactions (NCI) index [J. Am. Chem. Soc. 2010, 132, 6498]. This index has the dual advantages of being generally transferable to diverse chemical applications and being very fast to compute, since it can be calculated from promolecular densities. Thus, NCI analysis is applicable to large systems, including proteins and DNA, where analysis of non-covalent interactions is of great potential value. Here, we describe the NCI computational algorithms and their implementation for the analysis and visualization of weak interactions, using both self-consistent fully quantum-mechanical, as well as promolecular, densities. A wide range of options for tuning the range of interactions to be plotted is also presented. To demonstrate the capabilities of our approach, several examples are given from organic, inorganic, solid state, and macromolecular chemistry, including cases where NCI analysis gives insight into unconventional chemical bonding. The NCI code and its manual are available for download at http://www.chem.duke.edu/~yang/software.htm. PMID:21516178

  10. Chalcogen-bonded complexes. Selenium-bound adducts of NH3, H2O, PH3, and H2S with OCSe, SCSe, and CSe2.

    PubMed

    Ramasami, Ponnadurai; Ford, Thomas A

    2015-02-01

    Recent ab initio investigations of some complexes formed between carbon dioxide and its analogues carbonyl sulfide, carbonyl selenide, carbon disulfide, and thiocarbonyl selenide and the common bases ammonia, water, phosphine, and hydrogen sulfide have revealed significant differences between the properties of those complexes bound through the oxygen atom of the electron acceptor and their counterparts in which the interaction takes place through a sulfur atom. In each case the interaction is weak, but the structures, interaction energies, and vibrational spectra of the complexes show some regular variations in behavior as the base and the acid are systematically changed. The adducts bound through sulfur present examples of the type of non-covalent interaction known as the chalcogen bond. In this paper we extend the range of electron acceptors to include carbon diselenide, and we explore the effects of substituting selenium for sulfur as the acceptor atom in the complexes of OCSe, SCSe, and CSe2. These adducts are also classified as chalcogen-bonded complexes, and have many features in common with the sulfur-bonded species, but also exhibit some noticeable differences between the two series. PMID:25644370

  11. The stabilizing effects of immobilization in D-amino acid oxidase from Trigonopsis variabilis

    PubMed Central

    Dib, Iskandar; Nidetzky, Bernd

    2008-01-01

    Background Immobilization of Trigonopsis variabilis D-amino acid oxidase (TvDAO) on solid support is the key to a reasonably stable performance of this enzyme in the industrial process for the conversion of cephalosporin C as well as in other biocatalytic applications. Results To provide a mechanistic basis for the stabilization of the carrier-bound oxidase we analyzed the stabilizing effects of immobilization in TvDAO exposed to the stress of elevated temperature and operational conditions. Two different strategies of immobilization were used: multi-point covalent binding to epoxy-activated Sepabeads EC-EP; and non-covalent oriented immobilization of the enzyme through affinity of its N-terminal Strep-tag to Strep-Tactin coated on insoluble particles. At 50°C, the oriented immobilizate was not stabilized as compared to the free enzyme. The structure of TvDAO was stabilized via covalent attachment to Sepabeads EC-EP but concomitantly, binding of the FAD cofactor was weakened. FAD release from the enzyme into solution markedly reduced the positive effect of immobilization on the overall stability of TvDAO. Under conditions of substrate conversion in a bubble-aerated stirred tank reactor, both immobilization techniques as well as the addition of the surfactant Pluronic F-68 stabilized TvDAO by protecting the enzyme from the deleterious effect of gas-liquid interfaces. Immobilization of TvDAO on Sepabeads EC-EP however stabilized the enzyme beyond this effect and led to a biocatalyst that could be re-used in multiple cycles of substrate conversion. Conclusion Multi-point covalent attachment of TvDAO on an isoluble porous carrier provides stabilization against the denaturing effects of high temperature and exposure to a gas-liquid interface. Improvement of binding of the FAD cofactor, probably by using methods of protein engineering, would further enhance the stability of the immobilized enzyme. PMID:18798979

  12. Northwest Outward Bound Instructor's Manual.

    ERIC Educational Resources Information Center

    Northwest Outward Bound School, Portland, OR.

    Instructor responsibilities, procedures for completing activities safely, and instructional methods and techniques are outlined to assist instructors in the Northwest Outward Bound School (Portland, Oregon) as they strive for teaching excellence. Information is organized into six chapters addressing: history and philosophy of Outward Bound; course…

  13. Fractional diffusion on bounded domains

    DOE PAGESBeta

    Defterli, Ozlem; D'Elia, Marta; Du, Qiang; Gunzburger, Max Donald; Lehoucq, Richard B.; Meerschaert, Mark M.

    2015-03-13

    We found that the mathematically correct specification of a fractional differential equation on a bounded domain requires specification of appropriate boundary conditions, or their fractional analogue. In this paper we discuss the application of nonlocal diffusion theory to specify well-posed fractional diffusion equations on bounded domains.

  14. Bounds for Asian basket options

    NASA Astrophysics Data System (ADS)

    Deelstra, Griselda; Diallo, Ibrahima; Vanmaele, Michèle

    2008-09-01

    In this paper we propose pricing bounds for European-style discrete arithmetic Asian basket options in a Black and Scholes framework. We start from methods used for basket options and Asian options. First, we use the general approach for deriving upper and lower bounds for stop-loss premia of sums of non-independent random variables as in Kaas et al. [Upper and lower bounds for sums of random variables, Insurance Math. Econom. 27 (2000) 151-168] or Dhaene et al. [The concept of comonotonicity in actuarial science and finance: theory, Insurance Math. Econom. 31(1) (2002) 3-33]. We generalize the methods in Deelstra et al. [Pricing of arithmetic basket options by conditioning, Insurance Math. Econom. 34 (2004) 55-57] and Vanmaele et al. [Bounds for the price of discrete sampled arithmetic Asian options, J. Comput. Appl. Math. 185(1) (2006) 51-90]. Afterwards we show how to derive an analytical closed-form expression for a lower bound in the non-comonotonic case. Finally, we derive upper bounds for Asian basket options by applying techniques as in Thompson [Fast narrow bounds on the value of Asian options, Working Paper, University of Cambridge, 1999] and Lord [Partially exact and bounded approximations for arithmetic Asian options, J. Comput. Finance 10 (2) (2006) 1-52]. Numerical results are included and on the basis of our numerical tests, we explain which method we recommend depending on moneyness and time-to-maturity.

  15. Covalent Binding of Flavins to RnfG and RnfD in the Rnf Complex from Vibrio cholerae

    PubMed Central

    Backiel, Julianne; Juárez, Oscar; Zagorevski, Dmitri V.; Wang, Zhenyu; Nilges, Mark J.; Barquera, Blanca

    2009-01-01

    Enzymes of the Rnf family are believed to be bacterial redox-driven ion pumps, coupling an oxidoreduction process to the translocation of Na+ across the cell membrane. Here we show for the first time that Rnf is a flavoprotein, with FMN covalently bound to threonine-175 in RnfG and a second flavin bound to threonine-187 in RnfD. Rnf subunits D and G are homologous to subunits B and C of Na+-NQR, respectively. Each of these Na+-NQR subunits includes a conserved S(T)GAT motif, with FMN covalently bound to the final threonine. RnfD and RnfG both contain the same motif, suggesting that they bind flavins in a similar way. In order to investigate this, the genes for RnfD and RnfG from Vibrio cholerae were cloned and expressed individually in that organism. In both cases the produced protein fluoresced under UV illumination on an SDS gel, further indicating the presence of flavin. However, analysis of the mutants RnfG-T175L, RnfD-T278L, and RnfD-T187V showed that RnfG-T175 and RnfD-T187 are the likely flavin ligands. This indicates that, in the case of RnfD, the flavin is bound, not to the SGAT sequence but to the final residues of a TMAT sequence, a novel variant of the flavin binding motif. In the case of RnfG, flavin analysis, followed by MALDI-TOF-TOF mass spectrometry, showed that an FMN is covalently attached to threonine-175, the final threonine of the S(T)GAT sequence. Studies by visible, EPR, and ENDOR spectroscopy showed that, upon partial reduction, the isolated RnfG produces a neutral semiquinone intermediate. The semiquinone species disappeared upon full reduction and was not observed in the denatured protein. A topological analysis combining reporter protein fusion and computer predictions indicated that the flavins in RnfG and RnfD are localized in the periplasmic space. In contrast, in NqrC and NqrB the flavins are located in a cytoplasmic loop. This topological analysis suggests that there may be mechanistic differences between the Rnf and Na

  16. COVALENT BINDING OF TRICHLOROETHYLENE TO PROTEINS IN HUMAN AND RAT HEPATOCYTES. (R826409)

    EPA Science Inventory

    The environmental contaminant and occupational solvent trichloroethylene is metabolized to a reactive intermediate that covalently binds to specific hepatic proteins in exposed mice and rats. In order to compare covalent binding between humans and rodents, primary hepatocyte c...

  17. Covalent attachment of functionalized lipid bilayers to planar waveguides for measuring protein binding to biomimetic membranes.

    PubMed Central

    Heyse, S.; Vogel, H.; Sänger, M.; Sigrist, H.

    1995-01-01

    A new method is presented for measuring sensitively the interactions between ligands and their membrane-bound receptors in situ using integrated optics, thus avoiding the need for additional labels. Phospholipid bilayers were attached covalently to waveguides by a novel protocol, which can in principle be used with any glass-like surface. In a first step, phospholipids carrying head-group thiols were covalently immobilized onto SiO2-TiO2 waveguide surfaces. This was accomplished by acylation of aminated waveguides with the heterobifunctional crosslinker N-succinimidyl-3-maleimidopropionate, followed by the formation of thioethers between the surface-grafted maleimides and the synthetic thiolipids. The surface-attached thiolipids served as hydrophobic templates and anchors for the deposition of a complete lipid bilayer either by fusion of lipid vesicles or by lipid self-assembly from mixed lipid/detergent micelles. The step-by-step lipid bilayer formation on the waveguide surface was monitored in situ by an integrated optics technique, allowing the simultaneous determination of optical thickness and one of the two refractive indices of the adsorbed organic layers. Surface coverages of 50-60% were calculated for thiolipid layers. Subsequent deposition of POPC resulted in an overall lipid layer thickness of 45-50 A, which corresponds to the thickness of a fluid bilayer membrane. Specific recognition reactions occurring at cell membrane surfaces were modeled by the incorporation of lipid-anchored receptor molecules into the supported bilayer membranes. (1) The outer POPC layer was doped with biotinylated phosphatidylethanolamine. Subsequent specific binding of streptavidin was optically monitored. (2) A lipopeptide was incorporated in the outer POPC monolayer. Membrane binding of monoclonal antibodies, which were directed against the peptide moiety of the lipopeptide, was optically detected. The specific antibody binding correlated well with the lipopepitde

  18. Ions colliding with clusters of fullerenes-Decay pathways and covalent bond formations

    SciTech Connect

    Seitz, F.; Zettergren, H.; Chen, T.; Gatchell, M.; Alexander, J. D.; Stockett, M. H.; Schmidt, H. T.; Cederquist, H.; Rousseau, P.; Chesnel, J. Y.; Capron, M.; Poully, J. C.; Mery, A.; Maclot, S.; Adoui, L.; Wang, Y.; Martin, F.; Rangama, J.; Domaracka, A.; Vizcaino, V. [CIMAP, UMR 6252, CEA and others

    2013-07-21

    We report experimental results for the ionization and fragmentation of weakly bound van der Waals clusters of n C{sub 60} molecules following collisions with Ar{sup 2+}, He{sup 2+}, and Xe{sup 20+} at laboratory kinetic energies of 13 keV, 22.5 keV, and 300 keV, respectively. Intact singly charged C{sub 60} monomers are the dominant reaction products in all three cases and this is accounted for by means of Monte Carlo calculations of energy transfer processes and a simple Arrhenius-type [C{sub 60}]{sub n}{sup +}{yields}C{sub 60}{sup +}+(n-1)C{sub 60} evaporation model. Excitation energies in the range of only {approx}0.7 eV per C{sub 60} molecule in a [C{sub 60}]{sub 13}{sup +} cluster are sufficient for complete evaporation and such low energies correspond to ion trajectories far outside the clusters. Still we observe singly and even doubly charged intact cluster ions which stem from even more distant collisions. For penetrating collisions the clusters become multiply charged and some of the individual molecules may be promptly fragmented in direct knock-out processes leading to efficient formations of new covalent systems. For Ar{sup 2+} and He{sup 2+} collisions, we observe very efficient C{sub 119}{sup +} and C{sub 118}{sup +} formation and molecular dynamics simulations suggest that they are covalent dumb-bell systems due to bonding between C{sub 59}{sup +} or C{sub 58}{sup +} and C{sub 60} during cluster fragmentation. In the Ar{sup 2+} case, it is possible to form even smaller C{sub 120-2m}{sup +} molecules (m= 2-7), while no molecular fusion reactions are observed for the present Xe{sup 20+} collisions.

  19. Cationic polymethacrylates with covalently linked membrane destabilizing peptides as gene delivery vectors.

    PubMed

    Funhoff, Arjen M; van Nostrum, Cornelus F; Lok, Martin C; Kruijtzer, John A W; Crommelin, Daan J A; Hennink, Wim E

    2005-01-01

    A membrane-disrupting peptide derived from the influenza virus was covalently linked to different polymethacrylates (pDMAEMA, pDAMA and the degradable pHPMA-DMAE, monomers depicted in Fig. 1) using N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) as coupling agent to increase the transfection efficiency of polyplexes based on these polymers. It was shown by circular dichroism (CD) measurements that the polymer-conjugated peptide was, as the free peptide, able to undergo a conformational change of a random coil to an alpha helix upon lowering the pH to 5.0. This indicates that the property of the peptide to destabilize the endosomal membrane was preserved after its conjugation to the cationic polymers. In line herewith, a liposome leakage assay revealed that the polymer-bound peptide has comparable activity as the free peptide. The DNA condensing properties of the synthesized polymer-peptide conjugates were studied with dynamic light scattering and zeta-potential measurements, and it was shown that small (100 to 250 nm), positively charged (+15 to +20 mV) particles were formed. In vitro transfection and toxicity was tested in COS-7 cells, and these experiments showed that the polyplexes with grafted peptide had a substantially higher transfection activity than the control polyplexes, while the toxicity remained unchanged. Cellular uptake of the polyplexes was visualized with confocal laser scanning microscopy, and no differences in cellular uptake could be determined between the peptide containing systems and the control formulation. This shows that the increased transfection activity is indeed due to a better endosomal escape of the peptide grafted polyplexes. This study demonstrates that it is possible to covalently conjugate an endosome disruptive peptide to cationic gene delivery polymers with preservation of its membrane destabilization activity, making these conjugates suitable for in vivo DNA delivery. PMID:15588908

  20. A Covalent Linker Allows for Membrane Targeting of An Oxylipin Biosynthetic Complex

    SciTech Connect

    Gilbert, N.C.; Niebuhr, M.; Tsuruta, H.; Bordelon, T.; Ridderbusch, O.; Dassey, A.; Brash, A.R.; Bartlett, S.G.; Newcomer, M.E.

    2009-05-18

    A naturally occurring bifunctional protein from Plexaura homomalla links sequential catalytic activities in an oxylipin biosynthetic pathway. The C-terminal lipoxygenase (LOX) portion of the molecule catalyzes the transformation of arachidonic acid (AA) to the corresponding 8R-hydroperoxide, and the N-terminal allene oxide synthase (AOS) domain promotes the conversion of the hydroperoxide intermediate to the product allene oxide (AO). Small-angle X-ray scattering data indicate that in the absence of a covalent linkage the two catalytic domains that transform AA to AO associate to form a complex that recapitulates the structure of the bifunctional protein. The SAXS data also support a model for LOX and AOS domain orientation in the fusion protein inferred from a low-resolution crystal structure. However, results of membrane binding experiments indicate that covalent linkage of the domains is required for Ca2+-dependent membrane targeting of the sequential activities, despite the noncovalent domain association. Furthermore, membrane targeting is accompanied by a conformational change as monitored by specific proteolysis of the linker that joins the AOS and LOX domains. Our data are consistent with a model in which Ca2+-dependent membrane binding relieves the noncovalent interactions between the AOS and LOX domains and suggests that the C2-like domain of LOX mediates both protein-protein and protein-membrane interactions.

  1. Mechanistic studies of two-dimensional covalent organic frameworks rapidly polymerized from initially homogenous conditions.

    PubMed

    Smith, Brian J; Dichtel, William R

    2014-06-18

    Covalent organic frameworks (COFs) are periodic two- and three-dimensional (2D and 3D) polymer networks with high surface areas, low densities, and designed structures. Despite intense interest in framework materials, the nucleation and growth processes of COFs, and even of more established metal-organic frameworks (MOFs), are poorly understood. The kinetics of COF growth under varied reaction conditions provides mechanistic insight needed to improve their crystallinity and rationally synthesize new materials. Such kinetic measurements are unprecedented and difficult to perform on typical heterogeneous COF reaction mixtures. Here we synthesize 2D boronate ester-linked COF-5 under conditions in which the monomers are fully soluble. These homogeneous growth conditions provide equal or better material quality compared to any previous report and enable the first rigorous studies of the early stages of COF growth. COF-5 forms within minutes, and the precipitation rate is readily quantified from optical turbidity measurements. COF-5 formation follows an Arrhenius temperature dependence between 60-90 °C with an activation energy of 22-27 kcal/mol. The measured rate law includes a second order in both boronic acid and catechol moieties, and inverse second order in MeOH concentration. A competitive monofunctional catechol slows COF-5 formation but does not redissolve already precipitated COF, indicating both dynamic covalent bond formation and irreversible precipitation. Finally, stoichiometric H2O provides a 4-fold increase in crystallite domain areas, representing the first rational link between reaction conditions and material quality. PMID:24892961

  2. Rapid Covalent Modification of Silicon Oxide Surfaces through Microwave-Assisted Reactions with Alcohols.

    PubMed

    Lee, Austin W H; Gates, Byron D

    2016-07-26

    We demonstrate the method of a rapid covalent modification of silicon oxide surfaces with alcohol-containing compounds with assistance by microwave reactions. Alcohol-containing compounds are prevalent reagents in the laboratory, which are also relatively easy to handle because of their stability against exposure to atmospheric moisture. The condensation of these alcohols with the surfaces of silicon oxides is often hindered by slow reaction kinetics. Microwave radiation effectively accelerates this condensation reaction by heating the substrates and/or solvents. A variety of substrates were modified in this demonstration, such as silicon oxide films of various thicknesses, glass substrates such as microscope slides (soda lime), and quartz. The monolayers prepared through this strategy demonstrated the successful formation of covalent surface modifications of silicon oxides with water contact angles of up to 110° and typical hysteresis values of 2° or less. An evaluation of the hydrolytic stability of these monolayers demonstrated their excellent stability under acidic conditions. The techniques introduced in this article were successfully applied to tune the surface chemistry of silicon oxides to achieve hydrophobic, oleophobic, and/or charged surfaces. PMID:27396288

  3. Cysteine-reactive covalent capture tags for enrichment of cysteine-containing peptides.

    PubMed

    Giron, Priscille; Dayon, Loïc; Mihala, Nikolett; Sanchez, Jean-Charles; Rose, Keith

    2009-11-01

    Considering the tremendous complexity and the wide dynamic range of protein samples from biological origin and their proteolytic peptide mixtures, proteomics largely requires simplification strategies. One common approach to reduce sample complexity is to target a particular amino acid in proteins or peptides, such as cysteine (Cys), with chemical tags in order to reduce the analysis to a subset of the whole proteome. The present work describes the synthesis and the use of two new cysteinyl tags, so-called cysteine-reactive covalent capture tags (C3T), for the isolation of Cys-containing peptides. These bifunctional molecules were specifically designed to react with cysteines through iodoacetyl and acryloyl moieties and permit efficient selection of the tagged peptides. To do so, a thioproline was chosen as the isolating group to form, after a deprotection/activation step, a thiazolidine with an aldehyde resin by the covalent capture (CC) method. The applicability of the enrichment strategy was demonstrated on small synthetic peptides as well as on peptides derived from digested proteins. Mass spectrometric (MS) analysis and tandem mass spectrometric (MS/MS) sequencing confirmed the efficient and straightforward selection of the cysteine-containing peptides. The combination of C3T and CC methods provides an effective alternative to reduce sample complexity and access low abundance proteins. PMID:19813279

  4. Covalent anchoring of chloroperoxidase and glucose oxidase on the mesoporous molecular sieve SBA-15.

    PubMed

    Jung, Dirk; Streb, Carsten; Hartmann, Martin

    2010-01-01

    Functionalization of porous solids plays an important role in many areas, including heterogeneous catalysis and enzyme immobilization. In this study, large-pore ordered mesoporous SBA-15 molecular sieves were synthesized with tetraethyl orthosilicate (TEOS) in the presence of the non-ionic triblock co-polymer Pluronic P123 under acidic conditions. These materials were grafted with 3-aminopropyltrimethoxysilane (ATS), 3-glycidoxypropyltrimethoxysilane (GTS) and with 3-aminopropyltrimethoxysilane and glutaraldehyde (GA-ATS) in order to provide covalent anchoring points for enzymes. The samples were characterized by nitrogen adsorption, powder X-ray diffraction, solid-state NMR spectroscopy, elemental analysis, diffuse reflectance fourier transform infrared spectroscopy and diffuse reflectance UV/Vis spectroscopy. The obtained grafted materials were then used for the immobilization of chloroperoxidase (CPO) and glucose oxidase (GOx) and the resulting biocatalysts were tested in the oxidation of indole. It is found that enzymes anchored to the mesoporous host by the organic moieties can be stored for weeks without losing their activity. Furthermore, the covalently linked enzymes are shown to be less prone to leaching than the physically adsorbed enzymes, as tested in a fixed-bed reactor under continuous operation conditions. PMID:20386667

  5. Mass Spectrometric Studies of Non-Covalent Binding Interactions Between Metallointercalators and DNA

    NASA Astrophysics Data System (ADS)

    Urathamakul, Thitima; Talib, Jihan; Beck, Jennifer L.; Ralph, Stephen F.

    Over the past 2 decades there has been increasing interest in metal complexes that bind non-covalently to DNA, driven in part by a host of potential applications for molecules that can accomplish this task with high affinity and selectivity. As a result many workers have used a wide variety of experimental techniques, several of which are discussed in other chapters of this book, to unravel the details of the precise intermolecular interactions involved. Here we discuss one of the most recent additions to the armory of techniques used by chemists to interrogate metal complex/DNA interactions. For the majority of its existence mass spectrometry (MS) has proven to be of enormous advantage to chemists by virtue of its ability to provide the molecular weights of compounds as well as structural information via fragmentation patterns. However, the high energies associated with many earlier MS techniques which result in fragmentation of covalent bonds, prevent its application for studying weaker intermolecular interactions. The advent of soft ionisation methods such as matrix assisted laser desorption ionisation (MALDI) and electrospray ionisation (ESI) has revolutionised mass spectrometric analysis of biomolecules, by allowing these normally fragile molecules to be introduced into the gas phase for analysis with minimal, if any, fragmentation. It was then recognised that ESI-MS, in particular, might be suitable for investigating non-covalent interactions between small molecules and either proteins or nucleic acids. This was confirmed by a number of early studies involving organic intercalators and minor groove binding ligands, prompting our interest in evaluating ESI-MS as a tool for studying non-covalent interactions between metal complexes and DNA. This chapter contains a discussion of the basic principles behind ESI-MS that enable it to introduce representative samples of solutions containing metal complexes and DNA into the gas phase for analysis. This will be

  6. Gold Nanoparticles for Nucleic Acid Delivery

    PubMed Central

    Ding, Ya; Jiang, Ziwen; Saha, Krishnendu; Kim, Chang Soo; Kim, Sung Tae; Landis, Ryan F; Rotello, Vincent M

    2014-01-01

    Gold nanoparticles provide an attractive and applicable scaffold for delivery of nucleic acids. In this review, we focus on the use of covalent and noncovalent gold nanoparticle conjugates for applications in gene delivery and RNA-interference technologies. We also discuss challenges in nucleic acid delivery, including endosomal entrapment/escape and active delivery/presentation of nucleic acids in the cell. PMID:24599278

  7. Drug bioactivation, covalent binding to target proteins and toxicity relevance.

    PubMed

    Zhou, Shufeng; Chan, Eli; Duan, Wei; Huang, Min; Chen, Yu-Zong

    2005-01-01

    A number of therapeutic drugs with different structures and mechanisms of action have been reported to undergo metabolic activation by Phase I or Phase II drug-metabolizing enzymes. The bioactivation gives rise to reactive metabolites/intermediates, which readily confer covalent binding to various target proteins by nucleophilic substitution and/or Schiff's base mechanism. These drugs include analgesics (e.g., acetaminophen), antibacterial agents (e.g., sulfonamides and macrolide antibiotics), anticancer drugs (e.g., irinotecan), antiepileptic drugs (e.g., carbamazepine), anti-HIV agents (e.g., ritonavir), antipsychotics (e.g., clozapine), cardiovascular drugs (e.g., procainamide and hydralazine), immunosupressants (e.g., cyclosporine A), inhalational anesthetics (e.g., halothane), nonsteroidal anti-inflammatory drugs (NSAIDSs) (e.g., diclofenac), and steroids and their receptor modulators (e.g., estrogens and tamoxifen). Some herbal and dietary constituents are also bioactivated to reactive metabolites capable of binding covalently and inactivating cytochrome P450s (CYPs). A number of important target proteins of drugs have been identified by mass spectrometric techniques and proteomic approaches. The covalent binding and formation of drug-protein adducts are generally considered to be related to drug toxicity, and selective protein covalent binding by drug metabolites may lead to selective organ toxicity. However, the mechanisms involved in the protein adduct-induced toxicity are largely undefined, although it has been suggested that drug-protein adducts may cause toxicity either through impairing physiological functions of the modified proteins or through immune-mediated mechanisms. In addition, mechanism-based inhibition of CYPs may result in toxic drug-drug interactions. The clinical consequences of drug bioactivation and covalent binding to proteins are unpredictable, depending on many factors that are associated with the administered drugs and patients

  8. Covalent bonding modulated graphene-metal interfacial thermal transport

    NASA Astrophysics Data System (ADS)

    Jiang, Tao; Zhang, Xueqiang; Vishwanath, Suresh; Mu, Xin; Kanzyuba, Vasily; Sokolov, Denis A.; Ptasinska, Sylwia; Go, David B.; Xing, Huili Grace; Luo, Tengfei

    2016-05-01

    We report the covalent bonding enabled modulation of the interfacial thermal conductance between graphene and metals Cu, Al, and Pt by controlling the oxidation of graphene. By combining comprehensive X-ray photoelectron spectroscopy (XPS) analysis and time-domain thermoreflectance measurements, we quantify the effect of graphene oxidation on interfacial thermal conductance. It was found that thermal conductance increases with the degree of graphene oxidation until a peak value is obtained at an oxygen/carbon atom percentage of ~7.7%. The maximum enhancement in thermal conductance was measured to be 55%, 38%, and 49% for interfaces between oxidized graphene and Cu, Al, and Pt, respectively. In situ XPS measurements show that oxygen covalently binds to Cu and graphene simultaneously, forming a highly efficient bridge to enhance the thermal transport. Our molecular dynamics simulations verify that strong interfacial covalent bonds are the key to the thermal conductance enhancement. This work provides valuable insights into the mechanism of functionalization-induced thermal conductance enhancement and design guidelines for graphene-based devices.We report the covalent bonding enabled modulation of the interfacial thermal conductance between graphene and metals Cu, Al, and Pt by controlling the oxidation of graphene. By combining comprehensive X-ray photoelectron spectroscopy (XPS) analysis and time-domain thermoreflectance measurements, we quantify the effect of graphene oxidation on interfacial thermal conductance. It was found that thermal conductance increases with the degree of graphene oxidation until a peak value is obtained at an oxygen/carbon atom percentage of ~7.7%. The maximum enhancement in thermal conductance was measured to be 55%, 38%, and 49% for interfaces between oxidized graphene and Cu, Al, and Pt, respectively. In situ XPS measurements show that oxygen covalently binds to Cu and graphene simultaneously, forming a highly efficient bridge to enhance

  9. Saturating the holographic entropy bound

    SciTech Connect

    Bousso, Raphael; Freivogel, Ben; Leichenauer, Stefan

    2010-10-15

    The covariant entropy bound states that the entropy, S, of matter on a light sheet cannot exceed a quarter of its initial area, A, in Planck units. The gravitational entropy of black holes saturates this inequality. The entropy of matter systems, however, falls short of saturating the bound in known examples. This puzzling gap has led to speculation that a much stronger bound, S < or approx. A{sup 3/4}, may hold true. In this note, we exhibit light sheets whose entropy exceeds A{sup 3/4} by arbitrarily large factors. In open Friedmann-Robertson-Walker universes, such light sheets contain the entropy visible in the sky; in the limit of early curvature domination, the covariant bound can be saturated but not violated. As a corollary, we find that the maximum observable matter and radiation entropy in universes with positive (negative) cosmological constant is of order {Lambda}{sup -1} ({Lambda}{sup -2}), and not |{Lambda}|{sup -3/4} as had hitherto been believed. Our results strengthen the evidence for the covariant entropy bound, while showing that the stronger bound S < or approx. A{sup 3/4} is not universally valid. We conjecture that the stronger bound does hold for static, weakly gravitating systems.

  10. Arrhenius plot behavior of a. gamma. -radiation-releasable, membrane-bound exonuclease

    SciTech Connect

    Mitchel, R.E.J.

    1981-11-01

    The activation energy of a membrane-bound exonuclease in Micrococcus radiodurans has been measured and the effect of ionizing radiation damage in this sytem explore. The Arrhenius plot for the native bound enzyme was found to be biphasic and the calculated activation energies and transition temperature for the enzymatic reaction were not changed when the enzyme was: (1) solubilized from the membrane with its covalently bound lipid anchor attached, (2) released from the membrane by ionizing radiation, which cleaves off the covalently attached lipid and converts the enzyme from a dimer to a monomer, (3) attached to the membrane after exposure to ionizing radiation under oxic or anoxic conditions, and (4) attached to the membrane in the presence of 10 mM CHCl/sub 3/. Since other membrane-bound enzymes have been shown to be sensitive to membrane perturbations while this one was not, the results suggest that various perturbants, including ionizing radiation, may have differential effects on such enzymes.

  11. Reactivity of Metal Ions Bound to Water-Soluble Polymers

    SciTech Connect

    Sauer, N.N.; Watkins, J.G.; Lin, M.; Birnbaum, E.R.; Robison, T.W.; Smith, B.F.; Gohdes, J.W.; McDonald, J.G.

    1999-06-29

    The intent of this work is to determine the effectiveness of catalysts covalently bound to polymers and to understand the consequences of supporting the catalysts on catalyst efficiency and selectivity. Rhodium phosphine complexes with functional groups for coupling to polymers were prepared. These catalyst precursors were characterized using standard techniques including IR, NMR, and elemental analysis. Studies on the modified catalysts showed that they were still active hydrogenation catalysts. However, tethering of the catalysts to polyamines gave systems with low hydrogenation activity. Analogous biphasic systems were also explored. Phosphine ligands with a surfactant-like structure have been synthesized and used to prepare catalytically active complexes of palladium. The palladium complexes were utilized in Heck-type coupling reactions (e.g. coupling of iodobenzene and ethyl acrylate to produce ethyl cinnamate) under vigorously stirred biphasic reaction conditions, and were found to offer superior performance over a standard water-soluble palladium catalyst under analogous conditions.

  12. Covalently Bound Clusters of Alpha-Substituted PDI-Rival Electron Acceptors to Fullerene for Organic Solar Cells.

    PubMed

    Wu, Qinghe; Zhao, Donglin; Schneider, Alexander M; Chen, Wei; Yu, Luping

    2016-06-15

    A cluster type of electron acceptor, TPB, bearing four α-perylenediimides (PDIs), was developed, in which the four PDIs form a cross-like molecular conformation while still partially conjugated with the BDT-Th core. The blend TPB:PTB7-Th films show favorable morphology and efficient charge dissociation. The inverted solar cells exhibited the highest PCE of 8.47% with the extraordinarily high Jsc values (>18 mA/cm(2)), comparable with those of the corresponding PC71BM/PTB7-Th-based solar cells. PMID:27219665

  13. A Structure-guided Approach to Creating Covalent FGFR Inhibitors

    PubMed Central

    Zhou, Wenjun; Hur, Wooyoung; McDermott, Ultan; Dutt, Amit; Xian, Wa; Picarro, Scott B.; Zhang, Jianming; Sharma, Sreenath V.; Brugge, Joan; Meyerson, Matthew; Settleman, Jeffrey; Gray, Nathanael S.

    2010-01-01

    Summary The fibroblast growth factor receptor tyrosine kinases (FGFR1, 2, 3, and 4) represent promising therapeutic targets in a number of cancers. We have developed the first potent and selective irreversible inhibitor of FGFR1, 2, 3, and 4 which we named FIIN-1 that forms a covalent bond with cysteine 486 located in the P-loop of the FGFR1 ATP-binding site. We demonstrate that the inhibitor potently inhibits Tel-FGFR1 transformed Ba/F3 cells (EC50 = 14 nM) as well as numerous FGFR-dependent cancer cell lines. A biotin-derivatized version of the inhibitor, FIIN-1-biotin, was shown to covalently label FGFR1 at Cys486. FIIN-1 is a useful probe of FGFR-dependent cellular phenomena and may provide a starting point of the development of therapeutically relevant irreversible inhibitors of wild-type and drug-resistant forms of FGFR kinases. PMID:20338520

  14. Fine-Tuning Covalent Inhibition of Bacterial Quorum Sensing.

    PubMed

    Amara, Neri; Gregor, Rachel; Rayo, Josep; Dandela, Rambabu; Daniel, Erik; Liubin, Nina; Willems, H Marjo E; Ben-Zvi, Anat; Krom, Bastiaan P; Meijler, Michael M

    2016-05-01

    Emerging antibiotic resistance among human pathogens has galvanized efforts to find alternative routes to combat bacterial virulence. One new approach entails interfering with the ability of bacteria to coordinate population-wide gene expression, or quorum sensing (QS), thus inhibiting the production of virulence factors and biofilm formation. We have recently developed such a strategy by targeting LasR, the master regulator of QS in the opportunistic human pathogen Pseudomonas aeruginosa, through the rational design of covalent inhibitors closely based on the core structure of the native ligand. We now report several groups of new inhibitors, one of which, fluoro-substituted ITC-12, displayed complete covalent modification of LasR, as well as effective QS inhibition in vitro and promising in vivo results. In addition to their potential clinical relevance, this series of synthetic QS modulators can be used as a tool to further unravel the complicated QS regulation in P. aeruginosa. PMID:26840534

  15. Covalent conjugation of multi-walled carbon nanotubes with proteins.

    PubMed

    Yi, Changqing; Qi, Suijian; Zhang, Dawei; Yang, Mengsu

    2010-01-01

    Linkage of proteins to carbon nanotubes (CNTs) is fundamentally important for applications of CNTs in medicinal and biological fields, as well as in biosensor or chemically modulated nanoelectronic devices. In this contribution, we provide a detailed protocol for the synthesis and characterization of covalent CNT-protein adducts. Functionalization of multiwalled carbon nanotubes (MWCNTs) with proteins has been achieved by the initial carboxylation of MWCNTs followed by amidation with the desired proteins. Attenuated total reflection Fourier transform infrared (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS) measurements validated the presence of a covalent linkage between MWCNTs and proteins. The visualization of proteins on the surface of MWCNTs was furthermore achieved using atomic force microscopy (AFM). The protein-conjugated nanocomposites can also be assembled into multidimensional addressable heterostructures through highly specific biomolecular recognition system (e.g., antibody-antigen). PMID:20422377

  16. Covalent organic frameworks (COFs): from design to applications.

    PubMed

    Ding, San-Yuan; Wang, Wei

    2013-01-21

    Covalent organic frameworks (COFs) represent an exciting new type of porous organic materials, which are ingeniously constructed with organic building units via strong covalent bonds. The well-defined crystalline porous structures together with tailored functionalities have offered the COF materials superior potential in diverse applications, such as gas storage, adsorption, optoelectricity, and catalysis. Since the seminal work of Yaghi and co-workers in 2005, the rapid development in this research area has attracted intensive interest from researchers with diverse expertise. This critical review describes the state-of-the-art development in the design, synthesis, characterisation, and application of the crystalline porous COF materials. Our own opinions on further development of the COF materials are also presented for discussion (155 references). PMID:23060270

  17. Reconstruction of Covalent Organic Frameworks by Dynamic Equilibrium.

    PubMed

    Gao, Qiang; Bai, Linyi; Zeng, Yongfei; Wang, Peng; Zhang, Xiaojing; Zou, Ruqiang; Zhao, Yanli

    2015-11-16

    Covalent organic frameworks (COFs) are periodic two- or three-dimensional polymeric networks with high surface areas, low density, and designed structures. Because COFs are normally prepared based on reversible formation of covalent bonds with relatively weak stability, their structures can be easily broken or damaged due to changes in the surrounding enviro